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 2015 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 Medicaid Manuals for coverage 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 15 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. 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 a multiplex mutation screening assay or next-generation sequencing (NGS). 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 MET amplification to predict response to crizotinib ROS1 rearrangements to predict response to crizotinib Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 2

3 RET rearrangements to predict response to cabozantinib 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. 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. 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 will be replaced by ICD-10 code sets. Health Net National Medical Policies will now include the preliminary ICD-10 codes in preparation for this transition. Please note that these may not be the final versions of the codes and Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 3

4 that will not be accepted for billing or payment purposes until the October 1, 2015 implementation date. 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 HCPCS Codes N/A 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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 4

5 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 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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 5

6 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 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. Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 6

7 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 (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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 7

8 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 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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 8

9 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 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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 9

10 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 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. Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 10

11 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 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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 11

12 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. 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 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 12

13 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. Clinicopathologic characteristics, predominant histologic subtype and cytomorphology were assessed in fusion-positive lung adenocarcinomas and compared to those harboring EGFR, KRAS, HER2 or BRAF mutations. There were 58 (5.1%) ALK fusions, 11 (1.0%) ROS1 fusions and 15 (1.3%) RET fusions. Tumors with ROS1 fusions had significantly larger diameter than ROS1 fusion-negative tumors, whereas all the 15 tumors harboring RET fusions were 3 cm in diameter. The three fusion genes were all more prevalent in solid-predominant adenocarcinoma. Compared to fusion-negative lung adenocarcinomas, tumors harboring a fusion gene had significantly higher prevalence of extracellular mucin, cribriform pattern, signet ring cells and hepatoid cytology. No significant difference in relapse-free survival and overall survival was observed between fusion-positive and fusion-negative patients. Investigators concluded the study showed fusion-positive lung adenocarcinomas had identifiable common and fusion-pattern specific clinicopathologic, histologic and cytologic features, offering implications for fusion genes screening. Kim et al (2014) retrospectively analyzed 162 consecutive never-smokers who underwent curative resection for stage IB to IIIA lung adenocarcinoma at a single institution. The authors concurrently analyzed mutations in the (EGFR and v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) genes, and investigated ALK rearrangements by fluorescence in situ hybridization assay. ROS1 rearrangement was also determined in all triple (EGFR/KRAS/ALK)-negative tumors. Of 162 never smokers with lung adenocarcinoma, 14 (8.6%) and 5 (3.1%) had ALK and ROS1 rearrangements, respectively. Nineteen of the 74 (25.7%) EGFR and KRAS mutationnegative patients were fusion-positive (ALK or ROS1 fusion). Fusion-positive patients tended to have shorter median disease-free survival (DFS) than fusion-negative patients (28.0 vs months). In multivariate analysis, fusion-positive patients had significantly poorer DFS than fusion-negative patients after adjustment for age, sex, T stage, N stage, and adjuvant chemotherapy use. The first recurrence sites were not significantly different between fusion-positive and fusion-negative patients in this study. Investigators concluded the study shows significantly poorer DFS of ALK or ROS1 fusion-positive lung adenocarcinoma in never-smokers after curative surgery. Warth et al (2014) sought to identify the clinicopathological characteristics of NSCLC with ROS1 expression and translocation. They screened 1478 NSCLCs with a ROS1- specific antibody, and tested positive cases with FISH. All positive cases were analysed for associated clinicopathological characteristics, including survival and molecular tumour composition. Sixty-eight cases (4.6%) showed ROS1 immunoreactivity, and ROS1 translocations were confirmed in nine cases (0.6%). ROS1 expression was predominantly found in female adenocarcinoma patients, in patients with low T stages, and in association with TTF1 and napsin expression, and certain histomorphological adenocarcinoma patterns (lepidic, acinar, and solid). ROS1 translocations occurred in conjunction with other driver mutations (EGFR, KRAS, and BRAF). ROS1 expression was found to be a stage-independent predictor of favorable survival. Investigators concluded ROS1 translocations are rare events in resected NSCLCs from Caucasian patients. IHC screening for ROS1 expression and Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 13

14 clinicopathological parameters, including female sex, early tumor stages, adenocarcinomas with TTF1 and/or napsin expression, and a distinct histomorphological growth pattern, strongly facilitate case enrichment. Molecularly driven multistep concepts might not be optimal for case selection. Go et al (2013) sought to evaluate the clinical and pathological characteristics of patients with NSCLC that harbored a ROS1 rearrangement. Four hundred fifty-one Korean patients with resected NSCLC (the resected group) were examined for ROS1 rearrangements using a tissue microarray and ROS1 fluorescence in situ hybridization and analyzed for clinical and pathological features. Sixty-four patients with advanced pulmonary adenocarcinoma with no known oncogenic aberrations (the advanced group) were also screened for ROS1 rearrangements. Of the 451 consecutive patients from the resected group, ROS1 rearrangements were detected in eight cases (1.8%). In the advanced group, an additional eight patients (12.5%) were identified as harboring ROS1 rearrangements. ROS1 rearrangement was detected exclusively in adenocarcinomas and occurred more frequently in women than in men. With the exception of one patient with an EGFR deletion mutation in exon 19, ROS1-positive adenocarcinomas in all patients revealed no alterations in ALK, EGFR, KRAS, or MET genes. Most ROS1-rearranged tumors showed solid and papillary patterns. Investigators concluded ROS1 rearrangements were detected in 1.8% of patients with resected NSCLC and were detected exclusively in adenocarcinomas, which is similar to the frequency detected in non-asian patients. We suggest that ROS1 screening in adenocarcinoma patients with no known oncogenic aberrations is an effective strategy to find ROS1 rearrangements in NSCLC. Tsuta et al (2014) investigated 1874 patients with carcinomas, including 1620 adenocarcinomas (ADCs), 203 squamous cell carcinomas (SCCs), 8 large cell carcinomas, and 43 sarcomatoid carcinomas (SACs). Fluorescence in situ hybridisation (FISH) and/or reverse transcription-pcr (RT-PCR) were performed to detect RET gene rearrangement. In all, 22 cases (1.2%) showed RET rearrangements; all cases were of ADC histology. Of the 22 patients, 19 possessed KIF5B-RET fusion genes, whereas 3 possessed CCDC6-RET fusion genes. The RETrearranged tumors were significantly more common in younger patients and tended to occur in patients with no history of smoking. In addition, RET rearrangements were not associated with gender, occupational history (particularly radioactive exposure), tumor size, lymph node status, tumor stage, or patient survival. The predominant growth pattern in RET-rearranged ADCs was lepidic in 6 cases, papillary in 9 cases, acinar in 2 cases, micropapillary in 1 case, and solid in 4 cases. Cells with cytoplasmic mucin production were at least focally present in 12 of the 22 (54.5%) RET-rearranged ADC cases. Among the 21 analysed RET-rearranged tumors, RET immunopositivity was observed in 15 cases (71.4%), and was significantly associated with RET rearrangement. Investigators concluded the RET rearrangements were observed in 1.2% of NSCLCs. All cases of RET rearrangement were ADCs. The RET rearrangements were more likely to be observed in younger patients. Although cytoplasmic mucin production was at least focally present in 54.5% of RET-rearranged ADCs, specific histological features were not detected. Chen et al (2014) identified clinical studies that examined the association between BRAF mutations and features of NSCLC in a systematic review up to October The effect size of clinical features was estimated by odds ratios (ORs) with 95% confidence interval (CI) for each study, using a fixed-effects or random-effects model. Ten studies with a total of 5599 NSCLC patients were included. There was a 3% (170/5599) BRAF mutation rate. BRAF mutations in NSCLC were significantly associated with adenocarcinomas (ADCs). There were no significant differences in gender, smoking and stage in patients with and without BRAF mutations. The Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 14

15 BRAFV600E mutation was more frequent in women than non-brafv600e mutations and was closely related to never smokers. Investigators concluded the findings have important implications for the prediction of the NSCLC sub-types more accurately combined with other genetic changes. Su et al (2014) evaluated the sensitivity and specificity of multigene mutation testing by the Snapshot assay in NSCLC. The authors retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which EGFR mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v- ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens, and the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay was a sensitive and easily customized assay for multigene mutation testing in clinical practice. Brustugun et al (2014) presented clinicopathological characteristics of nearly one thousand unselected NSCLC patients tested for the targetable V600E/K BRAFmutation. NSCLC routinely tested for EGFR-mutations at Oslo University Hospital in the period February 2011-July 2013 were tested for V600E/K BRAF-mutations using a PCR-based method. The authors found a BRAF-mutation frequency of 1.7% in the total cohort of 979 patients, and 2.3% among 646 adenocarcinomas. One of the BRAF-positive samples was also KRAS-mutated, and one had an ALK-translocation. None of 231 squamous cell carcinomas were BRAF-mutated. The proportion of neversmokers among BRAF-positives was high (29%). Authors concluded BRAF-mutation analysis should be part of the subtyping of non-squamous NSCLC. Cardarella et al (2013) examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Using DNA sequencing, they successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-v600e BRAF mutations identified from patients with NSCLC. Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-v600e, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-v600e Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 15

16 mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity. Investigators concluded BRAF mutations occur in 4% of NSCLCs and half are non-v600e. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC. Scientific Rationale Update June 2014 Several biomarkers have emerged as predictive and prognostic markers for NSCLC. A predictive biomarker is a biomolecule that is indicative of therapeutic efficacy; that is, there is an interaction between the biomolecule and therapy on patient outcome. A prognostic biomarker is a biomolecule that is indicative of patient survival independent of the treatment received; that is, the biomolecule is an indicator of the innate tumor aggressiveness. The most useful biomarkers for predicting the efficacy of targeted therapy in advanced non-small cell lung cancer (NSCLC) are somatic genome alterations known as driver mutations. These mutations occur in cancer cells within genes encoding for proteins critical to cell growth and survival. Whenever feasible, patients with advanced non-small cell lung cancer (NSCLC) should have tumor assessed for the presence of a driver mutation. Methods for screening NSCLC patients for driver mutations and other abnormalities are continually evolving and there is no one standard platform for testing. In general, several techniques have been used: Sequencing of the gene is the most comprehensive method for mutation testing. Direct sequencing can be used for discovery when the target abnormality is not known, but this process can be expensive and time-consuming. Furthermore, the mutation must be fairly prevalent among all the cells in the tissue sample in order to be detected by direct sequencing. Next-generation sequencing (NGS) overcomes many of the shortcomings of direct sequencing. This massively parallel approach, relying heavily on automation, data storage, and computational processing, allows quantitative analysis of infrequent alleles and simultaneous evaluation of multiple genes or even whole genomes. Allele-specific testing analyzes the DNA for a pre-defined abnormality. The raw DNA is typically amplified using polymerase chain reactions (PCR) before the search for the mutated allele is undertaken, allowing for rare signals to be detected with greater sensitivity. This method tends to be faster and cheaper, but only pre-specified targets can be identified. Thus allele-specific testing cannot be used to identify new abnormalities. Mass spectrometry, a method that analyzes short bits of DNA by mass and can detect when a segment is a different molecular weight than expected, equating to a mutation. This method also can only identify pre-defined abnormalities. Fluorescence in-situ testing (FISH) is typically used to detect gene translocations, amplifications, and other rearrangements. With FISH, DNA probes are used to interrogate the chromosomes for the presence or absence of a specific DNA sequence. Following denaturation, a fluorescent labeled DNA probe is directly hybridized with the chromosomes on the slide ; immediate detection of the fluorescent signal is possible via fluorescence microscopy. The most common driver mutations that have been identified in NSCLC for which targeted therapies specifically aimed at the driver mutation have been developed include EGFR mutation, ALK translocation, and RAS mutation. Others include ROS1 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 16

17 translocation (predict response to crizotinib (Xalkori) for the treatment of NSCLC)., Her2 mutation, BRAF mutation, MET expression, PIK3CA, AKT1, PTEN alterations, FGFR1 amplification, β-catenin mutation, RET translocation, DDR2 mutation, and MEK1 mutation. FGFR1 amplification (i.e., Fibroblast growth factor receptor-1) is a cell surface tyrosine kinase receptor that mediates cell survival and proliferation. Gene amplification of FGFR1 has been detected in 13 to 25 percent of squamous tumors. For patients with squamous cell carcinomas, FGFR1 amplification is associated with smoking and with worse overall survival. The most recent NCCN guidelines on NSCLC (3.2014) state, " Mutation screening assays for detecting multiple biomarkers simutaneously have been developed that can detect more than 50 point mutations including EGFR, however, these multiplex polymerase chain reaction (PCR) systems do not detect gene rearrangements, because they are not point mutations. ALK gene rearrangements can be detected using FISH. Next generation sequencing (NGS) can detect panels of mutations and gene rearrangements. Other driver mutations and gene rearrangements are being identified such as HER2 and BRAF mutation, ROS1 and RET gene rearrangements and MET amplification. Targeted agents are available for patients with NSCLC who have these genetic alterations. Per the guidelines, DNA mutational analysis is the preferred method to assess for EGFR status. As noted above various DNA mutation detection assays can be used to determine the EGFR mutation status in tumor cells. The NCCN guidelines recommend testing for genetic alterations using multiplex mutation screening assay/ngs to ensure patients receive the most appropriate treatment; patients may be eligible for clinical trials for some of these targeted agents. Testing for ALK gene rearrangements can be done with FISH or NGS. Tuononen et al (2013) reported that the development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The authors sought screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, they aimed to identify potential novel mutations by targeted NGS. They analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. They observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. They noted their results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material. de Biase et al (2013) reported that selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. The authors defined a next generation sequencing protocol targeted to EGFR exons suitable for the routine diagnosis of such clinical samples. The protocol was validated in an unselected series of 80 small biopsies Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 17

18 (n=14) and cytology (n=66) specimens representative of the material ordinarily submitted for diagnostic evaluation to three referral medical centers in Italy. Specimens were systematically evaluated for tumor cell number and proportion relative to non-neoplastic cells. They were analyzed in batches of amplicons per run, reaching an analytical sensitivity of 1% and obtaining an adequate number of reads, to cover all exons on all samples analyzed. Next generation sequencing was compared with Sanger sequencing. The latter identified 15 EGFR mutations in 14/80 cases (17.5%) but did not detected mutations when the proportion of neoplastic cells was below 40%. Next generation sequencing identified 31 EGFR mutations in 24/80 cases (30.0%). Mutations were detected with a proportion of neoplastic cells as low as 5%. All mutations identified by the Sanger method were confirmed. In 6 cases next generation sequencing identified exon 19 deletions or the L858R mutation not seen after Sanger sequencing, allowing the patient to be treated with tyrosine kinase inhibitors. In one additional case the R831H mutation associated with treatment resistance was identified in an EGFR wild type tumor after Sanger sequencing. Next generation sequencing is robust, cost-effective and greatly improves the detection of EGFR mutations. Its use should be promoted for the clinical diagnosis of mutations in specimens with unfavorable tumor cell content. The VeriStrat test (Biodesix Inc) was developed to identify patients most likely to respond to EGFR TKI therapy. The assay utilizes mass spectrometry, a technique that analyzes the mass-to-charge ratio of charged particles, to characterize the protein profile of pretreatment serum from NSCLC patients who are candidates for EGFR TKI therapy. A classification algorithm is then used to categorize patients according to the likelihood of treatment response. A VeriStrat good result indicates that the patient may respond to treatment with EGFR TKIs, while a VeriStrat poor result indicates that the patient is unlikely to benefit from this type of therapy. A limitation of VeriStrat testing is that approximately 1% to 4% of patients receive an indeterminate VeriStrat result (i.e., neither good nor poor ), the implications of which have not yet been determined. Akerley et al (2013) assessed the impact of a serum-based proteomic test for NSCLC on physician treatment recommendations. A multivariate, serum-based proteomic test (VeriStrat) is commercially available to assist physicians when determining treatment using epidermal growth factor receptor inhibitor (EGFRi) therapy, such as erlotinib (Tarceva), by stratifying patients into two categories: those with significantly better ('good') and those with significantly worse ('poor') outcomes following treatment with EGFRi therapy. All tests ordered from August 9, 2011 to November 26, 2012, were considered for this study. Pre- and post-test treatment recommendations were prospectively collected from ordering physicians on a voluntary basis. Only those tests that had both pre- and post-test treatment information were included in the analysis group. Main outcome measures include proportional change and correlation of treatment recommendations before and after receipt of the test results. Over the duration of the study, 724 physicians ordered 2854 tests. The analysis group comprised the 226 physicians who provided pre- and post-test treatment information (n = 403 tests). Following receipt of the test results, 90.3% (95% CI: %) of patients who tested as 'good' received erlotinib recommendations versus 9.6% (95% CI: %, p < ) of patients who tested as 'poor'. Ninety percent of post-test treatment recommendations positively correlated with test results, with 40% showing a change from pre-test considerations. The authors concluded among test orderers, serum-based proteomic mass spectrometry testing significantly influenced therapy recommendations in NSCLC. Usage patterns should be monitored as use expands. The study was limited by data based on physicians willing to submit recommendations and endpoint limited to therapy recommendations. Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 18

19 Stinchcombe et al (2013) reported that in a multicenter randomized phase II trial of gemcitabine (arm A), erlotinib (arm B), and gemcitabine and erlotinib (arm C), similar progression-free survival (PFS) and overall survival (OS) were observed in all arms. The authors performed an exploratory, blinded, retrospective analysis of plasma or serum samples collected as part of the trial to investigate the ability of VeriStrat (VS) to predict treatment outcomes. Ninety-eight patients were assessable, and the majority had stage IV disease (81%), adenocarcinoma histology (63%), reported current or previous tobacco use (84%), and 26% had a performance status (PS) of 2. In arm A, patients with VS Good (n = 20) compared with VS Poor status (n = 8) had similar PFS (hazard ratio [HR]: 1.21; p = 0.67) and OS (HR: 0.82; p = 0.64). In arm B, patients with VS Good (n = 26) compared with VS Poor (n = 12) had a statistically significantly superior PFS (HR: 0.33; p = 0.002) and OS (HR: 0.40; p = 0.014). In arm C, patients with VS Good (n = 17) compared with Poor (n = 1 5) had a superior PFS (HR: 0.42; p = 0.027) and a trend toward superior OS (HR: 0.48; p = 0.051). In the multivariate analysis for PFS, VS status was statistically significant (p = 0.011); for OS, VS status (p = 0.017) and PS (p = 0.005) were statistically significant. A statistically significant VS and treatment interaction (gemcitabine versus erlotinib) was observed for PFS and OS. The authors concluded Gemcitabine is the superior treatment for elderly patients with VS Poor status. First-line erlotinib for elderly patients with VS Good status may warrant further investigation. Akerley et al (2013) reported erlotinib alone or bevacizumab in combination with chemotherapy improve survival in patients with advanced NSCLC. The current trial of erlotinib and bevacizumab was designed as an alternative to initial conventional chemotherapy for advanced lung cancer and a platform to explore selection factors. Eligibility criteria included stage IIIB/IV or recurrent non-squamous, non-small cell lung cancer (NSNSCLC), no prior chemotherapy for metastatic disease, PS=0-1, and no history of brain metastases for the first 40 patients. An expansion cohort of an additional 10 patients allowed treated brain metastases. Patients received erlotinib 150 mg/day and bevacizumab 15 mg/kg/3 weeks until objective or symptomatic progression. Pretreatment serum was collected for blinded VeriStrat evaluation. Fifty patients were accrued. The median age was 65 years, 10 were octogenarians, 37 had PS=1, 25 were female and 12 were never-smokers. Histologies were adenocarcinoma in 26 and unspecified in 24. Partial responses were observed in 12 (24%), stable in 30 (60%) and progressive disease in 8 (16%). The median time on therapy was 15.5 weeks. The median survival was 50.4 weeks with 1 and 2 years survivals of 50% and 21%, respectively. Only 38% of eligible patients received second line therapy, most often due to decline in PS. VeriStrat analysis was performed in 42 subjects (Good 32, Poor 9, and Indeterminate 1). Significant differences based on VeriStrat signature were noted in PFS (Good=18.9 weeks, Poor=6.3 weeks, p=0.0035) and overall survival (Good=71.4 weeks, Poor=19.9 weeks, p=0.0015). The authors concluded survival in an unselected population of patients with NSNSCLC treated with bevacizumab and erlotinib approximated that expected with conventional chemotherapy. VeriStrat analyses distinguished patients who were likely or unlikely to benefit from this combination. Carbone et al (2012) investigated the predictive and prognostic effects of VeriStrat, a serum or plasma-based assay, on response and survival in a subset of patients enrolled on the NCIC Clinical Trials Group, BR.21 phase III trial of erlotinib versus placebo in previously treated advanced NSCLC patients. Pretreatment plasma samples were available for 441 of 731 enrolled patients and were provided as anonymized aliquots to Biodesix. The VeriStrat test was performed in a Clinical Laboratory Improvement Act-accredited laboratory at Biodesix, Inc. (Boulder, CO). The results (Good, Poor) were returned to NCIC Clinical Trials Group, which performed all the statistical analyses. VeriStrat testing was successful in 436 Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 19

20 samples (98.9%), with 61% classified as Good. VeriStrat was prognostic for overall survival in both erlotinib-treated patients and those on placebo, independent of clinical covariates. For VeriStrat Good patients, the median survival was 10.5 months on erlotinib versus 6.6 months for placebo (hazard ratio 0.63, 95% confidence interval , p = 0.002). For VeriStrat Poor patients, the median survival was 4 months for patients receiving erlotinib, and 3.1 months for placebo (hazard ratio: 0.77, 95% confidence interval , p = 0.11). VeriStrat was predictive for objective response (p = 0.002), but was not able to predict for differential survival benefit from erlotinib (interaction p = 0.48). Similar results were found for progression-free survival. The authors concluded they were able to confirm that VeriStrat is predictive of objective response to erlotinib. VeriStrat is prognostic for both OS and progression-free survival, independent of clinical features, but is not predictive of differential survival benefit versus placebo. Amann et al (2010) reported that serum proteomics and mutations in the EGFR and KRAS have been associated with benefit after therapy with EGFR-targeted therapies in non-small cell lung cancer, but all three have not been evaluated in any one study. The investigators analyzed analyzed available biospecimens from Eastern Cooperative Oncology Group 3503, a single-arm phase II study of erlotinib in firstline advanced lung cancer, for proteomics signatures in the previously described serum matrix-assisted laser desorption ionization proteomic classifier (VeriStrat) as well as for KRAS and EGFR mutations. Out of 137 enrolled patients, analyzable biologic samples were available on 102. Nine of 41 (22%) demonstrated KRAS mutations and 3 of 41 (7%) harbored EGFR mutations. VeriStrat classification identified 64 of 88 (73%) as predicted to have "good" and 24 of 88 (27%) predicted to have "poor" outcomes. A statistically significant correlation of VeriStrat status (p<0.001) was found with survival. EGFR mutations, but not KRAS mutations, also correlated with survival. The investigators concluded the previously defined matrixassisted laser desorption ionization predictor remains a potent and highly clinically significant predictor of survival after first-line treatment with erlotinib in patients with wild-type EGFR and independent of mutations in KRAS. Two studies of clinical validity also evaluated NSCLC patients for changes in VeriStrat classification over time (Kuiper et al., 2012; Lazzari et al., 2012). In the first study of 49 patients treated with erlotinib and sorafenib, VeriStrat status was evaluated before the start of therapy and again at 1 and 3 weeks after treatment initiation. Overall, 46% maintained their status at all 3 time points, 34% changed at either week 1 or at week 3, and 20% changed at week 1 and reverted back to their initial status at week 3 (Kuiper et al., 2012). In the second study, VeriStrat classification remained significantly associated with both OS and PFS when considering it a timedependent variable. However, 11.7% of patients had a change in VeriStrat classification over the course of treatment, including 4 patients who were VeriStrat poor at baseline and VeriStrat good after treatment. Notably, 2 of these 4 patients (2 of 31 [6.5%] patients from the initial cohort with a poor result at baseline) exhibited a good clinical response to EGFR TKI therapy, but would likely not have been treated with an EGFR TKI after receiving a pretreatment result of VeriStrat poor (Lazzari et al., 2012). The implications of changes in VeriStrat status are currently unclear and require further investigation. Whether VeriStrat status correlates with other biological traits or tumor markers is unclear. Several studies reported that VeriStrat classification was independent of other factors known to be associated with EGFR TKI treatment response, such as smoking history, histology, patient sex, and both EGFR and KRAS gene status (Taguchi et al., 2007; Amann et al., 2010; Carbone et al., 2012; Lazzari et al., 2012; Gautschi et al., 2013). However, another study reported that, while there was an association between VeriStrat classification and OS, it was not statistically Molecular Tumor Markers for Non-Small Cell Lung Cancer Jun 15 20