Additional clinical features of this patient include:

*Not an actual patient. Challenge your knowledge of biomarker testing in advanced NSCLC. Consider this case study of disease progression following chemotherapy, with insightful commentary from Dr Edward Kim. Recommendations in this hypothetical case study are based on clinical guidelines and practice standards. These recommendations do not take the place of independent assessment or medical judgment. Disease history: James R. is a 58-year-old African-American man who initially presented to his pulmonologist with c/o progressively worsening cough, hemoptysis, and DOE X 6 m. CXR showed diffuse L lobar mass. Abdominal and chest CT revealed 1.5 cm X 2.0 cm lesion in L upper lobe with metastatic involvement of the thoracic spine. CT-guided needle biopsy confirmed diagnosis of stage IV NSCLC adenocarcinoma. At time of diagnosis, James R. was treated by his oncologist with 4 cycles of IV platinum doublet therapy: carboplatin (AUC of 6.0 mg/min) plus paclitaxel (200 mg/m 2 d 1 q 3 w). Avastin was not recommended r/t recent PMH of hemoptysis. He experienced AEs during chemotherapy, including N&V (relieved by oral metoclopramide); mild neutropenia, which resolved without treatment; and grade 1 peripheral neuropathy, treated with OTC analgesics. James R. is referred to you after his 3-mo follow-up visit. CT/PET scans obtained revealed local regional recurrence in left lung and spine, as well as probable liver metastasis. His PS=1. All labs are WNL. Additional clinical features of this patient include: PE: Diminished breath sounds at the L base upon auscultation Remaining ROS WNL PMH: HTN X 10 yr, controlled with lisinopril 10 mg qd Chronic lower back pain SH: Married, 2 grown children Self-employed (auto mechanic) 15 pack-year smoking history, but quit ~5 years ago FH: Colon cancer in paternal grandfather Avastin is a registered trademark of Genentech, Inc 1

Questions: 1. Before prescribing a second-line treatment, would you order biomarker testing of tissue from the sample obtained at the initial CT-guided needle biopsy? 2. Which biomarkers would you order? 3. What is your preferred method for obtaining tissue for biomarker analysis? 4. Subsequent biomarker testing reveals that James R. is negative for EML4-ALK rearrangements and positive for an EGFR mutation (in-frame deletion in exon 19). Given this information, which second-line regimen would you use to treat this patient? 2

Answers: 1Question A) Yes Preferred answer commentary from Dr Edward Kim: Ideally, when we are approaching the management of lung cancer patients, we want to get the pathology, the histological diagnosis, and the staging. We also want to include in this initial work-up the presence or absence of certain biomarkers. We are now looking more at the patient individually: what is unique about their tumor characteristics. And once we can identify those characteristics that are unique to that patient s tumor, hopefully find the right treatment. B) No Testing for biomarkers may provide more information, which will guide treatment decisions. Patients with specific genetic alterations may then be able to receive targeted therapy. 1,2 NCCN and draft CAP/IASLC/AMP guidelines recommend testing of all nonsquamous tumors for EGFR mutations and ALK rearrangements, as both have been shown to have prognostic and predictive value. Both sets of guidelines recommend that testing should be performed immediately after establishing histology or before initiating therapy in a patient. 1,2 It is preferable to have tumor tissue from the first biopsy available for biomarker testing, so as to minimize the need for a second biopsy as well as potential patient risks and associated costs. 3 2Question A) KRAS To date, only 2 molecular markers have been found to have both prognostic and predictive value in advanced NSCLC: EGFR mutations and ALK rearrangements. Other biomarkers whose clinical relevance in NSCLC remains to be determined include KRAS, HER2, MET, and BRAF. 1,4-7 B) EGFR NCCN and draft CAP/IASLC/AMP guidelines recommend routine testing for EGFR mutations and ALK rearrangements in all adenocarcinomas and other advanced nonsquamous NSCLC tumors. 1,2 Specifically, NCCN Clinical Practice Guidelines for NSCLC Version 2.2012 (February 2012) recommend testing all recurrent or metastatic adenocarcinoma, large-cell carcinoma, and NSCLC NOS. 1 Draft CAP/IASLC/AMP Lung Cancer Biomarkers guidelines (November 2011) specify that EGFR and ALK testing should be performed on all NSCLC tumors that contain an adenocarcinoma component and are not confirmed as squamous. 2 Testing is recommended immediately after establishing histology or prior to initiating targeted therapy for a patient. 1,2 3

Answers (cont d): C) EML4-ALK EGFR mutations and ALK rearrangements are understood to be mutually exclusive. 2 Because EGFR mutations occur with greater frequency in nonsquamous NSCLC vs ALK rearrangements, EGFR testing should be prioritized when tissue samples are limited. 6,8 Additionally, due to the high frequency of EGFR mutations in adenocarcinoma, every lung cancer with mixed histology in which an adenocarcinoma component cannot be excluded should be tested for EGFR mutations. 2 D) B and C Preferred answer commentary from Dr Edward Kim We have identified 2 important markers in lung cancer, the first one being EGFR mutation and the other one ALK translocation. These 2 are important because companion drugs will allow us to treat patients, to improve their response rate, to improve their progressionfree survival. Which populations should we test as far as these markers? Ideally, I would test all non small-cell lung cancer patients. If we have to choose, then we focus on the nonsquamous patients. It s really best to get these markers up front, so we have all the decision-making ability at the beginning to determine the appropriate therapy. 3Question A) FNA Although FNA is primarily used for histologic evaluation, multiple passes of CT-guided FNA may collect sufficient tissue for biomarker testing. 9,10 However, CT-guided core needle biopsy may be the preferred procedure as it ensures collection of adequate quantity and quality of lung tissue. 11-13 B) Bronchial washing Molecular markers in NSCLC may be detected using samples obtained through bronchoalveolar lavage. However, the utility of different molecular assays using bronchoalveolar lavage is still under investigation. Current molecular assays are associated with low sensitivity. In addition, reference laboratories may require a higher minimum tumor percentage/number of tumor cells than those typically available through bronchial washing. 14-18 C) CT-guided core needle biopsy CT-guided core needle biopsy may be an optimal procedure, as it ensures collection of adequate quantity and quality of lung tissue. 11,12 Use of CT-guided core needle biopsy has been utilized in the BATTLE clinical trial program (up to 3 passes per biopsy). 19 Although FNA is primarily used for histologic evaluation, multiple passes of CT-guided FNA may also effectively collect tissue for biomarker testing. 10 4

Answers (cont d): D) A or C Preferred answer commentary from Dr Edward Kim: Several techniques can be used to acquire tissue for testing. One is CT-guided core needle biopsy and another is FNA. Both have been used effectively in clinical practice and in clinical trials. 9,11-13 What is most important is extracting good quality cytologic tissue with minimal necrotic tissue. It is also important for physicians who are biopsying (pulmonologists, IRs, or thoracic surgeons) to note that tissue requirements for biomarker analysis may exceed those for cytologic or histologic analysis. 9,12,13,20 4Question A) Single-agent docetaxel NCCN Clinical Practice Guidelines identify single-agent docetaxel as an established second-line therapy option. 1 However, in a phase 3, randomized, open-label trial in patients with advanced NSCLC previously treated with a platinum-based regimen (the INTEREST trial), patients in the EGFR M+ subgroup who were treated with gefitinib experienced longer PFS compared with docetaxel (P=0.001). In patients with EGFR M+ disease, ORRs were also higher when patients were treated with an EGFR TKI (erlotinib) vs docetaxel (42.1% vs 21.1%; P=0.04). 21 B) Single-agent pemetrexed Pemetrexed monotherapy is a viable second-line treatment option. It is recommended in the NCCN Clinical Practice Guidelines for patients with nonsquamous histology and performance status (PS) 0-2. In clinical trials in patients with adenocarcinoma and large-cell carcinoma, pemetrexed has demonstrated efficacy similar to second-line docetaxel, with fewer toxicities. Unlike docetaxel (studied in the INTEREST trial), single-agent pemetrexed has not yet been compared directly with EGFR TKIs. In NCCN Guidelines, EGFR TKI therapy (i.e., erlotinib) is the only second-line treatment recommended for patients with EGFR M+ disease regardless of patient performance score. 1,21 C) EGFR TKI inhibitor (i.e., erlotinib) Preferred answer commentary from Dr Edward Kim: When we test patients, we know that those patients who harbor an EGFR mutation will do better than those patients who don t. We also know that when we treat these patients whose tumors harbor this EGFR mutation with an EGFR tyrosine kinase inhibitor, this is predictive of reliable response rate and a better progression-free survival as opposed to chemotherapy. 5

Case summary: It is recommended to perform biomarker testing for molecular markers such as EGFR and ALK early in the management of patients with advanced NSCLC; this enables physicians to offer tailored therapy to appropriate patients in the front-line setting. Both NCCN and draft CAP/IASLC/AMP guidelines support the routine testing of all nonsquamous tumors for EGFR mutations and ALK rearrangements immediately after establishing histology. 1,2 In the present case study, while biomarker testing was not conducted prior to initial treatment, obtaining biomarker status information at a later time remained important for determining appropriate treatment options in the second-line setting. For patients with EGFR mutations who are being treated with second-line therapy, EGFR TKIs have shown benefit in terms of both progression-free survival and overall response rate. 1,5,22-26 With effective targeted therapy available, physicians have a key opportunity to utilize biomarker testing to individualize and optimize treatment decisions for appropriate patients with advanced NSCLC. Another key point highlighted by this case study relates to the method of obtaining tissue for biomarker analysis. To confirm the initial diagnosis, the patient underwent a CT-guided needle biopsy, which may be an optimal procedure as it ensures collection of adequate quantity and quality of lung tissue for biomarker testing. 11,12 Thus, there remained available tissue for biomarker analysis at a later time beyond the initial treatment stage. Having an adequate quantity and quality of tissue upon initial biopsy can minimize the need for a second biopsy as well as potential patient risks and associated costs. 3 References on following page. 6

References: 1. National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology: non-small cell lung cancer, version 2/2012. http://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf. Accessed December 29, 2011. 2. College of American Pathologists (CAP)/International Association for the Study of Lung Cancer (IASLC) /Association of Molecular Pathology (AMP) expert panel. Lung cancer biomarkers guideline draft recommendations. http://capstaging.cap.org/apps/docs/membership/transformation/new/lung_public_comment_supporting_materials. pdf. Accessed December 29, 2011. 3. Wiener RS, Schwartz LM, Woloshin S, Welch HG. Population-based risk for complications after transthoracic needle lung biopsy of a pulmonary nodule: an analysis of discharge records. Ann Intern Med. 2011;155(3):137-144. 4. Rosell R, Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009;361(10):958-967. 5. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361(10):947-957. 6. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med. 2010;363(18):1693-1703. 7. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med. 2008;359(13):1367-1380. 8. Kris MG, Johnson BE, Kwiatkowski DJ, et al. Identification of driver mutations in tumor specimens from 1,000 patients with lung adenocarcinoma: the NCI s Lung Cancer Mutation Consortium (LCMC). Abstract CRA7506. J Clin Oncol. 2011;29(June 20 suppl). http://meeting.ascopubs.org/cgi/content/abstract/29/18_ suppl/cra7506?sid=84701cef-e846-4865-9c01-bfdd7afe5871. Accessed December 29, 2011. 9. Billah S, Stewart J, Staerkel G, Chen S, Gong Y, Guo M. EGFR and KRAS mutations in lung carcinoma: molecular testing by using cytology specimens. Cancer Cytopathol. 2011;119(2):111-117. 10. van Eijk R, Licht J, Schrumpf M, et al. Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qpcr. PLoS One. 2011;6(3):e17791. 11. Pao W, Kris MG, Iafrate AJ, et al. Integration of molecular profiling into the lung cancer clinic. Clin Cancer Res. 2009;15(17):5317-5322. 12. Shaw AT, Hayes DN, Martins R. The importance of histology and molecular testing (EGFR and EML4-ALK) in the initial evaluation of advanced non-small cell lung cancer. http://www.asco.org/ascov2/home/education%20&%20training/educational%20book/pdf%20 Files/2011/zds00111000292.pdf. Accessed December 28, 2011. 13. Felip E, Gridelli C, Baas P, et al. Metastatic non-small-cell lung cancer: consensus on pathology and molecular tests, first-line, second-line, and third-line therapy: 1st ESMO Consensus Conference in Lung Cancer; Lugano 2010. Ann Oncol. 2011;22(7):1507-1519. 14. Ahrendt SA, Chow JT, Xu L-H, et al. Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer. J Natl Cancer Inst. 1999;91(4):332-339. 15. Topaloglu O, Hoque MO, Tokumaru Y, et al. Detection of promoter hypermethylation of multiple genes in the tumor and bronchoalveolar lavage of patients with lung cancer. Clin Cancer Res. 2004;10(7):2284-2288. 16. ResponseDx. Specimen Preparation & Requirements. Response Genetics website. www.responsegenetics.com. Accessed February 7, 2012. 17. Diagnostic Services Specimen Requirements. Clarient Inc. website. http://www.clarientinc.com/ordering.aspx. Accessed February 7, 2012. 18. Lung Cancer Mutation Panel. Quest Diagnostics website. http://www.questdiagnostics.com/hcp/intguide/jsp /showintguidepage.jsp?fn=ts_lungcancermutation_panel.htm. Accessed February 7, 2012. 19. Kim ES, Herbst RS, Wistuba II, et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discovery. 2011:1(1);44-53. 20. Nakajima T, Yasufuku K, Suzuki M, et al. Assessment of epidermal growth factor receptor mutation by endobronchial ultrasound-guided transbronchial needle aspiration. Chest. 2007;132(2):597-602. 21. Douillard J-Y, Shepherd FA, Hirsh V, et al. Molecular predictors of outcome with gefitinib and docetaxel in previously treated non small-cell lung cancer: data from the randomized phase III INTEREST trial. J Clin Oncol. 2010;28(5):744-752. 22. Fukuoka M, Wu YL, Thongprasert S, et al. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS). J Clin Oncol. 2011;29(21):2866-2874. 23. Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;362(25):2380-2388. 24. Mitsudomi T, Morita S, Yatabe Y, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010;11(2):121-128. 25. Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 2011;12(8):735-742. 26. Rosell R, Gervais R, Vergnenegre A, et al. Erlotinib versus chemotherapy (CT) in advanced non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations: Interim results of the European erlotinib versus chemotherapy (EURTAC) phase III randomized trial. ASCO 2011, Abstract 7503. http://www.asco.org/ascov2/meetings/abstracts?&vmview=abst_detail_view&confid=102&abstract ID=78285. Accessed December 9, 2011. Copyright 2012. Boehringer Ingelheim Pharmaceuticals, Inc. All rights reserved. (4/12) OC182910PROF 7