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1 Reflex testing of resected stage I through III lung adenocarcinomas for EGFR and KRAS mutation: Report on initial experience and clinical utility at a single center Sandra P. D Angelo, MD, Bernard Park, MD, Christopher G. Azzoli, MD, Mark G. Kris, MD, Valerie Rusch, MD, Marc Ladanyi, MD, and Maureen F. Zakowski, MD Objective: The genes KRAS and EGFR have emerged as potential targets for therapy in lung adenocarcinoma; mutations in these genes can be found in almost half of patients. In anticipation of the clinical importance of molecularly defined adenocarcinoma subgroups for the treatment of patients with resected stages I through III lung adenocarcinoma, the Memorial Sloan Kettering Cancer Center (MSKCC) Departments of Surgery and Pathology have collaborated since 2006 to conduct reflex testing of tumor specimens for EGFR and KRAS mutations. Methods: Using established methods, the identification of EGFR exon 19 deletions and exon 21 L858R mutations was performed. In samples lacking these 2 sensitizing EGFR mutations, KRAS analysis was done. Results: We studied a total of 1831 patients who had stage I through IV lung adenocarcinomas and detected 448 KRAS and 364 EGFR mutations. Of these patients, a subset of 855 (78%) patients with stages I through III adenocarcinoma of the lung who underwent curative surgical resection at MSKCC were tested. In patients with early stage disease, 158 EGFR mutations and 207 KRAS mutations were detected. Conclusions: The results of the first 3 years of reflex testing at MSKCC reported here demonstrate the feasibility, clinical utility, and potential of this approach. This information allowed for enrollment of patients into clinical trials to explore mutation-specific, directed therapy and led to retrospective studies related to patient outcome. In addition, it may inform selection of chemotherapy for recurrent disease and may help to distinguish multiple primary tumors from metastatic disease. (J Thorac Cardiovasc Surg 2011;141:476-80) Earn CME credits at Despite successful surgery, half of patients with resected non small-cell lung cancer (NSCLC) recur and die within 5 years. 1 In patients with resected stage II through III NSCLC, 4 months of cisplatin-based chemotherapy improves survival rates by nearly 20% (relative risk reduction), with an absolute risk reduction of 10% to 13%. 2 Pathologic stage is the only prospectively validated clinical factor used to select patients for adjuvant chemotherapy. 3 More effective adjuvant therapies are sorely needed. The most promising molecular markers for the selection of treatment are somatic mutations in lung adenocarcinomas. From Memorial Sloan Kettering Cancer Center, New York, NY. Disclosures: Authors have nothing to disclose with regard to commercial support. Received for publication April 23, 2010; revisions received June 24, 2010; accepted for publication Aug 1, 2010; available ahead of print Oct 8, Address for reprints: Sandra P. D Angelo, MD, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY ( dangelos@mskcc.org) /$0.00 Published by Elsevier Inc. on behalf of The American Association for Thoracic Surgery doi: /j.jtcvs Epidermal growth factor receptor (EGFR) is a signaling protein attached to the cell membrane. Ligand binding to the extracellular portion of the EGFR protein leads to increases in cellular proliferation, motility, adhesion, invasion, blocking of apoptosis, and resistance to chemotherapy. 4 Erlotinib is an EGFR tyrosine kinase inhibitor currently approved for patients with metastatic NSCLC. Of all clinically relevant EGFR mutations, 90% are either missense mutations in exon 21 or deletions in exon Patients with deletions in exon 19 have better prognoses than patients with missense mutations in exon 21 when treated with erlotinib and gefinitib. 6 EGFR mutations are more common in people who have never smoked, in Asians, and in women with adenocarcinoma. 7 Patients with advanced NSCLC and EGFR exon 19 or 21 mutations have been noted to have high rates of radiologic response to gefitinib. 8 A recent trial in East Asian nonsmokers or former light smokers with stage IV adenocarcinoma evaluated gefitinib against carboplatin and paclitaxel as first-line chemotherapy. In the subgroup of patients with EGFR mutations, patients treated with gefitinib had higher objective responses 71% as opposed to 1% and longer progression-free survival. 9 These trials exemplify the importance of EGFR mutations as predictors of response and clinical benefit from tyrosine kinase inhibitors in patients with stage IV lung adenocarcinoma. 476 The Journal of Thoracic and Cardiovascular Surgery c February 2011

2 General Thoracic Surgery Abbreviations and Acronyms ACCP ¼ American College of Chest Physicians NSCLC ¼ non small-cell lung cancer MSKCC ¼ Memorial Sloan Kettering Cancer Center PCR ¼ polymerase chain reaction KRAS genes encode guanosine triphosphate-binding proteins that regulate cell growth, differentiation, and apoptosis. 10 KRAS mutations occur in 20% to 30% of lung adenocarcinomas. 11 More than 80% occur in exon 2 of KRAS in codon 12, with the remaining mutations occurring in codons 13 and 61. Although detected in some who have never smoked, they are found more often in current and former cigarette smokers and have been associated with poor prognoses in several studies. 12,13 In stage IV patients, KRAS mutations predict lack of response to EGFR tyrosine kinase inhibitors and lower efficacy of chemotherapy when combined with erlotinib. 14 The results of a prospective clinical trial of adjuvant chemotherapy for resected stage IB-II NSCLC did not demonstrate a benefit from adjuvant chemotherapy in the KRAS mutant subgroup (HR, 0.95; 95% CI; P ¼.87). 15 At MSKCC, routine reflex testing for EGFR and KRAS mutations was instituted for all patients with resected lung adenocarcinomas in Justification for this reflex testing is outlined in Table 1. Although it does not guide standard practice, the molecular information has been used to enroll patients in clinical trials of novel adjuvant therapy specifically targeting EGFR and KRAS. 3 Knowing that as much as 50% of patients with resected NSCLC would experience recurrence, we believed that the results of the molecular tests could inform the selection of chemotherapy for recurrent disease. EGFR/KRAS testing has been used to assist in distinguishing multiple primary tumors from metastatic tumors. 16 Resected specimens routinely provide excess tissue for molecular profiling. In contrast, patients with advanced NSCLC are usually diagnosed with a fine needle aspirate, limiting available tissue for analysis. Universal testing of all resected lung adenocarcinomas for EGFR and KRAS mutations has allowed for unique research efforts, including retrospective studies related to outcome and prospective studies of novel agents. Here we report the results of the first 3 years of reflex testing conducted by the pathology department of our institution. MATERIALS AND METHODS We tested all patients who underwent lung resection because of adenocarcinoma. EGFR/KRAS mutations are extremely uncommon in large cell carcinomas, small cell carcinomas, and squamous cell carcinomas. 17 At the time of gross prosection, a slice of tumor was frozen and retained along with a corresponding section for paraffin embedding and hematoxylin and eosin (H&E) staining. Initially, DNA was extracted from frozen tissue. After observing that several cases lacked sufficient tumoral DNA when the frozen tissues were used, we modified the process and instead extracted DNA from formalin-fixed paraffin-embedded tissue to ensure that a sufficient percentage of tumor cells (target >50%) was present in the tissue from which the DNA was prepared. After microscopic examination confirmed the diagnosis of adenocarcinoma, tissue was sent to our CLIA-certified molecular diagnostic laboratory in the Department of Pathology for extraction of DNA and identification of EGFR exon 19 deletions and exon 21 L858R mutations by polymerase chain reaction (PCR) assay. Nonsequencing-based PCR assays are used to detect these mutations. 18 In samples lacking these 2 sensitizing EGFR mutations, KRAS analysis is done by direct sequencing of exon 2 using PCR products. Molecular profiling for EGFR/KRAS has been approved by the New York State Department of Health. A molecular diagnostic report is created and forwarded to the patient s medical record for inspection by the patient s surgeon and medical oncologist (Figure 1). RESULTS Between 2006 and April 2009, tumors of 1831 patients with stage I through IV adenocarcinoma of the lung underwent reflex testing, and 448 KRAS and 364 EGFR mutations were detected. Of those patients, 1097 with stages I through III adenocarcinoma of the lung underwent curative surgical resection at MSKCC. A subset of 855 patients (78%) were tested. In the patients in the early stages, 158 EGFR mutations and 207 KRAS mutations were detected (Table 2). The group included 291 males and 564 females; 594 patients were in stage I; 111 were in stage II; and 147 were in stage III. In 2006, 124 patients underwent mutation testing; 19% had EGFR mutations, and 23% had KRAS mutations (Table 3). In 2007, 324 patients underwent mutation testing; 14% had EGFR mutations, and 20% had KRAS mutations. In 2008, 293 patients underwent mutation testing; 25% had EGFR mutations, and 30% had KRAS mutations. As of April 2009, 114 patients have undergone mutation testing; 25% haveegfr mutations and 30% have KRAS mutations (Table 3). Already, 15% of these patients have experienced recurrences, and genetic information from their resected specimens has been used to select therapy. DISCUSSION Reflex testing of adenocarcinomas for EGFR and KRAS mutations at MSKCC over the past 3 years reveals some interesting observations. Nearly 78% of patients with earlystage disease have undergone mutation testing. The majority of patients with resected adenocarcinoma are females, at a nearly 2 to 1 ratio. It is unclear whether this trend is the result of demographics unique to our patient population, is related to the preponderance of adenocarcinoma in females versus males, or is related to differing smoking habits in females and males. This sex distribution is not seen in our advanced-stage patients; in a database of 1081 cases of stage IV adenocarcinomas, 59% were women. It is interesting that in a recent report concerning a large group of patients that was screened for lung cancer, it was found that the prevalence of screen-detected early The Journal of Thoracic and Cardiovascular Surgery c Volume 141, Number 2 477

3 TABLE 1. Clinical utility of reflex testing in early-stage (I through III) adenocarcinoma It informs selection of chemotherapy for recurrent disease. It may help to distinguish multiple primary tumors from metastatic disease. It allows for enrollment of patients into clinical trials to explore mutationspecific therapy. It encourages additional research efforts, including retrospective studies related to patient outcome. TABLE 2. Patient characteristics Male Female Stage I Stage II Stage III Total tested cancers was higher in women than in men, even though women had less exposure to smoking. 19 Most resections for NSCLC at MSKCC occur at stage I, which is a stage of the disease for which no routine adjuvant therapy exists. 20 They are performed in a population of patients for whom opportunities to participate in clinical trial are especially important. Counted together, EGFR and KRAS mutation testing identifies a driver mutation in nearly half of the cases of resected adenocarcinomas at our institution. To date, 15% of patients who have undergone reflex testing of resected adenocarcinoma have experienced recurrence of disease. Given the short median follow-up, it is no surprise that the majority of patients with early recurrence were found to have lymph-node metastases (ie, stage II through III) at the time of surgery. In these patients, the molecular characteristics of their tumors were immediately available to inform the selection of therapy for recurrent, stage IV disease. An alternative approach would be to test for these mutations at the time of recurrence. FIGURE 1. Reflex testing flowchart Patients at MSKCC receive postoperative, cisplatinbased chemotherapy according to established standards of care. 21 Pathologic stage is the most important factor in selecting patients for adjuvant therapy. Previous data have supported the role of adjuvant cisplatin-based chemotherapy in patients with stage II through III NSCLC. The higher the stage, the more benefit is derived from adjuvant chemotherapy. 22 Other than stage, no clinical factors are predictive of the benefits of treatment. Historically, criteria developed by Martini and Melamed and the American College of Chest Physicians (ACCP) have been used to distinguish multiple primary lung tumors from metastatic lesions. 23,24 These criteria often provide conflicting results. A recent report interpreted 7 patients tumors using the Martini-Melamed criteria, the ACCP criteria, and comprehensive histologic subtyping. When the Martini-Melamed criteria were applied, 6 of 7 patients were determined to have had multiple primary lung tumors. According to the ACCP criteria, 3 patients had multiple primaries, and 3 had metastases; the seventh patient s case was indeterminate. However, based on comprehensive histologic subtyping, all of the patients had multiple primary lung tumors. Mutational testing also suggested that the tumors were multiple primary lung adenocarcinomas. This case series demonstrates that molecular profiling can assist in clarifying clinical issues and in resolving conflicts among existing clinical criteria. 16 Reflex testing has allowed for retrospective studies of the impact of gefitinib and erlotinib as adjuvant therapy for patients with stage I through III adenocarcinomas of the lung that harbor an EGFR mutation. We have reported improved disease-free survival rates in patients with completely resected stage I through III adenocarcinomas with EGFR mutations who were treated with perioperative gefitinib or erlotinib compared to individuals who did not receive treatment with EGFR TKI. 25 We evaluated 150 patients (112 women, 38 men) who had completely resected stage I through III lung adenocarcinoma and whose resection specimens contained EGFR-activating mutations in exon 19 or 21. Of those patients, 42 (28%) received cytotoxic chemotherapy, and 48 (32%) received either erlotinib (n ¼ 26) or gefitinib (n ¼ 22) postoperatively. The median time on a TKI was 16 months. The median disease-free survival (DFS) was 43 months in the patients who received TKI as 478 The Journal of Thoracic and Cardiovascular Surgery c February 2011

4 General Thoracic Surgery TABLE 3. Results through April Resected adenocarcinomas Total tested Percent tested EGFR mutant KRAS mutant EGFR/KRAS wild type Recurred 30 (24%) 61 (19%) 38 (13%) 3 (3%) Stage I 13 (17%) 35 (15%) 19 (9%) 1 (1%) Stage II 6 (38%) 11 (28%) 7 (17%) 1 (1%) Stage III 11 (38%) 15 (30%) 12 (25%) 2 (20%) opposed to 31 months in those who did not. After controlling for stage, we found that individuals who received adjuvant gefitinib or erlotinib had a better DFS (HR, 0.56; 95% CI, ; P ¼.157) than the non-tki group. Currently a multicenter phase-2 trial is evaluating the role of adjuvant erlotinib in patients with resected adenocarcinomas. This combined experience may serve as the basis for recommending perioperative treatment with erlotinib, based on the reflex identification of an EGFR mutation in all patients following resection. Reflex testing for EGFR and KRAS mutation at our institution has allowed for studies of the prognostic significance of these molecular markers. We evaluated 1000 patients (618 women) who had stage I (732), stage II (121), or stage III (147) lung adenocarcinomas. Median follow-up was 16 months. After adjustment for stage, there was a trend toward better survival rates in EGFR mutation-positive patients (n ¼ 145) compared to wild type (n ¼ 588) (HR, 0.76; 95% CI, ; P ¼.3). There was a trend toward worse survival rates in patients with KRAS mutation (n ¼ 267) compared to wild type (HR, 1.20; 95% CI, ; P ¼.4). The 3-year survival proportions for each group were: EGFR mutation 80% (95% CI, ); KRAS mutation 73% (95% CI, ); wild type 73% (95% CI, ). 26 Reflex testing has allowed us to select patients for research studies of mutation-specific adjuvant therapy. Currently, we have enrolled 23 patients in a trial of GI-4000 (GlobeImmune, Inc., Louisville, Colo); recombinant, heat-inactivated yeast (Saccharomyces cerevisiae) engineered to express 1 of 3 mutated KRAS oncoproteins (G12C, G12V, and G12D, representing 85% of the specific KRAS mutations detected in outpatients). 24,27 Patients with KRAS mutations are immunized with a vaccine, and immune response is recorded. As mentioned earlier, patients with EGFR exon 19 or exon 21 mutations are offered the opportunity to enroll in a phase II trial of erlotinib as an adjuvant therapy. To date, 17 patients have been enrolled in this trial. Mutations in EGFR and KRAS are found in one third of lung adenocarcinomas. We have demonstrated that these molecular lesions can be readily identified in resected specimens by reflex testing. As we have recently seen with the identification of the EML4-ALK fusion gene, 28 new genetic lesions with therapeutic implications will emerge and can be readily incorporated into the reflex testing process. Our experience over the past 3 years has demonstrated that reflex testing is feasible. The data obtained through this mechanism provide patients and scientists opportunities for research, and they can also aid in clinical decision making. References 1. Ravdin PM, Davis G. Prognosis of patients with resected non-small cell lung cancer: impact of clinical and pathologic variables. Lung Cancer. 2006;52: Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP, Vansteenkiste J. Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Engl J Med. 2004;350: Azzoli CG, Park BJ, Pao W, Zakowski M, Kris MG. Molecularly tailored adjuvant chemotherapy for resected non-small cell lung cancer: a time for excitement and equipoise. 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