Lung cancer is the leading cause of cancer-related

Original Articles Correlation of Mutation Status With Predominant Histologic Subtype of Adenocarcinoma According to the New Lung Adenocarcinoma Classification of the International Association for the Study of Lung Cancer/ American Thoracic Society/European Respiratory Society Celina Villa, MD; Philip T. Cagle, MD; Melissa Johnson, MD; Jyoti D. Patel, MD; Anjana V. Yeldandi, MD; Rishi Raj, MD; Malcolm M. DeCamp, MD; Kirtee Raparia, MD Context. Epidermal growth factor receptor () mutations have been identified as predictors of response to tyrosine kinase inhibitors in non small cell lung cancer. Objective. To investigate the relationship of mutation status to the histologic subtype of adenocarcinoma according to the new International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) classification. Design. We screened mutation in 200 consecutive lung adenocarcinoma resection specimens diagnosed between 2008 and 2011. Results. Among 200 lung adenocarcinomas, mutations were identified in 41 tumors (20.5%). The mean age in the -mutant group was 64.8 years and this group consisted of 78% females and 22% males. Most patients with -positive lung cancers were never-smokers (51%) as compared to 8% with -negative cancers (P,.001). Lung cancer is the leading cause of cancer-related mortality in both men and women in the United States. It is projected that in 2013, lung cancer will account for 26% and 28% of cancer-related deaths in the female and male populations, respectively. 1 Historically, lung cancer has been Accepted for publication December 27, 2013. Published as an Early Online Release February 26, 2014. From the Department of Pathology (Drs Villa, Yeldandi, and Raparia), the Division of Medical Oncology, and Robert H. Lurie Comprehensive Cancer Center (Drs Johnson and Patel), the Division of Pulmonary, and Critical Care Medicine (Dr Raj); and the Division of Thoracic Surgery, and Robert H. Lurie Comprehensive Cancer Center (Dr DeCamp), Northwestern University Feinberg School of Medicine, Chicago, Illinois; and the Department of Pathology & Genomic Medicine, The Methodist Hospital, Houston, Texas (Dr Cagle). The authors have no relevant financial interest in the products or companies described in this article. The study was presented at the United States and Canadian Academy of Pathology 2012; March 17 23, 2012; Vancouver, British Columbia, Canada. Reprints: Kirtee Raparia, MD, Department of Pathology, 303 East Chicago Avenue, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 (e-mail: kraparia@northwestern.edu). The most common mutations identified in our population were deletions in exon 19 (22 patients) and L858R in exon 21 (12 patients). Five patients had double mutations. The predominant pattern of adenocarcinoma was lepidic (44%) in -mutant lung cancers as compared to 69% with acinar pattern in wild-type lung cancers (P,.001). Of 22 minimally invasive adenocarcinomas, 8 (36%) had mutations, accounting for 20% of adenocarcinomas with mutations (P,.05). Conclusions. Based on the new IASLC/ATS/ERS classification, the predominant subtype of adenocarcinoma was lepidic (44%) in -mutant lung cancers (P,.001). However, histologic subtype should not be used to exclude patients from tyrosine kinase inhibitor therapy, since mutations are found in lung adenocarcinomas of other subtypes. (Arch Pathol Lab Med. 2014;138:1353 1357; doi: 10.5858/arpa.2013-0376-OA) divided into 2 major categories by histologic feature and response to conventional therapies: non small cell lung carcinoma (NSCLC) and small cell lung carcinoma. NSCLC includes 3 major cell types (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma), and these can be further divided into various subtypes or variants. 2 NSCLC accounts for most (~85%) of the lung cancers, with lung adenocarcinoma being the most common subtype in the Western population. Recently the International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) proposed an international multidisciplinary lung adenocarcinoma classification. This system divides adenocarcinoma into adenocarcinoma in situ (5-year survival: 100%), minimally invasive adenocarcinoma (5-year survival: 100%), and invasive adenocarcinoma. Invasive adenocarcinoma includes lepidic, acinar, papillary, micropapillary, and solid predominant patterns and mucinous, colloid, fetal, and enteric variants. 3 Development of invasive lung carcinoma is a result of sequential accumulation of genetic and epigenetic changes that are also reflected in the morphology of these tumors. Arch Pathol Lab Med Vol 138, October 2014 Mutation and Survival in Lung Adenocarcinoma Villa et al 1353

Lung cancer is now considered a heterogeneous group of diseases, and mutational profilingoflungadenocarcinoma is a routine practice in thoracic oncology. The discovery of activating mutations in the epidermal growth factor receptor () of lung adenocarcinoma tumors, and the subsequent recognition that this biomarker predicted high response rates and prolonged progression-free survival after treatment with tyrosine kinase inhibitors (TKIs), led to a dramatic revolution in the treatment of patients with lung cancer. 4 7 The College of American Pathologists (CAP), the IASLC, and the Association for Molecular Pathology recently published evidence-based recommendations for the molecular testing of lung cancers for mutations that specify that testing should be performed for all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors. 8 Although several studies published in the recent past correlate adenocarcinoma subtypes with mutations, we present the largest study to date of sequential pulmonary adenocarcinomas, correlating mutation status with the proposed IASLC/ATS/ERS adenocarcinoma classification in a Western population. MATERIALS AND METHODS The Institutional Review Board at the Northwestern University, Feinberg School of Medicine, Chicago, Illinois, approved the study. Patient Selection and Histologic Analysis Consecutive pulmonary adenocarcinomas obtained only from resection specimens and screened for mutations/deletions in exons 18, 19, 20, and 21 between the years 2008 and 2011 in the Department of Pathology, Northwestern Memorial Hospital (Chicago, Illinois), were included in the study. The staging of the lung cancers included stage I (n ¼ 132), stage 2 (n ¼ 24), stage 3(n¼31), and stage 4 (n ¼ 13) according to the 7th edition of AJCC (American Joint Committee on Cancer) Cancer Staging Manual. 9 Clinical parameters, including smoking history, were collected from the medical records. Patients with smoking history of less than 20 pack-years were considered light smokers. 10 We classified all the adenocarcinomas according to the recent IASLC/ ATS/ERS international multidisciplinary lung adenocarcinoma classification. Molecular Analysis The mutational analysis was performed on DNA samples extracted from surgically resected specimens of lung adenocarcinomas by using polymerase chain reaction (PCR) and Qiagen PyroMark 24 sequencer (Qiagen, Valencia, California). Formalin-fixed, paraffin embedded blocks of tumor tissue (50 lm) were treated with Hemo-De (Scientific Safety Solvents, Keller, Texas) and then digested overnight at 568 in a Proteinase K and lysate solution. Genomic DNA was extracted from the digested lysate by using the Qiagen QIAsymphony DNA extractor. The Qiagen Pyro Kit provides quantitative measurements of mutations in the gene at codons, 719, 768, 790, 858, and 861, as well as deletions and complex mutations in exon 19. Ten nanograms of DNA were added to 4 separate PCR reactions using pyro-specific primers for codons 719, 768, and 790, 858 861, and exon 19). The 4 PCR products were then immobilized to Steptavidin Sepharose High-Performance beads (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania) and added to 5 different pyrosequencing reactions (codons 719, 768, 790, 858 861, and exon 19). The products were purified on the PyroMark worktable and then added to the PyroMark Q24 pyrosequencer. The results were analyzed by using the Pyro- Mark Q24 software (Qiagen). 11 Statistical Analysis The relationships between gene mutation status and the clinicopathologic features, including subtype according to the IASLC/ATS/ERS proposed classification, were analyzed by using Fisher exact test for categorical variables and the Wilcoxon ranksum test for continuous variables. RESULTS Mutations and Demographics The frequency of mutations and no mutations (wild type) in 200 tumors was 20.5% and 79.5%, respectively. The mean age in the -mutant group was 64.8 years (age range, 39 to 86 years) and was similar to that of patients with wild-type tumors. As expected, mutations were more common in tumors sampled from female (78%) than male patients (22%). Most patients whose tumors had mutation were never-smokers (51%) as compared to 8% in patients with wild-type tumors (P,.001) (Figure 1; Table 1). Mutations and Histologic Subtypes Eight (36%) of 22 minimally invasive adenocarcinomas had mutations, representing 20% of the lung cancers with mutant gene as compared to 9% of the lung cancers with wild-type gene (P ¼.05) (Figure 2). The predominant pattern seen in the -mutant positive lung cancer group was lepidic (44%) as compared to patients with wild-type gene, whose tumors exhibited acinar pattern in 69% and lepidic pattern in 15% of cases; P,.001 (Figure 3). We also analyzed the histologic patterns in adenocarcinomas in relation to exon 19 deletions versus exon 21 mutations, and found no specific pattern associated with either of these specific mutations. The patient with T790M mutations had predominant acinar histologic pattern. Thyroid transcription factor 1 (TTF-1) positivity was seen in all the -mutant positive lung cancers tested (P ¼.05). The clinical and pathologic findings in both mutant and wild-type pulmonary adenocarcinomas are summarized in Table 2. Types of Mutations The mutations identified in our population were L858R in exon 21 (n ¼ 12 ), E746 deletions in exon 19 (n ¼ 12), L747 deletions in exon 19 (n ¼ 5), other deletions in exon 19 (n ¼ 5), G719A in exon 18 (n ¼ 2), S768 in exon 20 (n ¼ 2), L861Q mutation in exon 21 (n ¼ 4), T790M in exon 20 (n ¼ 1), and E709 in exon 18 (n ¼ 1). Five cancers had double mutation (Table 3). COMMENT Although there have been several studies in the recent past attempting to identify the histologic correlates of lung adenocarcinomas with mutations, the relationship between mutations and subtype of adenocarcinoma remains unclear. Studies suggest that mutations are found in 5% to 20% of white patients and 20% to 60% of Asian patients with lung adenocarcinomas. 12 19 Although mutations are frequently observed in never-smoker females with invasive adenocarcinoma having a predominant lepidic pattern, a significant percentage have also been noted in acinar and papillary variants of adenocarcinoma. 5,14,17,20 26 A study involving white patients identified that all the tumors with mutations were nonmucinous, well-differentiated, TTF-1 positive adenocarcinomas. 22 Rekhtman et al 27 also 1354 Arch Pathol Lab Med Vol 138, October 2014 Mutation and Survival in Lung Adenocarcinoma Villa et al

Table 1. Demographics of Patients With Mutant and Wild-Type Lung Cancers Characteristics Mutant Wild Type P Value Total patients, No. (%) 41 (20.5) 159 (79.5) Age, y Mean 64.8 66.5.65 Median 67 67 SD 11.9 9.6 Range 39 86 43 91 Age, No. (%) 65 y 17 (42) 71 (45).71.66 y 24 (58) 88 (55) Sex, No. (%) Male 9 (22) 50 (31).23 Female 32 (78) 109 (69) Smoking status, No. (%) Light smokers 10 (24) 26 (16),.001 Heavy smokers 10 (25) 120 (76) Never-smokers 21 (51) 13 (8) Follow-up duration, mo Mean 32.3 31.0.92 Median 32 30.0 SD 22.5 22.6 Range 0.7 135 0.03 175 Abbreviation:, epidermal growth factor receptor gene. Figure 1. Patients smoking history and mutation status: 56% of patients in the -mutant group were never-smokers as compared to 14% in the wild-type group; P,.001. Figure 2. Minimally invasive adenocarcinoma (MIA) and mutation status: MIA was seen in 8 -mutant lung cancers (36%) representing 20% of the lung cancers with mutant gene as compared to 9% of the lung cancers with wild-type gene; P ¼.05. showed that in a group of 35 patients (19%) who were positive for mutation and were never-/light smokers, the mutations were associated with nonmucinous adenocarcinoma with lepidic and papillary patterns that expressed TTF-1. A study in Chinese patients 25 showed that mutations were closely associated with the micropapillary and lepidic subtypes. Russell et al 23 showed that mutations were identified in 22% of patients with stage III (N2) lung adenocarcinoma and were associated with acinar and micropapillary predominant tumors. Our study similarly shows a 20.5% frequency of mutations, occurring commonly in females and never-smokers (51% of patients). The common mutations in lung adenocarcinoma in our series, L858R in exon 21 that was present in 12 patients (29%) and deletions in exon 19 (including E746 and L747) in 34 patients (54%), paralleled results from previous studies. In addition, 5 patients (12%) in our study had double mutations and 3 patients had rare exon 18 mutations. exon 21 mutations were commonly associated with lepidic predominant adenocarcinomas and exon 20 mutations with solid histology in the past studies. 28,29 But we did not find any association between the histologic patterns and tumors with exon 19 deletions versus exon 21 mutations. However, 1 patient with T790M mutation in our study had predominant acinar histologic pattern. Histologic features of lung adenocarcinoma may play a role in predicting the outcome in patients with mutation who are treated with TKIs. Yoshida et al 30 have shown that patients with mutations with predominant Figure 3. Predominant pattern of adenocarcinoma and mutation status: mutant-positive lung cancers were lepidic (44%) as compared to tumors with wild-type gene, whose tumors exhibited acinar pattern in 69% and lepidic pattern in 15%; P,.001. Arch Pathol Lab Med Vol 138, October 2014 Mutation and Survival in Lung Adenocarcinoma Villa et al 1355

Table 2. Clinical and Pathologic Characteristics of Mutant and Wild-Type Pulmonary Adenocarcinomas Characteristics Mutant, No. (%) Wild Type, No. (%) P Value Tumor size, cm,2 23 (56) 99 (62).25 2 3 11 (27) 24 (15) 3 5 6 (15) 24 (15).5 1 (2) 12 (8) Predominant pattern Lepidic 18 (44) 23 (15),.001 Acinar 17 (42) 109 (69) Papillary 2 (5) 5 (3) Solid 3 (7) 20 (13) Micropapillary 0 0 Variants of adenocarcinoma Mucinous 1 (2) 2 (1).50 MIA Yes 8 (20) 14 (9).05 No 33 (80) 145 (91) TTF-1 positivity Positive 32 (100) 101 (89).05 Negative 0 (0) 12 (11) Pleural status PI present 5 (12) 29 (18).35 PI absent 36 (88) 130 (82) Nodal status N0 32 (84) 109 (78).52 N1 2 (5) 16 (11) N2 3 (8) 14 (10) N3 1 (3) 1 (1) Patients developing brain metastasis 2 (5) 13 (8).47 Abbreviations:, epidermal growth factor receptor gene; MIA, minimally invasive adenocarcinoma; PI, pleural invasion (visceral); TTF-1, thyroid transcription factor 1. solid pattern of adenocarcinoma have significantly worse overall response to TKIs. Others 17,23,31 have shown that the new classification correlates with patient prognosis and provides important prognostic information. Our study is the largest study of sequential pulmonary adenocarcinomas correlating mutation status with the Table 3. Types of Mutations in 41 Patients Positive for Mutations Mutation Exon No. of Patients G719A 18 2 E709 18 1 E746-A750 del 19 12 L747- P753 del 19 3 L747-S752 del 19 1 L747-T751 del 19 1 Other deletions in exon 19 19 5 T790M 20 1 S768 20 2 A767-V769 20 1 A767-Y764 20 1 L858R 21 12 L861Q 21 4 Double mutation 5 proposed IASLC/ATS/ERS adenocarcinoma classification. Based on the proposed classification, adenocarcinomas with predominant lepidic pattern are more commonly associated with mutations (P,.001). However, subtype should not be used to exclude patients from TKI therapy, since mutations are found in lung adenocarcinomas of other subtypes. References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63(1):11 30. 2. Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARCPress; 2004. World Health Organization Classification of Tumours; vol 10. 3. Travis WD, Brambilla E, Noguchi M, et al. International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;6(2):244 285. 4. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from never smokers and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004; 101(36):13306 13311. 5. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129 2139. 6. Paez JG, Janne PA, Lee JC, et al. mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676):1497 1500. 7. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361(10):947 957. 8. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med. 2013;137(6):828 860. 9. Edge S, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, eds. AJCC Cancer Staging Manual. 7th ed. Chicago, IL: Springer; 2009. 10. Pham D, Kris MG, Riely GJ, et al. Use of cigarette-smoking history to estimate the likelihood of mutations in epidermal growth factor receptor gene exons 19 and 21 in lung adenocarcinomas. J Clin Oncol. 2006;24(11):1700 1704. 11. Rho JK, Choi YJ, Lee JK, et al. The role of MET activation in determining the sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors. Mol Cancer Res. 2009;7(10):1736 1743. 12. Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol. 2011;12(2):175 180. 13. Sequist LV, Bell DW, Lynch TJ, Haber DA. Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer. J Clin Oncol. 2007;25(5):587 595. 14. Tokumo M, Toyooka S, Kiura K, et al. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res. 2005;11(3):1167 1173. 15. Li C, Sun Y, Fang Z, et al. Comprehensive analysis of epidermal growth factor receptor gene status in lung adenocarcinoma. J Thorac Oncol. 2011;6(6): 1016 1021. 16. Liam CK, Wahid MI, Rajadurai P, Cheah YK, Ng TS. Epidermal growth factor receptor mutations in lung adenocarcinoma in malaysian patients. J Thorac Oncol. 2013;8(6):766 772. 17. Yoshizawa A, Sumiyoshi S, Sonobe M, et al. Validation of the IASLC/ATS/ ERS lung adenocarcinoma classification for prognosis and association with and KRAS gene mutations: analysis of 440 Japanese patients. J Thorac Oncol. 2013;8(1):52 61. 18. Bauml J, Mick R, Zhang Y, et al. Frequency of and KRAS mutations in patients with non small cell lung cancer by racial background: do disparities exist? Lung Cancer. 2013;81(3):347 353. 19. Boch C, Kollmeier J, Roth A, et al. The frequency of and KRAS mutations in non-small cell lung cancer (NSCLC): routine screening data for central Europe from a cohort study. BMJ Open. 2013;3(4). 20. Sun PL, Seol H, Lee HJ, et al. High incidence of mutations in Korean men smokers with no intratumoral heterogeneity of lung adenocarcinomas: correlation with histologic subtypes, /TTF-1 expressions, and clinical features. J Thorac Oncol. 2012;7(2):323 330. 21. Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res. 2004;64(24):8919 8923. 22. Leary AF, Castro DG, Nicholson AG, et al. Establishing an mutation screening service for non-small cell lung cancer sample quality criteria and candidate histological predictors. Eur J Cancer. 2012;48(1):61 67. 23. Russell PA, Barnett SA, Walkiewicz M, et al. Correlation of mutation status and survival with predominant histologic subtype according to the new IASLC/ ATS/ERS lung adenocarcinoma classification in stage III (N2) patients. J Thorac Oncol. 2013;8(4):461 468. 24. Mazieres J, Rouquette I, Lepage B, et al. Specificities of lung adenocarcinoma in women who have never smoked. J Thorac Oncol. 2013;8(7):923 929. 1356 Arch Pathol Lab Med Vol 138, October 2014 Mutation and Survival in Lung Adenocarcinoma Villa et al

25. Song Z, Zhu H, Guo Z, Wu W, Sun W, Zhang Y. Correlation of mutation and predominant histologic subtype according to the new lung adenocarcinoma classification in Chinese patients. Med Oncol. 2013;30(3):645. 26. Shim HS, Lee da H, Park EJ, Kim SH. Histopathologic characteristics of lung adenocarcinomas with epidermal growth factor receptor mutations in the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society lung adenocarcinoma classification. Arch Pathol Lab Med. 2011;135(10):1329 1334. 27. Rekhtman N, Ang DC, Riely GJ, Ladanyi M, Moreira AL. KRAS mutations are associated with solid growth pattern and tumor-infiltrating leukocytes in lung adenocarcinoma. Mod Pathol. 2013;26(10):1307 1319. 28. Lee HJ, Kim YT, Kang CH, et al. Epidermal growth factor receptor mutation in lung adenocarcinomas: relationship with CT characteristics and histologic subtypes. Radiology. 2013;268(1):254 264. 29. Arcila ME, Nafa K, Chaft JE, et al. exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics. Mol Cancer Ther. 2013;12(2):220 229. 30. Yoshida T, Ishii G, Goto K, et al. Solid predominant histology predicts tyrosine kinase inhibitor response in patients with mutation-positive lung adenocarcinoma. J Cancer Res Clin Oncol. 2013;139(10):1691 1700. 31. Tsuta K, Kawago M, Inoue E, et al. The utility of the proposed IASLC/ATS/ ERS lung adenocarcinoma subtypes for disease prognosis and correlation of driver gene alterations. Lung Cancer. 2013;81(3):371 376. Arch Pathol Lab Med Vol 138, October 2014 Mutation and Survival in Lung Adenocarcinoma Villa et al 1357