CHEST
Original Research LUNG CANCER
Comprehensive Analysis of Oncogenic Mutations in Lung Squamous Cell Carcinoma With Minor Glandular Component Yunjian Pan, MD; Rui Wang, MD; Ting Ye, MD; Chenguang Li, MD; Haichuan Hu, MD; Yongfu Yu, MS; Yang Zhang, MD; Lei Wang, MD; Xiaoyang Luo, MD; Hang Li, MD; Yuan Li, MD; Lei Shen, MD; Yihua Sun, MD; and Haiquan Chen, MD, FCCP
Background: The mutations in oncogenic genes, such as EGFR, ALK, BRAF, HER2, DDR2, RET, and AKT1, defined subsets of non-small cell lung cancers (NSCLCs) with potential sensitivity to targeted therapies. At present, the mutational spectrum, prevalence, and clinicopathologic characteristics in squamous cell carcinomas with minor (, 10%) glandular component (SQCC-mGCs) are not well established. Methods: Three hundred ten surgically resected lung squamous cell carcinoma (SQCC) specimens were collected. The histology of all cases was reevaluated using hematoxylin-eosin and immunohistochemistry staining. EGFR, KRAS, HER2, BRAF, PIK3CA, AKT1, and DDR2 mutations, as well as ALK and RET rearrangements, were examined in 310 SQCCs by directed sequencing. Results: Ninety-five SQCC-mGCs (30.6%) and 215 pure SQCCs (69.4%) were identified. Of the 95 SQCC-mGCs, 26 (27.4%; 95% CI, 18.7%-37.4%) were found to harbor known oncogenic mutations, including 10 with EGFR, seven with KRAS, three with PIK3CA, one with BRAF, one with HER2, one each with EGFR/PIK3CA and KRAS/PIK3CA double mutations, and two with EML4-ALK fusions. Ten of 215 pure SQCCs (4.7%; 95% CI, 2.3%-8.4%) harbored mutations, including seven with PIK3CA, and each with AKT1, DDR2, and EGFR. No RET rearrangements were detected in SQCCs. SQCC-mGCs had a significantly higher rate of mutations in known oncogenic genes than that in pure SQCCs (27.4% vs 4.7%, P , .001). All KRAS mutations occurred in SQCC-mGCs. Conclusions: Our results demonstrated that oncogenic mutations in EGFR, KRAS, BRAF, HER2, and ALK were extremely rare or absent in patients with pure SQCC, whereas SQCC-mGC had a relatively high frequency of EGFR, ALK, or KRAS mutations. Prospective identification of these known oncogenic mutations in SQCC-mGC before the initiation of treatment is an essential step to identify which patient could benefit from targeted therapies. CHEST 2014; 145(3):473–479 Abbreviations: cDNA 5 complementary DNA; CK 5 cytokeratin; EGFR-TKI 5 epidermal growth factor receptor tyrosine kinase inhibitor; IHC 5 immunohistochemistry; NSCLC 5 non-small cell lung cancer; OS 5 overall survival; PCR 5 polymerase chain reaction; RFS 5 relapse-free survival; SQCC 5 squamous cell carcinoma; SQCC-mGC 5 squamous cell carcinoma with minor glandular component; TTF-1 5 thyroid transcription factor-1
understanding of the genetic alterations Detailed that drive subsets of lung cancers has led to the
development of targeted agents, which have changed the treatment landscape.1-6 Oncogenic mutations occur in genes that encode signaling kinases crucial for cellular proliferation and survival.7 Cancer cells might depend on the mutant kinase for survival and die when it is inactivated. Therefore, these mutant kinases
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could be exploited for therapeutic target, which is best illustrated by EGFR. Treatment with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) leads to dramatic regression of tumors and improved survival in patients whose tumor harbored EGFR mutations.3,8,9 More recently, patients with ALK rearrangement demonstrated dramatic responses to crizotinib,1 which has been recommended by the US Food and CHEST / 145 / 3 / MARCH 2014
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Drug Administration as first-line therapy for patients with advanced-stage non-small cell lung cancer (NSCLC) with ALK rearrangement. Except EGFR and ALK, the established driver genes in NSCLCs also include KRAS, BRAF, HER2, RET, AKT1, PIK3CA, and DDR2.10 EGFR, KRAS, HER2, and BRAF mutations, as well as ALK and RET rearrangements, were found much more frequently in lung adenocarcinomas,1,7,11-13 whereas mutations in PIK3CA, AKT1, and DDR2 were found mainly in squamous cell carcinomas (SQCCs).7,10 Except for KRAS, all the other known mutant kinases could be targeted by agents being used in the clinic or evaluated in clinical trials.7 More than 80% of lung adenocarcinomas from East Asian populations can be defined by known oncogenic mutations, such as EGFR, KRAS, HER2, BRAF, or ALK.12 However, the mutation profiles in lung SQCCs were still largely unclear.14 The frequency of EGFR and KRAS mutations in lung SQCCs ranged from 1% to 15% and 1% to 9%,15-21 respectively. A study showed that lung SQCCs without any glandular component (pure SQCC) by microscopic examination and immunohistochemistry (IHC) biomarker verification were all negative for EGFR and KRAS mutations, suggesting that the variation of their mutation rates in SQCCs might be caused by the glandular component in SQCCs.22 However, its accurate prevalence and clinicopathologic and mutational characteristics remain unknown. In this study, tumor with , 10% glandular component is classified as SQCC with minor glandular component (SQCC-mGC), and SQCC without any glandular component is identified as pure SQCC. The mutational status of known oncogenic genes, including EGFR, KRAS, HER2, BRAF, PIK3CA, AKT1, DDR2, ALK, and RET, were examined in 310 SQCCs. Manuscript received November 1, 2012; revision accepted September 16, 2013. Affiliations: From the Department of Thoracic Surgery (Drs Pan, R. Wang, Ye, C. Li, Hu, Zhang, L. Wang, Luo, H. Li, Sun, and Chen), and the Department of Pathology (Drs Y. Li and Shen), Fudan University Shanghai Cancer Center; and the Department of Oncology (Drs Pan, R. Wang, Ye, C. Li, Hu, Zhang, L. Wang, Luo, H. Li, Y. Li, Shen, Sun, and Chen), Shanghai Medical College, and the Department of Biostatistics (Mr Yu), School of Public Health, Fudan University, Shanghai, China. Drs Pan, R. Wang, Ye, and C. Li contributed to this work equally and should be considered co-first authors. Drs Chen and Sun contributed to this work equally. Funding/Support: This study was supported by the National Natural Science Foundation of China [Grant 81101761, 81172218] and the Key Construction Program of the National “985” Project Grant [985-YFX0102]. Correspondence to: Haiquan Chen, MD, FCCP, Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, 270 Dong’An Rd, Shanghai 200032, China; e-mail: hqchen1@ yahoo.com © 2014 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.12-2679 474
Materials and Methods Specimen Collection A total of 324 cases reported as SQCC were included, and the frozen samples and corresponding formalin-fixed, paraffinembedded tumor blocks (at least two blocks for each case) were consecutively collected from August 2007 to August 2011 in Fudan University Shanghai Cancer Center. Written informed consent was obtained from each patient. Each specimen contained at least 50% tumor cells. The study was approved by the Committee for Ethical Review of Research (Fudan University Shanghai Cancer Center IRB# 090977-1). Only cases with confirmed SQCC diagnosis by hematoxylin-eosin and IHC staining were selected for mutational analysis. Clinicopathologic data were obtained from electronic medical records. Pathologic Reassessment Tumor blocks were cut into 4-mm-thick sections. All sections were stained with hematoxylin-eosin and IHC (at least two for each case). Morphologic examinations were performed by two pathologists (Y. Li and L. Shen), as shown in Figure 1. Well-differentiated tumors with typical keratinization and/or intercellular bridges were only stained with thyroid transcription factor-1 (TTF-1) and cytokeratin (CK) 7. Moderate and poorly differentiated tumors were stained with a panel of P63, CK5/6, TTF-1, and CK7. Primary antibodies included mouse anti-human P63 monoclonal antibody (4A4 clone; Maixin Technology Co, Ltd) (working concentration 1 mg/mL), mouse anti-human TTF-1 monoclonal antibody (8G7G3/1 clone; Shanghai Long Island Biotec Co, Ltd) (working concentration 1 mg/mL), mouse anti-human CK5/6 monoclonal antibody (D5/16 B4 clone; Dako) (working concentration 10 mg/mL), and mouse anti-human CK7 monoclonal antibody (OV-TL 12/30 clone; Maixin Technology Co, Ltd) (working concentration 2 mg/mL). Antigen retrieval was performed using sodium citrate buffer (10 mM, pH 5 6.0) (Solarbio). Slides were treated with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. Primary antibodies were applied and incubated for 60 min. The DAB Envision Kit was used (Real Envision Detection Kit; Gene Tech). For TTF-1 and CK7 staining, intensity (0, 11, 21, 31) and percentage of immunoreactive tumor cells were recorded as follows: 31, strong staining intensity in , 10% tumor cells; 21, moderate staining intensity in , 10% tumor cells; 11, faint or weak staining intensity in , 10% tumor cells; and 0, no staining. Tumors with 31 and 21 intensity in , 10% tumor cells were defined as TTF-1 or CK7-positive. Tumors with . 10% TTF-1 and/or CK7-positive tumor cells were excluded in the following study. Pure SQCC was defined by p63 and/or CK5/6-diffuse with TTF-1/CK7 double-negative staining. Reverse Transcription Polymerase Chain Reaction and Mutation Analysis Frozen tumor specimens were dissected, and RNA/DNA was coextracted following the standard instructions of the RNA/DNA isolation kit (Tiangen Biotech Co, Ltd). Single-stranded RNA of each sample is reverse transcribed into complementary DNA (cDNA) by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc). EGFR (exons 18-21), HER2 (exons 18-21), KRAS (exons 2-3), BRAF (exons 11-15), AKT1 (exons 2-3), PIK3CA (exon 9 and exon 20), and DDR2 (whole coding exons) were amplified with KOD Plus Neo DNA polymerase (Toyobo Co, Ltd). For detection of EML4-ALK, KIF5B-RET, and CCDC6-RET fusions, primers were designed to cover all known fusion variants. Polymerase chain reaction (PCR) was performed in a 25-mL reaction tube on Mastercycler pro PCR apparatus (Eppendorf AG). RNase-free water was used as a PCR-negative control. Thermocycler Original Research
Figure 1. Microscopic features of squamous cell carcinoma (SQCC) with minor glandular component with hematoxylin-eosin staining under pathologists’ review. The top shows an image of SQCC with acinar component (magnification, 3200). Two components (marked with red dotted boxes) are shown in amplified images on the bottom (magnification, 3400). settings were as following: 94°C for 2 min; 98°C for 10 s, 61°C for 30 s, 68°C for 30 s/kb, 40 cycles; and 68°C for 5 min. PCR products were qualified using agarose gel electrophoresis and directly sequenced. Sequences were analyzed using Chromas Lite, version 2.4 (Technelysium Pty Ltd) and Vector NTI 11.0 (Life Technologies Corp) to detect mutations/rearrangements by comparing with the standard gene sequences. For cDNA-screened mutations, subsequent DNA confirmations were conducted to exclude pseudomutations. Reverse transcription PCR primers used for cDNA screening are listed in e-Table 1.
Clinicopathologic characteristics of 310 patients are shown in Table 1. Of 95 SQCC-mGCs, 61 cases (64.2%) were TTF-1 and/or CK7-positive and 34 cases (35.8%) were TTF-1 and CK7 double negative, which were diagnosed solely on the glandular component by microscopic examination (e-Fig 1). All pure SQCCs showed TTF-1 and CK7 double-negative staining. Positive and negative control subjects of P63, CK5/6, TTF-1, and CK7 are shown in e-Figure 2.
Statistical Analysis The association between mutations and clinicopathologic parameters were analyzed by x2 (when no cell of a contingency table has expected count , 5) or Fisher exact tests (when any cell of a contingency table has expected count , 5). For multivariate analyses, logistic regression model was used. The Kaplan-Meier method was used to estimate relapse-free survival (RFS) and overall survival (OS), and the differences were compared using the log-rank test. All data were analyzed using the Statistical Package for the Social Sciences, version 16.0 software (IBM) or Prism 5.0 (GraphPad Software, Inc). The two-sided significance level was set at P , .05.
Results Patient Enrollment A total of 310 cases met eligibility for this study, including 95 SQCC-mGCs and 215 pure SQCCs. journal.publications.chestnet.org
Mutational Spectrum of SQCCs In total, 36 patients out of 310 with SQCCs were found to harbor mutations (11.6%; 95% CI, 8.3%15.7%). Twenty-six patients with SQCC-mGC harbored 28 known oncogenic mutations (27.4%; 95% CI, 18.7%-37.4%), including 10 EGFR, seven KRAS, three PIK3CA, one EGFR/PIK3CA, one KRAS/PIK3CA, one BRAF, one HER2 mutation, and two EML4-ALK fusions. Mutations predominantly occurred in TTF-1 and/or CK7-positive samples (22 of 61, 36.1%; 95% CI, 24.2%-49.4%), including eight EGFR, six KRAS, one BRAF, one HER2, one EGFR/PIK3CA, one KRAS/ PIK3CA, two PIK3CA mutations, and two EML4-ALK fusions. Of the 34 TTF-1 and CK7 double-negative CHEST / 145 / 3 / MARCH 2014
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Table 1—Detailed Clinicopathologic Characteristics of 310 Lung Squamous Cell Carcinomas Variable Total patients Age, y , 60 ⱖ 60 Sex Male Female Smoking Never smoker Smoker Location Central type Peripheral type Differentiation Moderate to well Poor Tumor size T1 T2-3 LN metastasis N0 N1-3 TNM stage Stage I Stage II-III
No. (%) 310 135 (43.5) 175 (56.5) 283 (91.3) 27 (8.7) 60 (19.4) 250 (80.6) 220 (71.0) 90 (29.0) 154 (49.7) 156 (50.3) 87 (28.1) 223 (71.9) 169 (54.5) 141 (45.5) 131 (42.3) 179 (57.7)
Never smokers are patients who smoked , 100 cigarettes in their lifetime. LN 5 lymph node.
Figure 2. Mutational profiles of pure SQCC, SQCC, and SQCCmGC. SQCC-mGC 5 SQCC with minor glandular component. See Figure 1 legend for expansion of other abbreviation.
iate logistic regression analysis, which confirmed that minor glandular component was the only independent and significant factor to predict tumors harboring EGFR mutations (Table 3). KRAS mutations were found exclusively in tumors that harbored a minor glandular component. We also found that both EGFR and KRAS mutations were significantly higher in SQCC-mGCs than in pure SQCCs (e-Table 4). No correlation was found between PIK3CA mutation status and clinicopathologic characteristics in patients with SQCCs. Clinical Outcome
SQCC-mGCs, four were found to harbor mutations (four of 34, 11.8%; 95% CI, 3.3%-27.5%), including two EGFR, one KRAS, and one PIK3CA mutation. Ten patients with pure SQCC (10 of 215, 4.7%; 95% CI, 2.3%-8.4%) were found to harbor known oncogenic mutations, including seven PIK3CA, one AKT1, one DDR2, and one EGFR mutation. No RET rearrangements are detected in either group. SQCC-mGCs had a significantly higher rate of mutations in known oncogenic genes than that in pure SQCCs (27.4% vs 4.7%, P , .001) (Fig 2). Clinicopathologic characteristics of patients with oncogenic mutations listed in e-Table 2. Primary data of 310 SQCCs were shown in e-Table 3. Correlation Between Clinicopathologic Characteristics and Major Oncogenic Mutations (EGFR, KRAS, PIK3CA) There were 12 EGFR mutations, eight KRAS mutations, and 12 PIK3CA mutations in this cohort. As shown in Table 2, there was a significantly higher rate of EGFR mutations in women or never smokers, which is similar to that in lung adenocarcinomas.5,11-13,15,23 Univariate analysis showed that EGFR mutations were found more frequently in tumor with minor glandular component or located in peripheral lung. The joint effect of these factors was examined using multivar476
The survival data were available in 239 patients with SQCC. The 2-year overall survival for patients with EGFR, KRAS, and PIK3CA mutations was 82.5% (95% CI, 46.1%-95.3%), 50.0% (95% CI, 11.1%-80.4%), and 66.7% (95% CI, 19.5%-90.4%), respectively. The number of AKT1, HER2, RET, and DDR2 mutations was too small for further analysis. Patients with late-stage disease (stage II-III) had a worse RFS and OS than patients with early-stage SQCC (stage I). There were no significant differences in RFS and OS between patients with EGFR mutation and wild type. The results of the univariate survival analysis are shown in e-Table 5. There were no statistically significant differences in RFS or OS for patients with SQCC with EGFR mutations compared with KRAS, PIK3CA, or wild type (e-Fig 3). Treatment Effect With EGFR-TKIs Five patients with SQCC received EGFR-TKI treatment when the disease relapsed. Three were treated with erlotinib (Tarceva) and two with gefitinib (Iressa). Detailed characteristics and survival data of the five patients are listed in Table 4. Only the patient with EGFR-activating mutation (exon 19 microdeletion) showed partial response in tumor when treated with gefitinib. Patients with wild-type EGFR experienced either stable disease or progression of disease. Original Research
Table 2—The Association Between Clinicopathologic Characteristics and Mutational Status of EGFR, KRAS, and PIK3CA in 310 Lung Squamous Cell Carcinomas EGFR Variable
KRAS
Mut
Wild
P Value
12
298
… .292
7 5
128 170
7 5
276 22
7 5
53 245
5 7
215 83
3 9
151 147
6 6
163 135
4 8
127 171
11 1
84 214
3 5
86 38
Total patients Age, y , 60 ⱖ 60 Sex Male Female Smoking Never smoker Smoker Location Central type Peripheral type Differentiation Moderate to well Poor LN metastasis N0 N1-3 TNM stage Stage I Stage II-III Glandular component With Without Recurrence Local Metastasis
PIK3CA
Mut
Wild
P Value
8
302
… .709
4 4
131 171
8 0
275 27
1 7
59 243
6 2
214 88
4 4
150 152
5 3
164 138
4 4
127 175
8 0
87 215
1 2
88 41
.002
Mut
Wild
P Value
12
298
… .893
5 7
130 168
11 1
272 26
3 9
57 241
6 6
214 84
9 3
145 153
7 5
162 136
5 7
126 172
5 7
90 208
2 2
87 41
1.000
.003
1.000
1.00
.045
.708
1.00
.081
.114
1.00
.749
.074
.732
.523
.787
.726
, .001
.966
, .001
.112
.523
.247
.596
Never smokers are patients who smoked , 100 cigarettes in their lifetime. Mut 5 mutant type; Wild 5 wild type. See Table 1 legend for expansion of other abbreviation.
Discussion Oncogenic mutations in EGFR, KRAS, BRAF, HER2, ALK, and RET have been well studied in lung adenocarcinomas.7,11,13,23 It still remains controversial whether SQCCs harbored these mutations.18,22,24 In this study, we performed comprehensive and concurrent analyses of major known oncogenic mutations in a large cohort of patients with SQCC, aiming to illustrate the spectrum of mutations in SQCC-mGCs and pure SQCCs. According to the World Health Organization’s classification, adenosquamous lung carcinomas were defined as tumors showing both glandular and squamous components, with each component composing at least
10% of the tumor.25 Although 10% is an arbitrary cutoff that is not based on any biologic and genetic evidences, pathologists tend to set it as the common criterion to separate adenosquamous lung carcinomas from SQCC-mGC.26-28 SQCC-mGCs also have two distinct cell components. However, their mutation profile and clinicopathologic characteristics were completely unknown. We found that 27.4% of SQCC-mGCs harbored known oncogenic mutations in EGFR, KRAS, BRAF, HER2, PIK3CA, and ALK. To our knowledge, the current study represents the first comprehensive mutational analysis of these known driver genes in a large cohort of patients with SQCC-mGCs. The diagnosis of SQCC-mGCs can be hardly made in small samples, such as biopsy or cytologic specimens,
Table 3—Multivariate Analysis of Clinicopathologic Parameters That Might Affect the Presence of EGFR Mutations Variable Sex Smoking Location Glandular component
Category
OR
95% CI
P Value
Male/female Smoker/never smoker Central/peripheral Present/absent
0.33 0.29 0.30 27.92
0.06-1.86 0.06-1.47 0.08-1.12 3.41-228.21
.208 .135 .073 .002
Never smokers are patients who smoked , 100 cigarettes in their lifetime. journal.publications.chestnet.org
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Table 4—Clinicopathologic Characteristics and Survival Data of Patients with EGFR-TKI Treatment ID
Pathology
Age, y/Sex
Smoking
Mutation
TKI Treatment
Responsea
RFS
OS
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Pure SCC Pure SCC SCC-mGC Pure SCC Pure SCC
55/M 50/M 49/F 43/M 68/F
Smoker Smoker Never Never Never
Wild type Wild type EGFR LREATSPK deletion Wild type Wild type
Erlotinib Erlotinib Gefitinib Erlotinib Gefitinib
PD PD PR SD PD
14.5 4.5 17.5 12.7 6.2
37.3 15 27.4 24.01 8.31
Never smokers are patients who smoked , 100 cigarettes in their lifetime. “1” indicates censored data. EGFR-TKI 5 epidermal growth factor receptor-tyrosine kinase inhibitor; F 5 female; M 5 male; OS 5 overall survival; PD 5 progressive disease; PR 5 partial response; RFS 5 relapsefree survival; SQCC 5 squamous cell carcinoma; SQCC-mGC 5 squamous cell carcinoma with minor glandular component; SD 5 stable disease; TKI 5 tyrosine kinase inhibitor. aResponse Evaluation Criteria In Solid Tumors (RECIST), version 1.1.
due to the insufficient tissue for further evaluation. Thus, there is a high probability that SQCC-mGC would be misdiagnosed as pure SQCC. In current study, the whole tumor from each patient was collected by surgical resection, and SQCC-mGCs were diagnosed by a combined strategy of morphologic examination and IHC biomarker staining. We also collected at least two tumor blocks for each case to minimize the possibility of incomplete sampling, especially in large tumors. Our results showed that SQCC-mGC was a common disease, accounting for nearly one-third of SQCCs. We found that 19 of 95 patients (20%) with SQCCmGCs harbored EGFR, BRAF, HER2, ALK, or PIK3CA mutations/fusions, which were druggable targets for anticancer therapy. Erlotinib/gefitinib specifically targets the EGFR tyrosine kinase and has showed a significant survival benefit for patients with NSCLC with activating EGFR mutations.9,29 Advanced NSCLCs with ALK fusion are highly sensitive to the ALK inhibitor crizotinib, which is, thus, recommended by the US Food and Drug Administration as first-line therapy for patients whose tumor contains this genetic abnormality. Thus, prospective identification of these druggable mutant kinases in SQCC-mGCs could lead to rationally chosen specific targeted therapy. In our study, the major known oncogenic mutations are investigated in pure SQCCs, which were verified by comprehensive pathologic assessment. We demonstrated that pure SQCCs were lacking EGFR and KRAS mutations, which is consistent with previous studies.22 We also found that one patient with pure SQCC (one of 215) had EGFR mutation. Although all samples in the current study were collected by surgical resection and verified by histologic assessment, we still could not exclude the possibility that SQCC with glandular component might be misdiagnosed as pure SQCC as a result of incomplete sampling of the tumor. In conclusion, our analyses demonstrated that oncogenic mutations in EGFR, KRAS, BRAF, HER2, and ALK were extremely rare or absent in patients with pure SQCC, whereas SQCC-mGC had relatively high frequency of EGFR, ALK, or KRAS mutations. Thus, 478
prospective identification of these oncogenic mutations in SQCC-mGC before the initiation of treatment is an essential step to identify which patient might benefit from targeted therapies. Acknowledgments Author contributions: Dr Chen is the guarantor of the manuscript. Dr Pan: contributed to conception and study design, acquisition and analysis of data, and writing and revision of the manuscript. Dr R. Wang: contributed to conception and study design, acquisition and analysis of data, and writing and revision of the manuscript. Dr Ye: contributed to conception and study design, acquisition and analysis of data, and writing and revision of the manuscript. Dr C. Li: contributed to acquisition of data and writing and revision of the manuscript. Dr Hu: contributed to acquisition of data and writing and revision of the manuscript. Mr Yu: contributed to analysis of data and revision of the manuscript. Dr Zhang: contributed to acquisition of data and revision of the manuscript. Dr L. Wang: contributed to acquisition of data and revision of the manuscript. Dr Luo: contributed to acquisition of data and revision of the manuscript. Dr H. Li: contributed to acquisition of data and revision of the manuscript. Dr Y. Li: contributed to analysis of data and revision of the manuscript. Dr Shen: contributed to analysis of data and revision of the manuscript. Dr Sun: contributed to conception and study design, review and revision of the manuscript. Dr Chen: contributed to conception and study design, analysis of data, and review and revision of the manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Other contributions: We thank David Garfield, MD, for his great help in language editing. We also thank Qiong Lu, MD; Shilei Liu, MD; and Shihua Yao, MD, for their technical support. Additional information: The e-Figures and e-Tables can be found in the “Supplemental Materials” area of the online article.
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2. 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. 3. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatinpaclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009; 361(10):947-957. 4. Paez JG, Jänne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676):1497-1500. 5. 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. 6. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448(7153):561-566. 7. Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol. 2011;12(2):175-180. 8. Mitsudomi T, Morita S, Yatabe Y, et al; West Japan Oncology Group. 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. 9. 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. 10. Hammerman PS, Sos ML, Ramos AH, et al. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. Cancer Discov. 2011;1(1): 78-89. 11. Li C, Fang R, Sun Y, et al. Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers. PLoS ONE. 2011;6(11):e28204. 12. Seo JS, Ju YS, Lee WC, et al. The transcriptional landscape and mutational profile of lung adenocarcinoma. Genome Res. 2012;22(11):2109-2119. 13. Sun Y, Ren Y, Fang Z, et al. Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases. J Clin Oncol. 2010;28(30): 4616-4620. 14. Heist RS, Sequist LV, Engelman JA. Genetic changes in squamous cell lung cancer: a review. J Thorac Oncol. 2012;7(5): 924-933. 15. Chou TY, Chiu CH, Li LH, et al. Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer. Clin Cancer Res. 2005;11(10): 3750-3757. 16. Kim KS, Jeong JY, Kim YC, et al. Predictors of the response to gefitinib in refractory non-small cell lung cancer. Clin Cancer Res. 2005;11(6):2244-2251.
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17. Le Calvez F, Mukeria A, Hunt JD, et al. TP53 and KRAS mutation load and types in lung cancers in relation to tobacco smoke: distinct patterns in never, former, and current smokers. Cancer Res. 2005;65(12):5076-5083. 18. Miyamae Y, Shimizu K, Hirato J, et al. Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma. Oncol Rep. 2011;25(4):921-928. 19. Pallis AG, Voutsina A, Kalikaki A, et al. ‘Classical’ but not ‘other’ mutations of EGFR kinase domain are associated with clinical outcome in gefitinib-treated patients with non-small cell lung cancer. Br J Cancer. 2007;97(11):1560-1566. 20. Park SH, Ha SY, Lee JI, et al. Epidermal growth factor receptor mutations and the clinical outcome in male smokers with squamous cell carcinoma of lung. J Korean Med Sci. 2009; 24(3):448-452. 21. Vachtenheim J, Horáková I, Novotná H, Opáalka P, Roubková H. Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung. Clin Cancer Res. 1995;1(3):359-365. 22. Rekhtman N, Paik PK, Arcila ME, et al. Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of lung: lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations. Clin Cancer Res. 2012; 18(4):1167-1176. 23. Gao B, Sun Y, Zhang J, et al. Spectrum of LKB1, EGFR, and KRAS mutations in chinese lung adenocarcinomas. J Thorac Oncol. 2010;5(8):1130-1135. 24. Ohtsuka K, Ohnishi H, Fujiwara M, et al. Abnormalities of epidermal growth factor receptor in lung squamous-cell carcinomas, adenosquamous carcinomas, and large-cell carcinomas: tyrosine kinase domain mutations are not rare in tumors with an adenocarcinoma component. Cancer. 2007;109(4):741-750. 25. World Health Organization. WHO histological classification of tumours of the lung. In: Travis WB, Brambilla A, MüllerHermelinck HK, Harris CC, eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press; 2004:10. 26. Jia XL, Chen G. EGFR and KRAS mutations in Chinese patients with adenosquamous carcinoma of the lung. Lung Cancer. 2011;74(3):396-400. 27. Kang SM, Kang HJ, Shin JH, et al. Identical epidermal growth factor receptor mutations in adenocarcinomatous and squamous cell carcinomatous components of adenosquamous carcinoma of the lung. Cancer. 2007;109(3):581-587. 28. Sasaki H, Endo K, Yukiue H, Kobayashi Y, Yano M, Fujii Y. Mutation of epidermal growth factor receptor gene in adenosquamous carcinoma of the lung. Lung Cancer. 2007;55(1): 129-130. 29. 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-smallcell lung cancer in Asia (IPASS). J Clin Oncol. 2011;29(21): 2866-2874.
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Original Research LUNG CANCER
Comprehensive Analysis of Oncogenic Mutations in Lung Squamous Cell Carcinoma With Minor Glandular Component Yunjian Pan, MD; Rui Wang, MD; Ting Ye, MD; Chenguang Li, MD; Haichuan Hu, MD; Yongfu Yu, MS; Yang Zhang, MD; Lei Wang, MD; Xiaoyang Luo, MD; Hang Li, MD; Yuan Li, MD; Lei Shen, MD; Yihua Sun, MD; and Haiquan Chen, MD, FCCP
Background: The mutations in oncogenic genes, such as EGFR, ALK, BRAF, HER2, DDR2, RET, and AKT1, defined subsets of non-small cell lung cancers (NSCLCs) with potential sensitivity to targeted therapies. At present, the mutational spectrum, prevalence, and clinicopathologic characteristics in squamous cell carcinomas with minor (, 10%) glandular component (SQCC-mGCs) are not well established. Methods: Three hundred ten surgically resected lung squamous cell carcinoma (SQCC) specimens were collected. The histology of all cases was reevaluated using hematoxylin-eosin and immunohistochemistry staining. EGFR, KRAS, HER2, BRAF, PIK3CA, AKT1, and DDR2 mutations, as well as ALK and RET rearrangements, were examined in 310 SQCCs by directed sequencing. Results: Ninety-five SQCC-mGCs (30.6%) and 215 pure SQCCs (69.4%) were identified. Of the 95 SQCC-mGCs, 26 (27.4%; 95% CI, 18.7%-37.4%) were found to harbor known oncogenic mutations, including 10 with EGFR, seven with KRAS, three with PIK3CA, one with BRAF, one with HER2, one each with EGFR/PIK3CA and KRAS/PIK3CA double mutations, and two with EML4-ALK fusions. Ten of 215 pure SQCCs (4.7%; 95% CI, 2.3%-8.4%) harbored mutations, including seven with PIK3CA, and each with AKT1, DDR2, and EGFR. No RET rearrangements were detected in SQCCs. SQCC-mGCs had a significantly higher rate of mutations in known oncogenic genes than that in pure SQCCs (27.4% vs 4.7%, P , .001). All KRAS mutations occurred in SQCC-mGCs. Conclusions: Our results demonstrated that oncogenic mutations in EGFR, KRAS, BRAF, HER2, and ALK were extremely rare or absent in patients with pure SQCC, whereas SQCC-mGC had a relatively high frequency of EGFR, ALK, or KRAS mutations. Prospective identification of these known oncogenic mutations in SQCC-mGC before the initiation of treatment is an essential step to identify which patient could benefit from targeted therapies. CHEST 2014; 145(3):473–479 Abbreviations: cDNA 5 complementary DNA; CK 5 cytokeratin; EGFR-TKI 5 epidermal growth factor receptor tyrosine kinase inhibitor; IHC 5 immunohistochemistry; NSCLC 5 non-small cell lung cancer; OS 5 overall survival; PCR 5 polymerase chain reaction; RFS 5 relapse-free survival; SQCC 5 squamous cell carcinoma; SQCC-mGC 5 squamous cell carcinoma with minor glandular component; TTF-1 5 thyroid transcription factor-1
understanding of the genetic alterations Detailed that drive subsets of lung cancers has led to the
development of targeted agents, which have changed the treatment landscape.1-6 Oncogenic mutations occur in genes that encode signaling kinases crucial for cellular proliferation and survival.7 Cancer cells might depend on the mutant kinase for survival and die when it is inactivated. Therefore, these mutant kinases
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could be exploited for therapeutic target, which is best illustrated by EGFR. Treatment with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) leads to dramatic regression of tumors and improved survival in patients whose tumor harbored EGFR mutations.3,8,9 More recently, patients with ALK rearrangement demonstrated dramatic responses to crizotinib,1 which has been recommended by the US Food and CHEST / 145 / 3 / MARCH 2014
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Drug Administration as first-line therapy for patients with advanced-stage non-small cell lung cancer (NSCLC) with ALK rearrangement. Except EGFR and ALK, the established driver genes in NSCLCs also include KRAS, BRAF, HER2, RET, AKT1, PIK3CA, and DDR2.10 EGFR, KRAS, HER2, and BRAF mutations, as well as ALK and RET rearrangements, were found much more frequently in lung adenocarcinomas,1,7,11-13 whereas mutations in PIK3CA, AKT1, and DDR2 were found mainly in squamous cell carcinomas (SQCCs).7,10 Except for KRAS, all the other known mutant kinases could be targeted by agents being used in the clinic or evaluated in clinical trials.7 More than 80% of lung adenocarcinomas from East Asian populations can be defined by known oncogenic mutations, such as EGFR, KRAS, HER2, BRAF, or ALK.12 However, the mutation profiles in lung SQCCs were still largely unclear.14 The frequency of EGFR and KRAS mutations in lung SQCCs ranged from 1% to 15% and 1% to 9%,15-21 respectively. A study showed that lung SQCCs without any glandular component (pure SQCC) by microscopic examination and immunohistochemistry (IHC) biomarker verification were all negative for EGFR and KRAS mutations, suggesting that the variation of their mutation rates in SQCCs might be caused by the glandular component in SQCCs.22 However, its accurate prevalence and clinicopathologic and mutational characteristics remain unknown. In this study, tumor with , 10% glandular component is classified as SQCC with minor glandular component (SQCC-mGC), and SQCC without any glandular component is identified as pure SQCC. The mutational status of known oncogenic genes, including EGFR, KRAS, HER2, BRAF, PIK3CA, AKT1, DDR2, ALK, and RET, were examined in 310 SQCCs. Manuscript received November 1, 2012; revision accepted September 16, 2013. Affiliations: From the Department of Thoracic Surgery (Drs Pan, R. Wang, Ye, C. Li, Hu, Zhang, L. Wang, Luo, H. Li, Sun, and Chen), and the Department of Pathology (Drs Y. Li and Shen), Fudan University Shanghai Cancer Center; and the Department of Oncology (Drs Pan, R. Wang, Ye, C. Li, Hu, Zhang, L. Wang, Luo, H. Li, Y. Li, Shen, Sun, and Chen), Shanghai Medical College, and the Department of Biostatistics (Mr Yu), School of Public Health, Fudan University, Shanghai, China. Drs Pan, R. Wang, Ye, and C. Li contributed to this work equally and should be considered co-first authors. Drs Chen and Sun contributed to this work equally. Funding/Support: This study was supported by the National Natural Science Foundation of China [Grant 81101761, 81172218] and the Key Construction Program of the National “985” Project Grant [985-YFX0102]. Correspondence to: Haiquan Chen, MD, FCCP, Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, 270 Dong’An Rd, Shanghai 200032, China; e-mail: hqchen1@ yahoo.com © 2014 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.12-2679 474
Materials and Methods Specimen Collection A total of 324 cases reported as SQCC were included, and the frozen samples and corresponding formalin-fixed, paraffinembedded tumor blocks (at least two blocks for each case) were consecutively collected from August 2007 to August 2011 in Fudan University Shanghai Cancer Center. Written informed consent was obtained from each patient. Each specimen contained at least 50% tumor cells. The study was approved by the Committee for Ethical Review of Research (Fudan University Shanghai Cancer Center IRB# 090977-1). Only cases with confirmed SQCC diagnosis by hematoxylin-eosin and IHC staining were selected for mutational analysis. Clinicopathologic data were obtained from electronic medical records. Pathologic Reassessment Tumor blocks were cut into 4-mm-thick sections. All sections were stained with hematoxylin-eosin and IHC (at least two for each case). Morphologic examinations were performed by two pathologists (Y. Li and L. Shen), as shown in Figure 1. Well-differentiated tumors with typical keratinization and/or intercellular bridges were only stained with thyroid transcription factor-1 (TTF-1) and cytokeratin (CK) 7. Moderate and poorly differentiated tumors were stained with a panel of P63, CK5/6, TTF-1, and CK7. Primary antibodies included mouse anti-human P63 monoclonal antibody (4A4 clone; Maixin Technology Co, Ltd) (working concentration 1 mg/mL), mouse anti-human TTF-1 monoclonal antibody (8G7G3/1 clone; Shanghai Long Island Biotec Co, Ltd) (working concentration 1 mg/mL), mouse anti-human CK5/6 monoclonal antibody (D5/16 B4 clone; Dako) (working concentration 10 mg/mL), and mouse anti-human CK7 monoclonal antibody (OV-TL 12/30 clone; Maixin Technology Co, Ltd) (working concentration 2 mg/mL). Antigen retrieval was performed using sodium citrate buffer (10 mM, pH 5 6.0) (Solarbio). Slides were treated with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. Primary antibodies were applied and incubated for 60 min. The DAB Envision Kit was used (Real Envision Detection Kit; Gene Tech). For TTF-1 and CK7 staining, intensity (0, 11, 21, 31) and percentage of immunoreactive tumor cells were recorded as follows: 31, strong staining intensity in , 10% tumor cells; 21, moderate staining intensity in , 10% tumor cells; 11, faint or weak staining intensity in , 10% tumor cells; and 0, no staining. Tumors with 31 and 21 intensity in , 10% tumor cells were defined as TTF-1 or CK7-positive. Tumors with . 10% TTF-1 and/or CK7-positive tumor cells were excluded in the following study. Pure SQCC was defined by p63 and/or CK5/6-diffuse with TTF-1/CK7 double-negative staining. Reverse Transcription Polymerase Chain Reaction and Mutation Analysis Frozen tumor specimens were dissected, and RNA/DNA was coextracted following the standard instructions of the RNA/DNA isolation kit (Tiangen Biotech Co, Ltd). Single-stranded RNA of each sample is reverse transcribed into complementary DNA (cDNA) by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc). EGFR (exons 18-21), HER2 (exons 18-21), KRAS (exons 2-3), BRAF (exons 11-15), AKT1 (exons 2-3), PIK3CA (exon 9 and exon 20), and DDR2 (whole coding exons) were amplified with KOD Plus Neo DNA polymerase (Toyobo Co, Ltd). For detection of EML4-ALK, KIF5B-RET, and CCDC6-RET fusions, primers were designed to cover all known fusion variants. Polymerase chain reaction (PCR) was performed in a 25-mL reaction tube on Mastercycler pro PCR apparatus (Eppendorf AG). RNase-free water was used as a PCR-negative control. Thermocycler Original Research
Figure 1. Microscopic features of squamous cell carcinoma (SQCC) with minor glandular component with hematoxylin-eosin staining under pathologists’ review. The top shows an image of SQCC with acinar component (magnification, 3200). Two components (marked with red dotted boxes) are shown in amplified images on the bottom (magnification, 3400). settings were as following: 94°C for 2 min; 98°C for 10 s, 61°C for 30 s, 68°C for 30 s/kb, 40 cycles; and 68°C for 5 min. PCR products were qualified using agarose gel electrophoresis and directly sequenced. Sequences were analyzed using Chromas Lite, version 2.4 (Technelysium Pty Ltd) and Vector NTI 11.0 (Life Technologies Corp) to detect mutations/rearrangements by comparing with the standard gene sequences. For cDNA-screened mutations, subsequent DNA confirmations were conducted to exclude pseudomutations. Reverse transcription PCR primers used for cDNA screening are listed in e-Table 1.
Clinicopathologic characteristics of 310 patients are shown in Table 1. Of 95 SQCC-mGCs, 61 cases (64.2%) were TTF-1 and/or CK7-positive and 34 cases (35.8%) were TTF-1 and CK7 double negative, which were diagnosed solely on the glandular component by microscopic examination (e-Fig 1). All pure SQCCs showed TTF-1 and CK7 double-negative staining. Positive and negative control subjects of P63, CK5/6, TTF-1, and CK7 are shown in e-Figure 2.
Statistical Analysis The association between mutations and clinicopathologic parameters were analyzed by x2 (when no cell of a contingency table has expected count , 5) or Fisher exact tests (when any cell of a contingency table has expected count , 5). For multivariate analyses, logistic regression model was used. The Kaplan-Meier method was used to estimate relapse-free survival (RFS) and overall survival (OS), and the differences were compared using the log-rank test. All data were analyzed using the Statistical Package for the Social Sciences, version 16.0 software (IBM) or Prism 5.0 (GraphPad Software, Inc). The two-sided significance level was set at P , .05.
Results Patient Enrollment A total of 310 cases met eligibility for this study, including 95 SQCC-mGCs and 215 pure SQCCs. journal.publications.chestnet.org
Mutational Spectrum of SQCCs In total, 36 patients out of 310 with SQCCs were found to harbor mutations (11.6%; 95% CI, 8.3%15.7%). Twenty-six patients with SQCC-mGC harbored 28 known oncogenic mutations (27.4%; 95% CI, 18.7%-37.4%), including 10 EGFR, seven KRAS, three PIK3CA, one EGFR/PIK3CA, one KRAS/PIK3CA, one BRAF, one HER2 mutation, and two EML4-ALK fusions. Mutations predominantly occurred in TTF-1 and/or CK7-positive samples (22 of 61, 36.1%; 95% CI, 24.2%-49.4%), including eight EGFR, six KRAS, one BRAF, one HER2, one EGFR/PIK3CA, one KRAS/ PIK3CA, two PIK3CA mutations, and two EML4-ALK fusions. Of the 34 TTF-1 and CK7 double-negative CHEST / 145 / 3 / MARCH 2014
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Table 1—Detailed Clinicopathologic Characteristics of 310 Lung Squamous Cell Carcinomas Variable Total patients Age, y , 60 ⱖ 60 Sex Male Female Smoking Never smoker Smoker Location Central type Peripheral type Differentiation Moderate to well Poor Tumor size T1 T2-3 LN metastasis N0 N1-3 TNM stage Stage I Stage II-III
No. (%) 310 135 (43.5) 175 (56.5) 283 (91.3) 27 (8.7) 60 (19.4) 250 (80.6) 220 (71.0) 90 (29.0) 154 (49.7) 156 (50.3) 87 (28.1) 223 (71.9) 169 (54.5) 141 (45.5) 131 (42.3) 179 (57.7)
Never smokers are patients who smoked , 100 cigarettes in their lifetime. LN 5 lymph node.
Figure 2. Mutational profiles of pure SQCC, SQCC, and SQCCmGC. SQCC-mGC 5 SQCC with minor glandular component. See Figure 1 legend for expansion of other abbreviation.
iate logistic regression analysis, which confirmed that minor glandular component was the only independent and significant factor to predict tumors harboring EGFR mutations (Table 3). KRAS mutations were found exclusively in tumors that harbored a minor glandular component. We also found that both EGFR and KRAS mutations were significantly higher in SQCC-mGCs than in pure SQCCs (e-Table 4). No correlation was found between PIK3CA mutation status and clinicopathologic characteristics in patients with SQCCs. Clinical Outcome
SQCC-mGCs, four were found to harbor mutations (four of 34, 11.8%; 95% CI, 3.3%-27.5%), including two EGFR, one KRAS, and one PIK3CA mutation. Ten patients with pure SQCC (10 of 215, 4.7%; 95% CI, 2.3%-8.4%) were found to harbor known oncogenic mutations, including seven PIK3CA, one AKT1, one DDR2, and one EGFR mutation. No RET rearrangements are detected in either group. SQCC-mGCs had a significantly higher rate of mutations in known oncogenic genes than that in pure SQCCs (27.4% vs 4.7%, P , .001) (Fig 2). Clinicopathologic characteristics of patients with oncogenic mutations listed in e-Table 2. Primary data of 310 SQCCs were shown in e-Table 3. Correlation Between Clinicopathologic Characteristics and Major Oncogenic Mutations (EGFR, KRAS, PIK3CA) There were 12 EGFR mutations, eight KRAS mutations, and 12 PIK3CA mutations in this cohort. As shown in Table 2, there was a significantly higher rate of EGFR mutations in women or never smokers, which is similar to that in lung adenocarcinomas.5,11-13,15,23 Univariate analysis showed that EGFR mutations were found more frequently in tumor with minor glandular component or located in peripheral lung. The joint effect of these factors was examined using multivar476
The survival data were available in 239 patients with SQCC. The 2-year overall survival for patients with EGFR, KRAS, and PIK3CA mutations was 82.5% (95% CI, 46.1%-95.3%), 50.0% (95% CI, 11.1%-80.4%), and 66.7% (95% CI, 19.5%-90.4%), respectively. The number of AKT1, HER2, RET, and DDR2 mutations was too small for further analysis. Patients with late-stage disease (stage II-III) had a worse RFS and OS than patients with early-stage SQCC (stage I). There were no significant differences in RFS and OS between patients with EGFR mutation and wild type. The results of the univariate survival analysis are shown in e-Table 5. There were no statistically significant differences in RFS or OS for patients with SQCC with EGFR mutations compared with KRAS, PIK3CA, or wild type (e-Fig 3). Treatment Effect With EGFR-TKIs Five patients with SQCC received EGFR-TKI treatment when the disease relapsed. Three were treated with erlotinib (Tarceva) and two with gefitinib (Iressa). Detailed characteristics and survival data of the five patients are listed in Table 4. Only the patient with EGFR-activating mutation (exon 19 microdeletion) showed partial response in tumor when treated with gefitinib. Patients with wild-type EGFR experienced either stable disease or progression of disease. Original Research
Table 2—The Association Between Clinicopathologic Characteristics and Mutational Status of EGFR, KRAS, and PIK3CA in 310 Lung Squamous Cell Carcinomas EGFR Variable
KRAS
Mut
Wild
P Value
12
298
… .292
7 5
128 170
7 5
276 22
7 5
53 245
5 7
215 83
3 9
151 147
6 6
163 135
4 8
127 171
11 1
84 214
3 5
86 38
Total patients Age, y , 60 ⱖ 60 Sex Male Female Smoking Never smoker Smoker Location Central type Peripheral type Differentiation Moderate to well Poor LN metastasis N0 N1-3 TNM stage Stage I Stage II-III Glandular component With Without Recurrence Local Metastasis
PIK3CA
Mut
Wild
P Value
8
302
… .709
4 4
131 171
8 0
275 27
1 7
59 243
6 2
214 88
4 4
150 152
5 3
164 138
4 4
127 175
8 0
87 215
1 2
88 41
.002
Mut
Wild
P Value
12
298
… .893
5 7
130 168
11 1
272 26
3 9
57 241
6 6
214 84
9 3
145 153
7 5
162 136
5 7
126 172
5 7
90 208
2 2
87 41
1.000
.003
1.000
1.00
.045
.708
1.00
.081
.114
1.00
.749
.074
.732
.523
.787
.726
, .001
.966
, .001
.112
.523
.247
.596
Never smokers are patients who smoked , 100 cigarettes in their lifetime. Mut 5 mutant type; Wild 5 wild type. See Table 1 legend for expansion of other abbreviation.
Discussion Oncogenic mutations in EGFR, KRAS, BRAF, HER2, ALK, and RET have been well studied in lung adenocarcinomas.7,11,13,23 It still remains controversial whether SQCCs harbored these mutations.18,22,24 In this study, we performed comprehensive and concurrent analyses of major known oncogenic mutations in a large cohort of patients with SQCC, aiming to illustrate the spectrum of mutations in SQCC-mGCs and pure SQCCs. According to the World Health Organization’s classification, adenosquamous lung carcinomas were defined as tumors showing both glandular and squamous components, with each component composing at least
10% of the tumor.25 Although 10% is an arbitrary cutoff that is not based on any biologic and genetic evidences, pathologists tend to set it as the common criterion to separate adenosquamous lung carcinomas from SQCC-mGC.26-28 SQCC-mGCs also have two distinct cell components. However, their mutation profile and clinicopathologic characteristics were completely unknown. We found that 27.4% of SQCC-mGCs harbored known oncogenic mutations in EGFR, KRAS, BRAF, HER2, PIK3CA, and ALK. To our knowledge, the current study represents the first comprehensive mutational analysis of these known driver genes in a large cohort of patients with SQCC-mGCs. The diagnosis of SQCC-mGCs can be hardly made in small samples, such as biopsy or cytologic specimens,
Table 3—Multivariate Analysis of Clinicopathologic Parameters That Might Affect the Presence of EGFR Mutations Variable Sex Smoking Location Glandular component
Category
OR
95% CI
P Value
Male/female Smoker/never smoker Central/peripheral Present/absent
0.33 0.29 0.30 27.92
0.06-1.86 0.06-1.47 0.08-1.12 3.41-228.21
.208 .135 .073 .002
Never smokers are patients who smoked , 100 cigarettes in their lifetime. journal.publications.chestnet.org
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Table 4—Clinicopathologic Characteristics and Survival Data of Patients with EGFR-TKI Treatment ID
Pathology
Age, y/Sex
Smoking
Mutation
TKI Treatment
Responsea
RFS
OS
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Pure SCC Pure SCC SCC-mGC Pure SCC Pure SCC
55/M 50/M 49/F 43/M 68/F
Smoker Smoker Never Never Never
Wild type Wild type EGFR LREATSPK deletion Wild type Wild type
Erlotinib Erlotinib Gefitinib Erlotinib Gefitinib
PD PD PR SD PD
14.5 4.5 17.5 12.7 6.2
37.3 15 27.4 24.01 8.31
Never smokers are patients who smoked , 100 cigarettes in their lifetime. “1” indicates censored data. EGFR-TKI 5 epidermal growth factor receptor-tyrosine kinase inhibitor; F 5 female; M 5 male; OS 5 overall survival; PD 5 progressive disease; PR 5 partial response; RFS 5 relapsefree survival; SQCC 5 squamous cell carcinoma; SQCC-mGC 5 squamous cell carcinoma with minor glandular component; SD 5 stable disease; TKI 5 tyrosine kinase inhibitor. aResponse Evaluation Criteria In Solid Tumors (RECIST), version 1.1.
due to the insufficient tissue for further evaluation. Thus, there is a high probability that SQCC-mGC would be misdiagnosed as pure SQCC. In current study, the whole tumor from each patient was collected by surgical resection, and SQCC-mGCs were diagnosed by a combined strategy of morphologic examination and IHC biomarker staining. We also collected at least two tumor blocks for each case to minimize the possibility of incomplete sampling, especially in large tumors. Our results showed that SQCC-mGC was a common disease, accounting for nearly one-third of SQCCs. We found that 19 of 95 patients (20%) with SQCCmGCs harbored EGFR, BRAF, HER2, ALK, or PIK3CA mutations/fusions, which were druggable targets for anticancer therapy. Erlotinib/gefitinib specifically targets the EGFR tyrosine kinase and has showed a significant survival benefit for patients with NSCLC with activating EGFR mutations.9,29 Advanced NSCLCs with ALK fusion are highly sensitive to the ALK inhibitor crizotinib, which is, thus, recommended by the US Food and Drug Administration as first-line therapy for patients whose tumor contains this genetic abnormality. Thus, prospective identification of these druggable mutant kinases in SQCC-mGCs could lead to rationally chosen specific targeted therapy. In our study, the major known oncogenic mutations are investigated in pure SQCCs, which were verified by comprehensive pathologic assessment. We demonstrated that pure SQCCs were lacking EGFR and KRAS mutations, which is consistent with previous studies.22 We also found that one patient with pure SQCC (one of 215) had EGFR mutation. Although all samples in the current study were collected by surgical resection and verified by histologic assessment, we still could not exclude the possibility that SQCC with glandular component might be misdiagnosed as pure SQCC as a result of incomplete sampling of the tumor. In conclusion, our analyses demonstrated that oncogenic mutations in EGFR, KRAS, BRAF, HER2, and ALK were extremely rare or absent in patients with pure SQCC, whereas SQCC-mGC had relatively high frequency of EGFR, ALK, or KRAS mutations. Thus, 478
prospective identification of these oncogenic mutations in SQCC-mGC before the initiation of treatment is an essential step to identify which patient might benefit from targeted therapies. Acknowledgments Author contributions: Dr Chen is the guarantor of the manuscript. Dr Pan: contributed to conception and study design, acquisition and analysis of data, and writing and revision of the manuscript. Dr R. Wang: contributed to conception and study design, acquisition and analysis of data, and writing and revision of the manuscript. Dr Ye: contributed to conception and study design, acquisition and analysis of data, and writing and revision of the manuscript. Dr C. Li: contributed to acquisition of data and writing and revision of the manuscript. Dr Hu: contributed to acquisition of data and writing and revision of the manuscript. Mr Yu: contributed to analysis of data and revision of the manuscript. Dr Zhang: contributed to acquisition of data and revision of the manuscript. Dr L. Wang: contributed to acquisition of data and revision of the manuscript. Dr Luo: contributed to acquisition of data and revision of the manuscript. Dr H. Li: contributed to acquisition of data and revision of the manuscript. Dr Y. Li: contributed to analysis of data and revision of the manuscript. Dr Shen: contributed to analysis of data and revision of the manuscript. Dr Sun: contributed to conception and study design, review and revision of the manuscript. Dr Chen: contributed to conception and study design, analysis of data, and review and revision of the manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Other contributions: We thank David Garfield, MD, for his great help in language editing. We also thank Qiong Lu, MD; Shilei Liu, MD; and Shihua Yao, MD, for their technical support. Additional information: The e-Figures and e-Tables can be found in the “Supplemental Materials” area of the online article.
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