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Original Research: Lung Cancer |

Comprehensive Analysis of Oncogenic Mutations in Lung Squamous Cell Carcinoma With Minor Glandular ComponentMutations in Lung Squamous Cell Carcinomas FREE TO VIEW

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; Haiquan Chen, MD, FCCP
Author and Funding Information

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.

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


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].

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2014;145(3):473-479. doi:10.1378/chest.12-2679
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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.

Figures in this Article

Detailed understanding of the genetic alterations that drive subsets of lung cancers has led to the development of targeted agents, which have changed the treatment landscape.16 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 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 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 DDR2.10EGFR, KRAS, HER2, and BRAF mutations, as well as ALK and RET rearrangements, were found much more frequently in lung adenocarcinomas,1,7,1113 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%,1521 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.

Specimen Collection

A total of 324 cases reported as SQCC were included, and the frozen samples and corresponding formalin-fixed, paraffin-embedded 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-μm-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.

Figure Jump LinkFigure 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, ×200). Two components (marked with red dotted boxes) are shown in amplified images on the bottom (magnification, ×400).Grahic Jump Location

Primary antibodies included mouse anti-human P63 monoclonal antibody (4A4 clone; Maixin Technology Co, Ltd) (working concentration 1 μg/mL), mouse anti-human TTF-1 monoclonal antibody (8G7G3/1 clone; Shanghai Long Island Biotec Co, Ltd) (working concentration 1 μg/mL), mouse anti-human CK5/6 monoclonal antibody (D5/16 B4 clone; Dako) (working concentration 10 μg/mL), and mouse anti-human CK7 monoclonal antibody (OV-TL 12/30 clone; Maixin Technology Co, Ltd) (working concentration 2 μg/mL). Antigen retrieval was performed using sodium citrate buffer (10 mM, pH = 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, 1+, 2+, 3+) and percentage of immunoreactive tumor cells were recorded as follows: 3+, strong staining intensity in < 10% tumor cells; 2+, moderate staining intensity in < 10% tumor cells; 1+, faint or weak staining intensity in < 10% tumor cells; and 0, no staining. Tumors with 3+ and 2+ 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-μL reaction tube on Mastercycler pro PCR apparatus (Eppendorf AG). RNase-free water was used as a PCR-negative control. Thermocycler 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.

Statistical Analysis

The association between mutations and clinicopathologic parameters were analyzed by χ2 (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.

Patient Enrollment

A total of 310 cases met eligibility for this study, including 95 SQCC-mGCs and 215 pure SQCCs. 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.

Table Graphic Jump Location
Table 1 —Detailed Clinicopathologic Characteristics of 310 Lung Squamous Cell Carcinomas

Never smokers are patients who smoked < 100 cigarettes in their lifetime. LN = lymph node.

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 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.

Figure Jump LinkFigure 2. Mutational profiles of pure SQCC, SQCC, and SQCC-mGC. SQCC-mGC = SQCC with minor glandular component. See Figure 1 legend for expansion of other abbreviation.Grahic Jump Location
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.5,1113,15,23 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 multivariate 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.

Table Graphic Jump Location
Table 2 —The Association Between Clinicopathologic Characteristics and Mutational Status of EGFR, KRAS, and PIK3CA in 310 Lung Squamous Cell Carcinomas

Never smokers are patients who smoked < 100 cigarettes in their lifetime. Mut = mutant type; Wild = wild type. See Table 1 legend for expansion of other abbreviation.

Table Graphic Jump Location
Table 3 —Multivariate Analysis of Clinicopathologic Parameters That Might Affect the Presence of EGFR Mutations

Never smokers are patients who smoked < 100 cigarettes in their lifetime.

Clinical Outcome

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.

Table Graphic Jump Location
Table 4 —Clinicopathologic Characteristics and Survival Data of Patients with EGFR-TKI Treatment

Never smokers are patients who smoked < 100 cigarettes in their lifetime. “+” indicates censored data. EGFR-TKI = epidermal growth factor receptor-tyrosine kinase inhibitor; F = female; M = male; OS = overall survival; PD = progressive disease; PR = partial response; RFS = relapse-free survival; SQCC = squamous cell carcinoma; SQCC-mGC = squamous cell carcinoma with minor glandular component; SD = stable disease; TKI = tyrosine kinase inhibitor.

a 

Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1.

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.2628 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, 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 SQCC-mGCs 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, 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.

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.

cDNA

complementary DNA

CK

cytokeratin

EGFR-TKI

epidermal growth factor receptor tyrosine kinase inhibitor

IHC

immunohistochemistry

NSCLC

non-small cell lung cancer

OS

overall survival

PCR

polymerase chain reaction

RFS

relapse-free survival

SQCC

squamous cell carcinoma

SQCC-mGC

squamous cell carcinoma with minor glandular component

TTF-1

thyroid transcription factor-1

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Figures

Figure Jump LinkFigure 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, ×200). Two components (marked with red dotted boxes) are shown in amplified images on the bottom (magnification, ×400).Grahic Jump Location
Figure Jump LinkFigure 2. Mutational profiles of pure SQCC, SQCC, and SQCC-mGC. SQCC-mGC = SQCC with minor glandular component. See Figure 1 legend for expansion of other abbreviation.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Detailed Clinicopathologic Characteristics of 310 Lung Squamous Cell Carcinomas

Never smokers are patients who smoked < 100 cigarettes in their lifetime. LN = lymph node.

Table Graphic Jump Location
Table 2 —The Association Between Clinicopathologic Characteristics and Mutational Status of EGFR, KRAS, and PIK3CA in 310 Lung Squamous Cell Carcinomas

Never smokers are patients who smoked < 100 cigarettes in their lifetime. Mut = mutant type; Wild = wild type. See Table 1 legend for expansion of other abbreviation.

Table Graphic Jump Location
Table 3 —Multivariate Analysis of Clinicopathologic Parameters That Might Affect the Presence of EGFR Mutations

Never smokers are patients who smoked < 100 cigarettes in their lifetime.

Table Graphic Jump Location
Table 4 —Clinicopathologic Characteristics and Survival Data of Patients with EGFR-TKI Treatment

Never smokers are patients who smoked < 100 cigarettes in their lifetime. “+” indicates censored data. EGFR-TKI = epidermal growth factor receptor-tyrosine kinase inhibitor; F = female; M = male; OS = overall survival; PD = progressive disease; PR = partial response; RFS = relapse-free survival; SQCC = squamous cell carcinoma; SQCC-mGC = squamous cell carcinoma with minor glandular component; SD = stable disease; TKI = tyrosine kinase inhibitor.

a 

Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1.

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