Journal of Surgical Oncology 2014;110:245–251
Simultaneous Diagnostic Platform of Genotyping EGFR, KRAS, and ALK in 510 Korean Patients With Non-Small-Cell Lung Cancer Highlights Significantly Higher ALK Rearrangement Rate in Advanced Stage TAE-JUNG KIM, MD, PhD,1 CHAN KWON PARK, MD,2 CHANG DONG YEO, MD,2 KIHOON PARK, MD,2 CHIN KOOK RHEE, MD, PhD,2 JUSANG KIM, MD,2 SEUNG JOON KIM, MD, PhD,2 SANG HAAK LEE, MD, PhD,2 KYO-YOUNG LEE, MD, PhD,1 AND HYOUNG-KYU YOON, MD, PhD2* 1
Department of Hospital Pathology, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea Department of Internal Medicine, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea
2
Background: Simultaneous genotyping has advantages in turnaround time and detecting the real mutational prevalence in unresectable non‐small‐ cell lung cancer (NSCLC), a group not previously genetically characterized. Methods: We developed simultaneous panel of screening EGFR and KRAS mutations by direct sequencing or PNA clamping, and ALK rearrangement by fluorescent in situ hybridization (FISH) in multicenter manner. Results: Of 510 NSCLC Korean patients, simultaneous genotyping identified mutations of EGFR (29.0%) and KRAS (8.6%) and rearrangement of ALK (9.2%). Seven patients had overlaps in mutations. Although several well‐known associations between genotypes and clinical characteristics were identified, we found no relationship between ALK rearrangement and sex or smoking history. Unlike the other genotype mutations, ALK rearrangement was associated with advanced disease. Among the ALK‐negative group, patients with 10–15% of ALK FISH split shared characteristics, such as younger age and advanced stage disease, more with the ALK‐positive group (>15% ALK FISH split) than 15%, the specimen is interpreted as positive; if the rate 10%, the specimen is interpreted as negative; and the rate 10–15%, the specimen is interpreted as equivocal (Fig. 1).
Direct Sequencing
ALK‐Immunohistochemistry
Genotyping EGFR and KRAS in 429 patients were analyzed by direct sequencing. Nucleotide sequencing of the kinase domain of EGFR
IHC was assayed in each hospital and the results were independently scored, regardless of ALK FISH results. Briefly, 4‐mm formalin‐fixed,
DNA Extraction
Journal of Surgical Oncology
Simultaneous Genotyping and ALK
247
Fig. 1. Specimen‐specific scoring algorithm of ALK fluorescent in situ hybridization.
paraffin‐embedded tumor tissues were submitted. Unstained paraffin‐ embedded sections were depleted of paraffin with xylene, rehydrated through a graded series of ethanol solutions, and subjected to immunohistochemical staining with a mouse monoclonal antibody (ALK1, 1:20, Dako, Carpinteria, CA). Heat‐induced antigen retrieval pretreatment was performed with Target Retrieval Solution pH 9.0 (Dako). Immune complexes were detected with the EnVision DAB system (Dako). Semi‐quantitative assessment was performed by estimating the staining intensity and percentage of tumor cells with positive cytoplasmic staining. Samples were then classified into four categories with negative (0), faint and/or doubtful heterogenous staining (1þ), moderate (2þ), and intense homogenous staining (3þ).
Statistical Analysis Statistics are provided for the clinical characteristics of the 510 patients tested. Demographic and disease characteristics were compared between patients with the mutant and WT gene using chi‐squared test and t‐test.
RESULTS TAT of simultaneous panel genotyping was determined by process improvement and genotyping method. Particularly, PNA clamping method enables us to gain dramatic improvement of 2 days TAT for detecting EGFR and KRAS mutation in comparison with 2 weeks TAT for direct sequencing. This cohort comprised 278 men and 232 women, and the mean patient age for the entire cohort was 66 years (range, 22–97 years). If total, 207 patients (40.6%) were smokers, and 303 (59.4%) never smoked. Histologically, the 510 NSCLCs comprised 456 adenocarcinomas, 24 squamous cell carcinomas, 16 adenosquamous carcinomas, 8 non‐small‐ cell carcinomas, not otherwise specified (NOS), 4 large‐cell carcinomas, and 2 sarcomatoid carcinomas. A high percentage of the study population (60.9%) had unresectable disease (Stages IIIB and IV). Most study specimens (75.3%) were obtained by non‐surgical biopsy (bronchoscopic biopsy, 16.1%; percutaneous needle aspiration biopsy, 46.5%; metastatic site biopsy, 9.6%; cell block from pleural effusion, 3.1%); the remainder were surgical resections (lobectomy, 21.2%; wedge resection, 3.5%). Journal of Surgical Oncology
Of the 510 patients studied, we identified 148 cases (29.0%) of EGFR mutation, 44 cases (8.6%) of KRAS mutation, and 47 cases (9.2%) of ALK rearrangement. Among the 24 cases of squamous cell carcinomas, none had the EGFR mutation, but 1 case of KRAS mutation and 1 case of ALK rearrangement were detected. When compared to WT of each gene, ALK rearranged patient was significantly associated with lower mean age (P ¼ 0.034) and younger age (60 years vs. >60 years) (P ¼ 0.019). On the other hand, EGFR and KRAS mutations showed no significant association with age. The prevalence of EGFR and KRAS mutations showed strong sex predilection compared with the WT of each gene: women were more likely to have an EGFR mutation (P < 0.001) and men a KRAS mutation (P ¼ 0.026). However, ALK rearrangement did not show a significant sex predilection (P ¼ 0.30). Mutations in EGFR and KRAS showed a strong predilection with smoking history: “never smokers” with EGFR; 38.9% (118/303) (P < 0.001), “ever smokers”; with KRAS; 12.1% (25/207) (P ¼ 0.022), but no significant relationship with ALK. Adenocarcinoma histology showed a strong association with EGFR mutation; 31.6% (144/456) (P < 0.001), but not with KRAS mutation or ALK rearrangement (P ¼ 0.40 and P ¼ 0.33, respectively). ALK rearrangement showed a strong association with advanced stage at initial diagnosis; 11.3% (42/369) of Stages III and IV patients, on the other hand, 4.3% (6/141) of Stages I and II patients (P ¼ 0.006), but EGFR and KRAS mutations were not associated with stage (P ¼ 0.08 and P ¼ 0.32, respectively). EGFR mutation and ALK rearrangement showed strong predilection with sample type: surgical resection with EGFR mutation (P < 0.001) and biopsy with ALK rearrangement (P ¼ 0.007) (Table I). Characteristics of overlaps in genotypes were demonstrated in Table II. Coexisting genetic alterations were detected in seven (1.4%) patients; five were EGFR mutations with ALK rearrangement, one was KRAS mutation with ALK rearrangement, and one was EGFR and KRAS. The overlaps comprised four men and three women. Six overlaps were adenocarcinoma histology and one was adenosquamous carcinoma histology. Six out of seven overlaps were never smokers and Stage IV. Among patients with ALK‐negative NSCLC, those with 10–15% of the tumor cells positive for ALK rearrangement shared more clinical characteristics with ALK rearrangement‐positive patients (>15% ALK FISH split) than with the ALK FISH negative (60 Sex Men Women Smoking history Never smoker Ever smoker Histology ADC SCC ADSQ Other NSCLC Clinical stage IA IB IIA IIB IIIA IIIB IV Type of sample Biopsy Bronchoscopic PCNAb Metastatic site Cell block Surgical resection Wedge resection Lobectomy
KRAS (N ¼ 44a)
ALK (N ¼ 47a)
Total, N (%)
N (%)
Pb
N (%)
Pb
N (%)
Pb
66 171 (33.5) 339 (66.5)
65.2 49 (33.6) 97 (66.4)
0.37c 0.99
64.2 15 (34.1) 29 (65.9)
0.67c 0.93
62.3 23 (48.9) 24 (51.1)
0.034c 0.019
278 (54.5) 232 (45.5)
47 (31.8) 101 (68.2)
Simultaneous Diagnostic Platform of Genotyping EGFR, KRAS, and ALK in 510 Korean Patients With Non-Small-Cell Lung Cancer Highlights Significantly Higher ALK Rearrangement Rate in Advanced Stage TAE-JUNG KIM, MD, PhD,1 CHAN KWON PARK, MD,2 CHANG DONG YEO, MD,2 KIHOON PARK, MD,2 CHIN KOOK RHEE, MD, PhD,2 JUSANG KIM, MD,2 SEUNG JOON KIM, MD, PhD,2 SANG HAAK LEE, MD, PhD,2 KYO-YOUNG LEE, MD, PhD,1 AND HYOUNG-KYU YOON, MD, PhD2* 1
Department of Hospital Pathology, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea Department of Internal Medicine, College of Medicine, Catholic University of Korea, Seoul, Republic of Korea
2
Background: Simultaneous genotyping has advantages in turnaround time and detecting the real mutational prevalence in unresectable non‐small‐ cell lung cancer (NSCLC), a group not previously genetically characterized. Methods: We developed simultaneous panel of screening EGFR and KRAS mutations by direct sequencing or PNA clamping, and ALK rearrangement by fluorescent in situ hybridization (FISH) in multicenter manner. Results: Of 510 NSCLC Korean patients, simultaneous genotyping identified mutations of EGFR (29.0%) and KRAS (8.6%) and rearrangement of ALK (9.2%). Seven patients had overlaps in mutations. Although several well‐known associations between genotypes and clinical characteristics were identified, we found no relationship between ALK rearrangement and sex or smoking history. Unlike the other genotype mutations, ALK rearrangement was associated with advanced disease. Among the ALK‐negative group, patients with 10–15% of ALK FISH split shared characteristics, such as younger age and advanced stage disease, more with the ALK‐positive group (>15% ALK FISH split) than 15%, the specimen is interpreted as positive; if the rate 10%, the specimen is interpreted as negative; and the rate 10–15%, the specimen is interpreted as equivocal (Fig. 1).
Direct Sequencing
ALK‐Immunohistochemistry
Genotyping EGFR and KRAS in 429 patients were analyzed by direct sequencing. Nucleotide sequencing of the kinase domain of EGFR
IHC was assayed in each hospital and the results were independently scored, regardless of ALK FISH results. Briefly, 4‐mm formalin‐fixed,
DNA Extraction
Journal of Surgical Oncology
Simultaneous Genotyping and ALK
247
Fig. 1. Specimen‐specific scoring algorithm of ALK fluorescent in situ hybridization.
paraffin‐embedded tumor tissues were submitted. Unstained paraffin‐ embedded sections were depleted of paraffin with xylene, rehydrated through a graded series of ethanol solutions, and subjected to immunohistochemical staining with a mouse monoclonal antibody (ALK1, 1:20, Dako, Carpinteria, CA). Heat‐induced antigen retrieval pretreatment was performed with Target Retrieval Solution pH 9.0 (Dako). Immune complexes were detected with the EnVision DAB system (Dako). Semi‐quantitative assessment was performed by estimating the staining intensity and percentage of tumor cells with positive cytoplasmic staining. Samples were then classified into four categories with negative (0), faint and/or doubtful heterogenous staining (1þ), moderate (2þ), and intense homogenous staining (3þ).
Statistical Analysis Statistics are provided for the clinical characteristics of the 510 patients tested. Demographic and disease characteristics were compared between patients with the mutant and WT gene using chi‐squared test and t‐test.
RESULTS TAT of simultaneous panel genotyping was determined by process improvement and genotyping method. Particularly, PNA clamping method enables us to gain dramatic improvement of 2 days TAT for detecting EGFR and KRAS mutation in comparison with 2 weeks TAT for direct sequencing. This cohort comprised 278 men and 232 women, and the mean patient age for the entire cohort was 66 years (range, 22–97 years). If total, 207 patients (40.6%) were smokers, and 303 (59.4%) never smoked. Histologically, the 510 NSCLCs comprised 456 adenocarcinomas, 24 squamous cell carcinomas, 16 adenosquamous carcinomas, 8 non‐small‐ cell carcinomas, not otherwise specified (NOS), 4 large‐cell carcinomas, and 2 sarcomatoid carcinomas. A high percentage of the study population (60.9%) had unresectable disease (Stages IIIB and IV). Most study specimens (75.3%) were obtained by non‐surgical biopsy (bronchoscopic biopsy, 16.1%; percutaneous needle aspiration biopsy, 46.5%; metastatic site biopsy, 9.6%; cell block from pleural effusion, 3.1%); the remainder were surgical resections (lobectomy, 21.2%; wedge resection, 3.5%). Journal of Surgical Oncology
Of the 510 patients studied, we identified 148 cases (29.0%) of EGFR mutation, 44 cases (8.6%) of KRAS mutation, and 47 cases (9.2%) of ALK rearrangement. Among the 24 cases of squamous cell carcinomas, none had the EGFR mutation, but 1 case of KRAS mutation and 1 case of ALK rearrangement were detected. When compared to WT of each gene, ALK rearranged patient was significantly associated with lower mean age (P ¼ 0.034) and younger age (60 years vs. >60 years) (P ¼ 0.019). On the other hand, EGFR and KRAS mutations showed no significant association with age. The prevalence of EGFR and KRAS mutations showed strong sex predilection compared with the WT of each gene: women were more likely to have an EGFR mutation (P < 0.001) and men a KRAS mutation (P ¼ 0.026). However, ALK rearrangement did not show a significant sex predilection (P ¼ 0.30). Mutations in EGFR and KRAS showed a strong predilection with smoking history: “never smokers” with EGFR; 38.9% (118/303) (P < 0.001), “ever smokers”; with KRAS; 12.1% (25/207) (P ¼ 0.022), but no significant relationship with ALK. Adenocarcinoma histology showed a strong association with EGFR mutation; 31.6% (144/456) (P < 0.001), but not with KRAS mutation or ALK rearrangement (P ¼ 0.40 and P ¼ 0.33, respectively). ALK rearrangement showed a strong association with advanced stage at initial diagnosis; 11.3% (42/369) of Stages III and IV patients, on the other hand, 4.3% (6/141) of Stages I and II patients (P ¼ 0.006), but EGFR and KRAS mutations were not associated with stage (P ¼ 0.08 and P ¼ 0.32, respectively). EGFR mutation and ALK rearrangement showed strong predilection with sample type: surgical resection with EGFR mutation (P < 0.001) and biopsy with ALK rearrangement (P ¼ 0.007) (Table I). Characteristics of overlaps in genotypes were demonstrated in Table II. Coexisting genetic alterations were detected in seven (1.4%) patients; five were EGFR mutations with ALK rearrangement, one was KRAS mutation with ALK rearrangement, and one was EGFR and KRAS. The overlaps comprised four men and three women. Six overlaps were adenocarcinoma histology and one was adenosquamous carcinoma histology. Six out of seven overlaps were never smokers and Stage IV. Among patients with ALK‐negative NSCLC, those with 10–15% of the tumor cells positive for ALK rearrangement shared more clinical characteristics with ALK rearrangement‐positive patients (>15% ALK FISH split) than with the ALK FISH negative (60 Sex Men Women Smoking history Never smoker Ever smoker Histology ADC SCC ADSQ Other NSCLC Clinical stage IA IB IIA IIB IIIA IIIB IV Type of sample Biopsy Bronchoscopic PCNAb Metastatic site Cell block Surgical resection Wedge resection Lobectomy
KRAS (N ¼ 44a)
ALK (N ¼ 47a)
Total, N (%)
N (%)
Pb
N (%)
Pb
N (%)
Pb
66 171 (33.5) 339 (66.5)
65.2 49 (33.6) 97 (66.4)
0.37c 0.99
64.2 15 (34.1) 29 (65.9)
0.67c 0.93
62.3 23 (48.9) 24 (51.1)
0.034c 0.019
278 (54.5) 232 (45.5)
47 (31.8) 101 (68.2)
