Tumor Biol. DOI 10.1007/s13277-015-3331-4
RESEARCH ARTICLE
The clinicopathological significance of ALK rearrangements and KRAS and EGFR mutations in primary pulmonary mucinous adenocarcinoma Yang Qu 1,2 & Nanying Che 2 & Dan Zhao 2 & Chen Zhang 2 & Dan Su 2 & Lijuan Zhou 2 & Lili Zhang 2 & Chongli Wang 2 & Haiqing Zhang 2 & Lixin Wei 1
Received: 2 January 2015 / Accepted: 12 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Primary pulmonary mucinous adenocarcinoma (PPMA) is one of the important subtypes of lung adenocarcinoma. Detection of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements and of KRAS and epidermal growth factor receptor (EGFR) mutations will help in diagnosing and predicting treatment outcome. The aim of this study was to investigate the clinicopathological significance of ALK rearrangements, KRAS and EGFR mutations in PPMA. ALK expression was detected immunohistochemically. KRAS and EGFR mutations were determined by the amplification refractory mutation system. Seventy-three patients of PPMA were enrolled. ALK rearrangements were detected in 34.2 % of patients and were more frequent in upper/middle lobe, stage III-IV, lymphatic permeation-positive patients and non-smokers. ALK rearrangements were significantly increased in the solid tumor predominant with mucin production subtype, and in special tissue structures, including signet ring cells, cribriform, and micropapillary patterns. KRAS mutations were observed in 23.3 % of patients and were more prevalent in invasive mucinous adenocarcinoma and lower lobe tumors. Only one case of ALK rearrangements harbored Yang Qu and Nanying Che contributed equally to this work. * Haiqing Zhang [email protected] * Lixin Wei [email protected] 1
Department of Pathology, The General Hospital of People’s Liberation Army, 28# Fuxing Road, Haidian District, Beijing 100853, China
2
Department of Pathology, Beijing Chest Hospital, Capital Medical University, 97# Machang, Tongzhou District, Beijing 101149, China
KRAS mutation, and no cases manifested with the coexistence of ALK rearrangements and EGFR mutations. KRAS and EGFR co-mutation was detected in one case. PPMA patients with ALK rearrangements or KRAS mutation represent a unique subtype in NSCLC. The results provide basis data for target therapy screening of PPMA patients.
Keywords EGFR . KRAS . ALK rearrangements . PPMA
Introduction Lung carcinoma is the leading death-threaten tumor worldwide, non-small cell lung cancer (NSCLC) accounts for approximately 80 % of all lung cancer cases. NSCLCs include squamous cell carcinomas, adenocarcinomas, and large cell carcinomas [1, 2]. Primary pulmonary mucinous adenocarcinoma (PPMA) is a group of subtypes of mucinproducing adenocarcinoma in the lung and was first identified in 1978 as a mucous cyst tumor of the lung [3]; other names like cystic mucinous adenocarcinoma, colloid adenocarcinoma, and mucinous bronchioloalveolar carcinoma were subsequently reported [4, 5]. According to the new International Association for the Study of Lung Cancer/ American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) multidisciplinary classification of lung adenocarcinoma published in 2011, mucinous bronchioloalveolar carcinoma was no longer used; invasive mucinous adenocarcinoma (formerly mucinous bronchioloalveolar carcinoma) was listed as independent variants subtype [6]. The concept of mucinous adenocarcinoma is still in transition, and the new classification system has been devised and generally accepted; accordingly, in this study, PPMA refers to all of the subtypes in the lung with the mucin-producing phenotype, including mucinous
Tumor Biol.
adenocarcinoma in situ (m-AIS) and mucinous minimally invasive adenocarcinoma (m-MIA), invasive mucinous adenocarcinoma, solid predominant with mucin production (SA) and colloid variant [6]. While every subtype has its own definite diagnostic standard in the new classification system, due to the histologic heterogeneity of PPMA, the clinically relevant pathological characteristics remain difficult to define for this disease. Molecularly targeted therapies allow for longer survival times and improved quality of life for lung cancer patients due to their ability to specifically target proteins expressed in cancer cells. In addition, these therapeutics have minimal sideeffects, unlike conventional therapies such as chemotherapy and radiotherapy. In NSCLC patients, KRAS and EGFR gene mutations and ALK rearrangements were frequently found [7, 8]. Remarkable successes have been archived in EGFR mutant patients with NSCLC by EGFR tyrosine kinase inhibitor (TKIs) treatment [9]. KRAS is a downstream target of EGFR. Therefore, KRAS and EGFR mutations are almost always mutually exclusive. Moreover, patients with mutant KRAS were unresponsive to EGFR TKIs [10, 11]. Several studies have shown that mutations in KRAS, but not in EGFR, were frequently detected in patients with invasive mucous adenocarcinoma [12, 13]. The echinoderm microtubule associated protein-like 4 (EML4) and ALK fusion gene (EML4-ALK) has recently been identified in a small subset of NSCLC patients. The EML4-ALK fusion gene is formed upon rearrangement from chromosomal inversion or translocation of chromosome 2p, and the encoded chimeric protein is dimerized and unliganded, leading to constitutive activation downstream signaling pathways that promote cell survival and proliferation [14]. ALK rearrangements was predominantly found in Asian, non-smoker lung carcinoma patients [15, 16] and were observed in 1.8–11.7 % of all NSCLC patients [17]. Lung adenocarcinomas with histologic features of extracellular mucin, signet ring cells, and cribriform pattern exhibited a high frequency of ALK rearrangements [18, 19]. The ALK inhibitor Crizotinib has been approved by the Food and Drug Administration (FDA) for use in the United States and by the Chinese FDA (CFDA). Identification of the patients harboring ALK rearrangements was indispensable for effectively treating these patients. Several studies have shown that ALK rearrangements and mutations in KRAS and EGFR are mutually exclusive in lung adenocarcinoma [20, 21]; however, the association among these genetic alterations in PPMA patients is unclear. The aim of this study was to examine the association between the clinicopathological characteristics of patients with PPMA and their ALK rearrangements status and KRAS and EGFR mutation status. The results of this study may help to identify a molecular target in the treatment of PPMA.
Materials and methods Patients and samples Samples were obtained from 73 patients diagnosed with PPMA via primary surgical resection at Beijing Thorax Hospital between January 2004 and December 2013. The research was reviewed and approved by the Ethics Committee of Beijing Thorax Hospital, and written informed consent forms were obtained from all patients. All patients who received neoadjuvant chemotherapy and/or radiotherapy prior to resection and those who had metastatic lung adenocarcinoma were excluded. Patients’ clinical and pathological characteristics were collected using a standardized case report form (Table 1). Tumor stages were classified according to the American Joint Committee on Cancer Staging System, 7th edition. Tumor tissue preparation All tumor tissue samples were fixed with 10 % formaldehyde and embedded in paraffin. Slides with 4 μm slices of tissue Table 1 Clinicopathologic parameters of the 73 PPMA patients included in this study Parameter Age (years) 3 Lobe Upper/middle Lower Stage I-II III-IV Histological subtype m-AIS and m-MIA Invasive mucinous adenocarcinoma Solid predominant with mucin production Colloid variant Pleural invasion Lymphatic permeation
n (%)
53 (72.6) 20 (27.4) 36 (49.3) 37 (50.7) 45 (61.6) 28 (38.4) 24 (32.9) 49 (67.1) 36 (49.3) 37 (50.7) 41 (56.2) 32 (43.8) 12 (16.4) 35 (48) 21 (28.7) 5 (6.8) 16 (21.9) 28 (34.6)
Tumor Biol.
were prepared for hematoxylin and eosin and ALK staining. Tumor tissue that was cut into 5 μm slices was obtained for the EGFR and KRAS gene mutation tests.
incubation, followed by 47 cycles of 95 °C for 25 s, and 93 °C for 25 s. Statistical analysis
Histological evaluation The slides with tumor sections were independently and blindly reviewed by two expert pathologists, and any discordant results were evaluated by a third pathologist. Lung adenocarcinomas were defined according to the 2011 IASLC/ATS/ ERS classification guidelines and were classified based on the identification of the following predominant histologic subtypes: m-AIS and m-MIA, invasive mucinous adenocarcinoma, solid predominant with mucin production, and colloid variant. Pleural invasion and lymphatic permeation were also documented. All adenocarcinomas were classified according to their predominant pattern as extracellular mucin, signet ring cells, cribriform, or micropapillary. Immunohistochemistry The immunohistochemical staining for ALK expression was performed in the Benchmark XT automatic staining module (Ventana Medical Systems, Tucson, AZ, USA) [22, 23]. Briefly, the 4-μm sections were de-paraffinized; antigen retrieval was performed, and samples were probed with the rabbit ALK monoclonal antibody (D5F3) (Cell Signaling Technology, Danvers, MA, USA). Detection was performed with the Optiview DAB Immunohistochemistry Detection kit with signal amplification (Roche, Bael, Switzerland) according to the manufacturer’s instructions. A rabbit monoclonal antibody raised against immunoglobulin G (Roche, Bael, Switzerland) was used as a negative control. Intensive brown staining with granular texture in the cytoplasm was considered positive for ALK, while faint cytoplasmic staining was considered negative. KRAS and EGFR mutation analysis Amplification refractory mutation system was performed to detect genetic mutations in KRAS and EGFR [24]. Tumor tissues were dissected from the unstained histological sections. DNA in the tumor tissues was isolated and purified using the DNA Tissue Isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. KRAS and EGFR mutations were subsequently identified using the Human KRAS and EGFR Mutation Detection kit (Amoy Diagnostics, Xiamen, China), which had been approved for clinical detection by the CFDA in China. Exons 18–21 in the EGFR gene and exons 12–13 in the KRAS gene were detected according to the kit’s instructions. Polymerase chain reaction was performed as follows: 95 °C for 5 min for the initial
Categorical variables were analyzed by the Chi-squared or Fisher’s exact tests in evaluating the relationship of the ALK rearrangements and KRAS and EGFR mutations with the clinicopathological parameters. Multivariate logistic regression was performed to predict the independent risk factors for ALK rearrangements. SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for all analyses. A p value of less than 0.05 was considered to be statistically significant.
Results Clinicopathological characteristics of the PPMA patients The clinicopathological data for the PPMA cases are summarized in Table 1. Of the 73 patients, 36 were men and 37 were women. Smokers accounted for 38.4 % of the cases (28/73, 25/36 men and 3/37 women). The maximum diameters of the tumors ranged from 1.0 to 15.0 cm. The histologic predominant patterns were invasive mucous (48 %, 35/73), solid predominant with mucin production (28.7 %, 21/73), m-AIS and m-MIA (16.4 %, 12/73), and colloid (6.8 %, 5/73). Twentyone-point-nine percent (16/73) of the patients exhibited pleural invasion, and 34.6 % (28/73) had lymphatic permeation (Table 1). Association between the clinicopathological characteristics and ALK rearrangements in patients with PPMA Expression of ALK was detected via immunostaining (Fig. 1). ALK rearrangements occurred in 34.2 % (25/73) of the PPMA patient samples (Table 2). The ALK rearrangement rate was significantly higher in non-smokers (46.7 %, 21/45) than among smokers (14.3 %, 4/28, p=0.017). ALK rearrangements were more likely to be found in tumors localized to the upper/middle pulmonary lobe (47.2 %, 17/36) than in the lower lobe of the lung (21.6 %, 8/37, p=0.019). Patients with stage III-IV disease had a significantly higher frequency of ALK rearrangements (50 %, 16/32) than patients with early stage (I-II) PPMA (22 %, 9/41, p=0.008). Fifty percent of PPMA patients with lymphatic permeation (14/28) had ALK rearrangements, which was significantly higher than those without lymphatic metastases (31.6 %, 18/57, p=0.024). In addition, there were no significant differences in ALK rearrangements with age, sex, tumor diameter, or pleural invasion (Table 2).
Tumor Biol. Fig. 1 Detection of ALK expression via immunohistochemistry. a An ALK-positive case showing the cancer portion as positive and the normal lung epithelium as negative. b A magnified image from subpanel a showing ALKpositive cells with strong granular cytoplasmic staining. c A negative control sample from the same case as shown in subpanel b, for which immunoglobulinG was utilized in place of the ALK antibody. d An ALK-negative case showing lack of ALK staining in the cytoplasm. Original magnifications: a: ×40; b–d: ×200
As shown in Table 2, the histologic predominant pattern with the most ALK rearrangements was the solid predominant with mucin production (71.4 %, 15/21, p=0.001). When comparing histologic features, patients who had signet ring cells had a significantly higher rate of ALK rearrangements (89.5 %, 17/19) than patients who did not have this histological feature (14.8 %, 8/54, p
RESEARCH ARTICLE
The clinicopathological significance of ALK rearrangements and KRAS and EGFR mutations in primary pulmonary mucinous adenocarcinoma Yang Qu 1,2 & Nanying Che 2 & Dan Zhao 2 & Chen Zhang 2 & Dan Su 2 & Lijuan Zhou 2 & Lili Zhang 2 & Chongli Wang 2 & Haiqing Zhang 2 & Lixin Wei 1
Received: 2 January 2015 / Accepted: 12 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Primary pulmonary mucinous adenocarcinoma (PPMA) is one of the important subtypes of lung adenocarcinoma. Detection of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements and of KRAS and epidermal growth factor receptor (EGFR) mutations will help in diagnosing and predicting treatment outcome. The aim of this study was to investigate the clinicopathological significance of ALK rearrangements, KRAS and EGFR mutations in PPMA. ALK expression was detected immunohistochemically. KRAS and EGFR mutations were determined by the amplification refractory mutation system. Seventy-three patients of PPMA were enrolled. ALK rearrangements were detected in 34.2 % of patients and were more frequent in upper/middle lobe, stage III-IV, lymphatic permeation-positive patients and non-smokers. ALK rearrangements were significantly increased in the solid tumor predominant with mucin production subtype, and in special tissue structures, including signet ring cells, cribriform, and micropapillary patterns. KRAS mutations were observed in 23.3 % of patients and were more prevalent in invasive mucinous adenocarcinoma and lower lobe tumors. Only one case of ALK rearrangements harbored Yang Qu and Nanying Che contributed equally to this work. * Haiqing Zhang [email protected] * Lixin Wei [email protected] 1
Department of Pathology, The General Hospital of People’s Liberation Army, 28# Fuxing Road, Haidian District, Beijing 100853, China
2
Department of Pathology, Beijing Chest Hospital, Capital Medical University, 97# Machang, Tongzhou District, Beijing 101149, China
KRAS mutation, and no cases manifested with the coexistence of ALK rearrangements and EGFR mutations. KRAS and EGFR co-mutation was detected in one case. PPMA patients with ALK rearrangements or KRAS mutation represent a unique subtype in NSCLC. The results provide basis data for target therapy screening of PPMA patients.
Keywords EGFR . KRAS . ALK rearrangements . PPMA
Introduction Lung carcinoma is the leading death-threaten tumor worldwide, non-small cell lung cancer (NSCLC) accounts for approximately 80 % of all lung cancer cases. NSCLCs include squamous cell carcinomas, adenocarcinomas, and large cell carcinomas [1, 2]. Primary pulmonary mucinous adenocarcinoma (PPMA) is a group of subtypes of mucinproducing adenocarcinoma in the lung and was first identified in 1978 as a mucous cyst tumor of the lung [3]; other names like cystic mucinous adenocarcinoma, colloid adenocarcinoma, and mucinous bronchioloalveolar carcinoma were subsequently reported [4, 5]. According to the new International Association for the Study of Lung Cancer/ American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) multidisciplinary classification of lung adenocarcinoma published in 2011, mucinous bronchioloalveolar carcinoma was no longer used; invasive mucinous adenocarcinoma (formerly mucinous bronchioloalveolar carcinoma) was listed as independent variants subtype [6]. The concept of mucinous adenocarcinoma is still in transition, and the new classification system has been devised and generally accepted; accordingly, in this study, PPMA refers to all of the subtypes in the lung with the mucin-producing phenotype, including mucinous
Tumor Biol.
adenocarcinoma in situ (m-AIS) and mucinous minimally invasive adenocarcinoma (m-MIA), invasive mucinous adenocarcinoma, solid predominant with mucin production (SA) and colloid variant [6]. While every subtype has its own definite diagnostic standard in the new classification system, due to the histologic heterogeneity of PPMA, the clinically relevant pathological characteristics remain difficult to define for this disease. Molecularly targeted therapies allow for longer survival times and improved quality of life for lung cancer patients due to their ability to specifically target proteins expressed in cancer cells. In addition, these therapeutics have minimal sideeffects, unlike conventional therapies such as chemotherapy and radiotherapy. In NSCLC patients, KRAS and EGFR gene mutations and ALK rearrangements were frequently found [7, 8]. Remarkable successes have been archived in EGFR mutant patients with NSCLC by EGFR tyrosine kinase inhibitor (TKIs) treatment [9]. KRAS is a downstream target of EGFR. Therefore, KRAS and EGFR mutations are almost always mutually exclusive. Moreover, patients with mutant KRAS were unresponsive to EGFR TKIs [10, 11]. Several studies have shown that mutations in KRAS, but not in EGFR, were frequently detected in patients with invasive mucous adenocarcinoma [12, 13]. The echinoderm microtubule associated protein-like 4 (EML4) and ALK fusion gene (EML4-ALK) has recently been identified in a small subset of NSCLC patients. The EML4-ALK fusion gene is formed upon rearrangement from chromosomal inversion or translocation of chromosome 2p, and the encoded chimeric protein is dimerized and unliganded, leading to constitutive activation downstream signaling pathways that promote cell survival and proliferation [14]. ALK rearrangements was predominantly found in Asian, non-smoker lung carcinoma patients [15, 16] and were observed in 1.8–11.7 % of all NSCLC patients [17]. Lung adenocarcinomas with histologic features of extracellular mucin, signet ring cells, and cribriform pattern exhibited a high frequency of ALK rearrangements [18, 19]. The ALK inhibitor Crizotinib has been approved by the Food and Drug Administration (FDA) for use in the United States and by the Chinese FDA (CFDA). Identification of the patients harboring ALK rearrangements was indispensable for effectively treating these patients. Several studies have shown that ALK rearrangements and mutations in KRAS and EGFR are mutually exclusive in lung adenocarcinoma [20, 21]; however, the association among these genetic alterations in PPMA patients is unclear. The aim of this study was to examine the association between the clinicopathological characteristics of patients with PPMA and their ALK rearrangements status and KRAS and EGFR mutation status. The results of this study may help to identify a molecular target in the treatment of PPMA.
Materials and methods Patients and samples Samples were obtained from 73 patients diagnosed with PPMA via primary surgical resection at Beijing Thorax Hospital between January 2004 and December 2013. The research was reviewed and approved by the Ethics Committee of Beijing Thorax Hospital, and written informed consent forms were obtained from all patients. All patients who received neoadjuvant chemotherapy and/or radiotherapy prior to resection and those who had metastatic lung adenocarcinoma were excluded. Patients’ clinical and pathological characteristics were collected using a standardized case report form (Table 1). Tumor stages were classified according to the American Joint Committee on Cancer Staging System, 7th edition. Tumor tissue preparation All tumor tissue samples were fixed with 10 % formaldehyde and embedded in paraffin. Slides with 4 μm slices of tissue Table 1 Clinicopathologic parameters of the 73 PPMA patients included in this study Parameter Age (years) 3 Lobe Upper/middle Lower Stage I-II III-IV Histological subtype m-AIS and m-MIA Invasive mucinous adenocarcinoma Solid predominant with mucin production Colloid variant Pleural invasion Lymphatic permeation
n (%)
53 (72.6) 20 (27.4) 36 (49.3) 37 (50.7) 45 (61.6) 28 (38.4) 24 (32.9) 49 (67.1) 36 (49.3) 37 (50.7) 41 (56.2) 32 (43.8) 12 (16.4) 35 (48) 21 (28.7) 5 (6.8) 16 (21.9) 28 (34.6)
Tumor Biol.
were prepared for hematoxylin and eosin and ALK staining. Tumor tissue that was cut into 5 μm slices was obtained for the EGFR and KRAS gene mutation tests.
incubation, followed by 47 cycles of 95 °C for 25 s, and 93 °C for 25 s. Statistical analysis
Histological evaluation The slides with tumor sections were independently and blindly reviewed by two expert pathologists, and any discordant results were evaluated by a third pathologist. Lung adenocarcinomas were defined according to the 2011 IASLC/ATS/ ERS classification guidelines and were classified based on the identification of the following predominant histologic subtypes: m-AIS and m-MIA, invasive mucinous adenocarcinoma, solid predominant with mucin production, and colloid variant. Pleural invasion and lymphatic permeation were also documented. All adenocarcinomas were classified according to their predominant pattern as extracellular mucin, signet ring cells, cribriform, or micropapillary. Immunohistochemistry The immunohistochemical staining for ALK expression was performed in the Benchmark XT automatic staining module (Ventana Medical Systems, Tucson, AZ, USA) [22, 23]. Briefly, the 4-μm sections were de-paraffinized; antigen retrieval was performed, and samples were probed with the rabbit ALK monoclonal antibody (D5F3) (Cell Signaling Technology, Danvers, MA, USA). Detection was performed with the Optiview DAB Immunohistochemistry Detection kit with signal amplification (Roche, Bael, Switzerland) according to the manufacturer’s instructions. A rabbit monoclonal antibody raised against immunoglobulin G (Roche, Bael, Switzerland) was used as a negative control. Intensive brown staining with granular texture in the cytoplasm was considered positive for ALK, while faint cytoplasmic staining was considered negative. KRAS and EGFR mutation analysis Amplification refractory mutation system was performed to detect genetic mutations in KRAS and EGFR [24]. Tumor tissues were dissected from the unstained histological sections. DNA in the tumor tissues was isolated and purified using the DNA Tissue Isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. KRAS and EGFR mutations were subsequently identified using the Human KRAS and EGFR Mutation Detection kit (Amoy Diagnostics, Xiamen, China), which had been approved for clinical detection by the CFDA in China. Exons 18–21 in the EGFR gene and exons 12–13 in the KRAS gene were detected according to the kit’s instructions. Polymerase chain reaction was performed as follows: 95 °C for 5 min for the initial
Categorical variables were analyzed by the Chi-squared or Fisher’s exact tests in evaluating the relationship of the ALK rearrangements and KRAS and EGFR mutations with the clinicopathological parameters. Multivariate logistic regression was performed to predict the independent risk factors for ALK rearrangements. SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for all analyses. A p value of less than 0.05 was considered to be statistically significant.
Results Clinicopathological characteristics of the PPMA patients The clinicopathological data for the PPMA cases are summarized in Table 1. Of the 73 patients, 36 were men and 37 were women. Smokers accounted for 38.4 % of the cases (28/73, 25/36 men and 3/37 women). The maximum diameters of the tumors ranged from 1.0 to 15.0 cm. The histologic predominant patterns were invasive mucous (48 %, 35/73), solid predominant with mucin production (28.7 %, 21/73), m-AIS and m-MIA (16.4 %, 12/73), and colloid (6.8 %, 5/73). Twentyone-point-nine percent (16/73) of the patients exhibited pleural invasion, and 34.6 % (28/73) had lymphatic permeation (Table 1). Association between the clinicopathological characteristics and ALK rearrangements in patients with PPMA Expression of ALK was detected via immunostaining (Fig. 1). ALK rearrangements occurred in 34.2 % (25/73) of the PPMA patient samples (Table 2). The ALK rearrangement rate was significantly higher in non-smokers (46.7 %, 21/45) than among smokers (14.3 %, 4/28, p=0.017). ALK rearrangements were more likely to be found in tumors localized to the upper/middle pulmonary lobe (47.2 %, 17/36) than in the lower lobe of the lung (21.6 %, 8/37, p=0.019). Patients with stage III-IV disease had a significantly higher frequency of ALK rearrangements (50 %, 16/32) than patients with early stage (I-II) PPMA (22 %, 9/41, p=0.008). Fifty percent of PPMA patients with lymphatic permeation (14/28) had ALK rearrangements, which was significantly higher than those without lymphatic metastases (31.6 %, 18/57, p=0.024). In addition, there were no significant differences in ALK rearrangements with age, sex, tumor diameter, or pleural invasion (Table 2).
Tumor Biol. Fig. 1 Detection of ALK expression via immunohistochemistry. a An ALK-positive case showing the cancer portion as positive and the normal lung epithelium as negative. b A magnified image from subpanel a showing ALKpositive cells with strong granular cytoplasmic staining. c A negative control sample from the same case as shown in subpanel b, for which immunoglobulinG was utilized in place of the ALK antibody. d An ALK-negative case showing lack of ALK staining in the cytoplasm. Original magnifications: a: ×40; b–d: ×200
As shown in Table 2, the histologic predominant pattern with the most ALK rearrangements was the solid predominant with mucin production (71.4 %, 15/21, p=0.001). When comparing histologic features, patients who had signet ring cells had a significantly higher rate of ALK rearrangements (89.5 %, 17/19) than patients who did not have this histological feature (14.8 %, 8/54, p
