BIOMEDICAL REPORTS 2: 481-489, 2014 Differential expression of CRKL and AXL genes in lung adenocarcinoma subtypes according to the epidermal growth factor receptor and anaplastic lymphoma kinase gene status DA-PING YU1, YU-JIE DONG2, HAI-QING ZHANG2, JING-HUI WANG3, YANG QU2, LI-JUAN ZHOU2, DAN SU2, LI-LI ZHANG2, DAN ZHAO2 and YI-RAN CAI2 Departments of 1Thoracic Surgery, 2Pathology and 3Oncology, Beijing Chest Hospital, Capital Medical University, Beijing 101149, P.R. China Received January 21, 2014; Accepted February 24, 2014 DOI: 10.3892/br.2014.261 Abstract. Non-small-cell lung cancer (NSCLC) is with lymph node infiltration in acinar ACs. ACs with EGFR the most common cause of cancer-related mortality. mutations exhibited high levels of AXL protein expression Adenocarcinoma (AC) is the predominant histological type of compared to those without mutations (P<0.001). Acinar AC NSCLC; however, AC consists of several subtypes. It has not with cribriform structure is a distinct subtype that frequently yet been determined whether there is a correlation of CRKL harbors ALK rearrangement. The activation of AXL may be and AXL expression with epidermal growth factor receptor one of the factors contributing to the invasion of acinar and (EGFR) and anaplastic lymphoma kinase (ALK) gene status in micropapillary ACs. lung AC. We assayed exons 18 through 21 of the EGFR gene by direct sequencing; ALK rearrangement and the expression Introduction of CRKL and AXL were detected by immunostaining. A total of 212 cases of AC were included in this study, diagnosed using Lung cancer is the leading cause of cancer-related mortality the novel classification system established by the International worldwide (1,2). Adenocarcinoma (AC) is the most common Association for the Study of Lung Cancer, the American histological subtype of non-small-cell lung cancer (NSCLC) Thoracic Society and the European Respiratory Society accounting for approximately half of all lung cancers (3). in 2011, including 69 acinar ACs, 17 lepidic predominant A previous study reported that >80% of lung ACs are diag- ACs (LPAs), 63 papillary, 14 mucinous, 17 micropapillary and nosed as mixed AC subtype according to the 2004 World 32 solid ACs. Of the 212 cases, 101 harbored EGFR mutations. Health Organization (WHO) classification (4). Therefore, The most common subtypes carrying delK745-S753 were comprehensive histological profiling is required through papillary and acinar ACs. ALK rearrangement was found in semi-quantitative evaluation of the percentages of the various 23 cases (11%) of lung ACs. Acinar and solid ACs were the histological components. To address such issues, a new classi- most frequent subtypes with ALK aberrance, particularly in fication based on a multidisciplinary approach tothe diagnosis acinar ACs with cribriform structure (4/5 cases, 80%). The of lung ACs was established by the International Association expression of CRKL was significantly different among the for the Study of Lung Cancer, the American Thoracic Society AC subtypes (P=0.01), with the highest and lowest expres- and the European Respiratory Society in 2011 (5). In the new sion levels of CRKL protein in papillary ACs and LPAs, nomenclature system, invasive ACs are classifiedaccording to respectively (P<0.05). AXL expression was also significantly the predominant histological pattern into specific subtypes, different among the AC subtypes (P=0.002) and was correlated such as lepidic [formerly the majority of mixed subtype tumors with non-mucinous bronchioloalveolar carcinoma (BAC)], mucinous (formerly mucinous BAC), acinar, papil- lary, solid and micropapillary ACs. Although widely divergent clinical, radiological, molecular and pathological data have Correspondence to: Dr Yi-Ran Cai, Department of been collected on lung AC, there remains some confusion (5). Pathology, Beijing Chest Hospital, Capital Medical University, 97 Ma Chang Street, Tongzhou, Beijing 101149, P.R. China Despite significant advances regarding the understanding of E-mail: [email protected] this type of tumor over the last few decades, there remains a need for universally accepted criteria for the classification Key words: lung adenocarcinoma, epidermal growth factor receptor of AC subtypes (6,7). As significant resources are invested on gene mutation, anaplastic lymphoma kinase gene rearrangement, trials investigating the molecular and therapeutic aspects of CRKL, AXL lung AC, the development of standardized criteria is crucial and may lead to improvements in patient care and prognosis. The various subtypes of AC may be associated with a different 482 YU et al: CRKL AND AXL EXPRESSION IN LUNG AC SUBTYPES BY EGFR AND ALK STATUS prognosis and the underlying mechanisms have not been therapy and determine the correlation of CRKL and AXL clearly determined. expression with the status of ALK and EGFR among the Mutations in exons 18 through 21 of the epidermal growth different histological subtypes of lung AC. We hypothesized factor receptor gene (EGFR) are associated with sensitivity that the characteristics of CRKL and AXL expression are to tyrosine kinase inhibitors (TKIs); therefore, it is crucial to correlated with multiple clinicopathological factors. Owing to elucidate the nature of these mutations. EGFR mutations are the presence of lung AC subtypes and targeted therapy with mainly classified as ‘classical’ activating mutations (del19 and gefitinib and crizotinib, it is crucial to evaluate the expression L858R) and as variants of unknown function; thus, further of CRKL and AXL in ACs by different EGFR and ALK status. analyses are required accordingly (8). Persistent EGFR signaling activation underlies tumor development in human Materials and methods lung ACs. Treatment with gefitinib or erlotinib achieves specific inhibition of the EGFR signaling pathway, leading to Tumor samples. A total of 212 primary lung AC samples apoptosis of cancer cells (9,10). from patients who had undergone surgery at the Beijing Echinoderm microtubule-associated protein-like 4 Chest Hospital (Beijing, China) between 2006 and 2012 (EML4)-anaplastic lymphoma kinase (ALK), a transforming were analyzed. All the sections were reviewed by 2 reference fusion gene resulting from ALK rearrangement, is a potent pathologists (Yi‑Ran Cai and Yu‑Jie Dong) to confirm the oncogenic driver and a promising therapeutic target in ACs diagnosis and predominance (>70%) of tumor tissues. The via the administration of crizotinib (11-13). The method for the ACs were reviewed following the new classification (5) and the detection of ALK rearrangement includes fluorescent in situ 2 observers were blinded to the outcomes. The clinicopatho- hybridization (FISH), immunohistochemistry (IHC) and logical characteristics, including age, gender and histological reverse transcription-polymerase chain reaction (RT-PCR). subtype were recorded. FISH is currently the only approved diagnostic test for ALK rearrangement for the detection of break-apart signals. DNA extraction, PCR amplification and direct sequencing for However, there are certain disadvantages to FISH, particularly EGFR mutations. Genomic DNA was extracted from that apparatuses are not always readily available in routine 50-100 mg tumor tissue scraped off formalin-fixed and diagnostic laboratories and subtle intrachromosomal rear- paraffin-embedded blocks according to the previously rangements may be difficult to interpret (14,15); therefore, described protocol (23). Briefly, PCR for exons 18 through 21 false-negative outcomes are inevitable. IHC has been consid- was performed with 100 ng template DNA in a 50-µl volume ered as an alternative to FISH, as it is able to detect ALK containing 0.75 U HotStarTaq DNA polymerase (Fermentas rearrangements. International Inc., Ontario, Canada), 5 µl PCR buffer, 0.8 µM It was previously reported that the CRKL is selectively deoxyribonucleotide triphosphate, 0.5 µM of each primer and upregulated in a number of malignant tumors, including 49, different concentrations of MgCl2, depending on the various 55, 67, 50, 50 and 63% of breast, lung, skin, ovarian and colon markers. The nucleic acid used for mutations was based on cancers, respectively (16). CRKL is a member of the human NM_005228.3. The primers were designed as follows: exon 18, Crk adapter protein family and is amplified in lung cancer forward 5'-CAACCAAGCTCTCTTGAGGATC-3' and reverse cells with enhanced expression. In addition, knockdown of 5'-CCCAGCCCAGAGGCCTGT-3'; exon 19, forward 5'-GCA CRKL in lung cancer cell lines is associated with a significant GCATGTGGCACCATCTC-3' and reverse 5'-AGAGCCATG decrease in the proliferation, progression, survival, motility GACCCCCACAC-3'; exon 20, forward 5'-CACACTGAC and invasiveness of lung cancer cells. These findings suggest GTGCCTCTCC-3' and reverse 5'-AGCAGGTACTGGGAG that the overexpression of CRKL may contribute to the onco- CCAAT-3'; and exon 21, forward 5'-TCTGTCCCTCACAGC genic phenotype in lung cancer (17). Although evidence favors AGGGTCT-3' and reverse 5'-GCTGGCTGACCTAAAGCC CRKL gene amplification in several human malignancies, ACC-3'. Amplification and sequencing of the exon fragments including lung cancer, the correlation between CRKL and were performed as previously described (23). The PCR prod- EGFR and the clinicopathological characteristics in lung ACs ucts were sequenced in the sense and antisense directions. has not been clearly determined. Only specimens with an identified mutation in both rounds AXL, a receptor tyrosine kinase, is associated with the were