Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes

  • Authors:
    • Yi‑Ran Cai
    • Yu‑Jie Dong
    • Hong‑Bo Wu
    • Da‑Ping Yu
    • Li‑Juan Zhou
    • Dan Su
    • Li Zhang
    • Xue‑Jing Chen
  • View Affiliations

  • Published online on: September 2, 2016     https://doi.org/10.3892/ol.2016.5080
  • Pages: 3312-3322
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Abstract

Non-small cell lung cancer (NSCLC) is a lethal cancer-related disease in population. Adenocarcinoma (AC) is subclassified into several subtypes based on the new classification by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society in 2011. Correlation between original expression of Crk‑like (CRKL) and anaplastic lymphoma receptor tyrosine kinase in diverse histological components of AC and epidermal growth factor receptor (EGFR) or ALK status was evaluated by immunohistochemistry and sequencing in present study. A total of 106 cases, including 83 patients (78.3%) with mixed‑type ACs, were assessed in the present study using eligible follow‑up data. The ACs consisted of 32 acinar, 12 papillary, 5 mucinous, 11 micropapillary and 46 solid‑predominant ACs. In total, 69.8% samples were composed of 2 or 3 histological components, with different expression levels of CRKL and AXL. ACs with EGFR mutation had a higher level of AXL expression compared with ACs without mutation (P=0.019). Multivariate survival analysis showed that AC subtypes and EGFR mutation subtypes were significantly associated with the progression‑free survival (PFS) time. Acinar AC was the subtype with the most notable PFS time (30.6 months), which was significantly different from the PFS time of papillary, mucinous, micropapillary and solid‑predominant ACs (hazard ratio, 0.4; 95% CI, 0.21‑0.75; P=0.005). Among the ACs with exon 19 mutation, the median PFS time (28.8 months) of patients with a lower level of AXL protein expression was increased compared with the PFS time of patients with the L858R mutation and wild‑type EGFR (9.1 months and 11 months, respectively; P=0.03), whereas no significant difference in ACs with an increased level of AXL expression. However, AC patients with higher level of CRKL expression had better PFS (28.8 months) than patients with the L858R mutation and wild‑type EGFR (9.1 months and 11.3 months, respectively). Exon 19 deletion is an important status that is associated with an improved response to conventional chemotherapy. The identification of EGFR mutations combined with CRKL and AXL status may potentially alter the way that lung AC is treated.

Introduction

Adenocarcinoma (AC) is the most common histological type of non-small cell lung cancer (NSCLC) (1). Overall, >80% of lung ACs are diagnosed with mixed ACs, according to the 2004 World Health Organization (WHO) classification (2). Therefore, a semiquantitative evaluation system for calculating the components is necessary. A novel classification based on a multidisciplinary approach to the diagnosis of lung ACs was established by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society in 2011 (3). In the novel nomenclature system, invasive ACs are classified by predominant components, such as lepidic (formerly most mixed subtype tumors with non-mucinous BAC), mucinous (formerly mucinous BAC), acinar, papillary, solid patterns and micropapillary ACs (3). Although multidisciplinary data from widely divergent clinical, radiologic, molecular and pathological spectra accounts for the attributes of lung AC, it remains unclear how to address the biological properties of lung AC. Despite marked advances in the understanding of this tumor in the past decades, the production of universally accepted criteria for AC subtypes is required (4,5). Previous studies have reported that the new classification is an independent predictor of overall survival (6,7).

Epidermal growth factor receptor (EGFR) mutations of exon 18 through exon 21 are reported to be associated with the sensitivity to tyrosine kinase inhibitors (TKIs); therefore, it is important to understand the nature of these mutations. EGFR mutations are mainly categorized into two groups, with ‘classical’ activating mutations including del19 and L858R. Additional analyses are required on other variants with unknown function (8). Tumor development in human lung ACs is increased by the activation of the EGFR signaling pathway. Therefore, target treatment with gefitinib leads to specific inhibition through apoptosis of cancer cells (9,10). A previous study on stage IB lung AC identified that micropapillary-predominant AC is the most common AC subtype with EGFR mutation, whereas solid-predominant AC has a lower frequency of EGFR mutation (11).

Echinoderm microtubule associated protein like 4 (EML4)-anaplastic lymphoma receptor tyrosine kinase (ALK), the major type of fusion gene resulting from ALK rearrangement, has been reported to be a potent oncogenic driver and a promising therapeutic target in ACs through the administration of crizotinib (1214). Methods to detect ALK rearrangement include fluorescent in situ hybridization (FISH), immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). FISH analysis is the only approved diagnostic test for the detection of the break-apart signal of ALK rearrangement. However, the disadvantages of the FISH test are that particular apparatuses are not always readily available in routine diagnostic laboratories, and subtle intrachromosomal rearrangement may be challenging to interpret; therefore, false-negative outcomes are inevitable. The subtle changes may be challenging to interpret by FISH analysis, and have led to false-negative results (15,16). IHC has been considered as an alternative to FISH for the detection of ALK rearrangement, and Ventana IHC for ALK fusion gene has been approved by the European Union (17).

Crk-like (CRKL) is upregulated in malignant tumors, including 49% of breast cancer, 55% of lung cancer, 67% of skin cancer, 50% of ovarian carcinoma, and 63% of colon carcinoma tumors (18). CRKL is a member of the human Crk adapter protein family and has been found to be amplified in lung cancer cells, with enhanced expression as a result of the amplification. In addition, knockdown of CRKL in lung cancer cell lines has led to a significant decrease in the proliferation, progression, survival, motility and invasiveness of lung cancer cells. All these data indicate that overexpression of CRKL may result in the oncogenic phenotype of lung cancer (19). Although evidence favors CRKL gene amplification in several human malignancies, including lung cancer, whether the expression of CRKL is associated with EGFR status in lung ACs remains to be elucidated.

AXL receptor tyrosine kinase (AXL) is confirmed to be associated with the carcinogenesis of numerous tumors. Elevated AXL expression and interaction with its ligand growth arrest-specific 6 (Gas6) have been associated with cell survival, proliferation, and migration in solid tumors (20,21). AXL is increasingly upregulated during the multistep process of esophageal carcinogenesis and is an adverse prognostic marker in esophageal AC (22). A previous study identified AXL activation as a novel mechanism of acquired resistance to EGFR inhibitors in non-small cell lung cancer (23). Overexpression of AXL is consistently manifested in prostate cancer cell lines and human prostate tumors. Blockage of AXL expression strongly inhibits the proliferation, migration and invasion of tumor cells, and therefore tumor growth (24).

The primary purpose of the present study is to analyze the correlation between the original expression levels of CRKL and AXL and status of the ALK and EGFR genes and the prognosis of different AC histological subtypes. Due to the presence of mixed AC components, it was important to evaluate the role of CRKL and AXL expression in AC subtypes combined with EGFR and ALK status.

Materials and methods

Study design

The present study is a retrospective review of 108 treatment-naive patients with AC, with samples consisting of 91 samples of resected primary lung cancer and 15 samples of metastatic nodules from advanced lung cancer from Beijing Chest Hospital (Beijing, China) between 2006 and 2012. All sample sections were evaluated by two pathologists to confirm the diagnosis and predominance (>70%) of tumor tissues. All slides were evaluated by pathologists based on the new classification (25) using a multi-headed microscope (objective, 40X; magnification, ×400) and the clinical stage was defined according to the 7th Edition of the TNM Classification of the Union for International Cancer Control (3,26). A mean number of 4.5 slides (range, 2–11 slides) were reviewed. Patients were examined at 3-month intervals for the first 2 years following treatment and at 6-month intervals thereafter. The progression-free survival (PFS) time was measured from the date of treatment to the date of the first documented disease progression. The data were collected from the medical record system of Beijing Chest Hospital. The evaluation of disease progression included a physical examination, computed tomography scan of the chest and abdomen, brain magnetic resonance imaging, and bone scintigraphy. The last follow-up date was January 1, 2014. In total, 27 patients were censored from the current evaluation due to incomplete follow-up data.

DNA extraction, polymerase chain reaction amplification and direct sequencing for EGFR mutation

Genomic DNA was extracted from 50–100-mg tumor tissues obtained from formalin-fixed and paraffin-embedded blocks. The procedures followed a previously described protocol (27). PCR for exons 18–21 was performed using 100 ng template DNA in 50 µl volumes containing 0.75 U Hotstart Taq DNA polymerase (Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA), 5 µl PCR buffer, 0.8 µM dNTP (Fermentas; Thermo Fisher Scientific, Inc.), 0.5 µM of each primer (Sangon Biotech Co., Ltd., Shanghai, China), and various concentrations of MgCl2, depending on varied markers. The nucleic acids used for the mutations were based on NM_005228.3. The primers were designed by Sangon Biotech Co., Ltd. as follows: Exon 18 forward, 5′-CAACCAAGCTCTCTTGAGGATC-3′ and reverse, 5′-CCCAGCCCAGAGGCCTGT-3′; exon 19 forward, 5′-GCAGCATGTGGCACCATCTC-3′ and reverse, 5′-AGAGCCATGGACCCCCACAC-3′; exon 20 forward, 5′-CACACTGACGTGCCTCTCC-3′ and reverse, 5′-AGCAGGTACTGGGAGCCAAT-3′; and exon 21 forward, 5′-TCTGTCCCTCACAGCAGGGTCT-3′ and reverse, 5′-GCTGGCTGACCTAAAGCCACC-3′. The amplification and sequencing of exon fragments were performed as previously described (27). PCR products were sequenced in sense and antisense directions. Only specimens in which a mutation was identified in the two rounds were recorded as mutation-positive.

IHC for CRKL, AXL gene and ALK rearrangement

Tissue sections (4 µm) were prepared from tissue microarray blocks, deparaffinized using xylene and rehydrated through an ethanol series to water. Slides were incubated with the rabbit polyclonal anti-CRKL (catalog no., ab151791; Abcam Inc., Cambridge, UK) and polyclonal goat anti-AXL (catalog no., AF154; R&D Systems, Inc., Minneapolis, MN, USA) antibodies using a MaxVision horseradish peroxidase-polymer system kit (catalog nos. 5030 and 5108; Maixin Bio, Fuzhou, China). Incubation with the primary antibodies was performed overnight at 4°C and at a 1:200 dilution. The MaxVision horseradish peroxidase-polymer system kit was used for immunostaining according to the manufacturer's instructions. Detection was accomplished using diaminobenzidine (DAB) (catalog no. CAS 7411-49-6; ImmunoCruz Staining System; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The slides were then counterstained in hematoxylin, and the stained tumor cells (≥1,000 cells), were scored by two independent observers. Cytoplasmic staining was considered positive for both CRKL and AXL. The immunoreactivity of carcinoma samples was semi-quantitatively evaluated by two aspects: Percentage of positive cells and staining. The staining strength was described as follows: 0, tumor cells were not stained; 1, light-yellow stained cells; 2, yellow stained cells; 3, brown stained cells. The observed area covered all histological patterns. The raw scores, ranging from 0 to 300, were calculated as follows: Percentage × staining strength. The final scores for statistical analysis were the average of raw scores from subtype components (Fig. 1A-D). Lower expression levels of AXL and CRKL were defined as a staining score ≤100, otherwise the tissues were classified as having a higher expression level.

IHC analysis for ALK rearrangement was completed by using Ventana method on a Benchmark XT autostainer (Roche Diagnostics, Indianapolis, IN, USA). This IHC method was an automatic staining by utilizing a ready-to-use primary anti-ALK rabbit monoclonal antibody (clone, D5F3; catalog no. 790–4794; Ventana Medical Systems, Inc., Tucson, AZ, USA). The staining procedure followed the Ventana ALK test protocol using an optiview amplification kit and an optiview DAB IHC detection kit. Presence or absence of ALK rearrangement was evaluated as positive or negative following the manufacturer's protocol (Roche Diagnostics). Neoplastic cells with diffuse dark brown cytoplasmic staining were classified as ALK rearrangement-positive; any other colors were classified as ALK rearrangement-negative (Fig. 1E).

Statistical analysis

CRKL and AXL expression was categorized into lower and higher levels of expression according to the aforementioned cut-off values. Association between CRKL and AXL expression were analyzed with clinicopathological factors by crosstab χ2 test or Fisher's exact test (Table I). The impact of the following factors on the progression-free survival (PFS) rates was also evaluated: Gender; age; smoking status; clinical stage; EGFR gene status; and ALK fusion gene status. These clinicopathological factors were used in univariate and multivariate analyses to determine whether they had a significant effect on PFS (Table II). The survival rates and pairwise comparisons were stratified by clinicopathological characteristics and calculated using the Kaplan-Meier method and log-rank test. All statistical tests were two-sided and P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed on SAS system for Windows, version 9.2 (SAS Institute Inc., Cary, NC, USA). The survival curves were plotted using GraphPad Prism, version 6 (GraphPad Software, Inc., La Jolla, CA, USA).

Table I.

Association between CRKL and AXL expression and clinicopathological characteristics.

Table I.

Association between CRKL and AXL expression and clinicopathological characteristics.

CRKL expression, n (%) AXL expression, n (%)


ClinicalLower levelHigher levelP-valueLower levelHigher levelP-value
Gender 0.53 0.7
  Male16 (32.7)33 (67.3) 21 (42.9)28 (57.1)
  Female15 (26.3)42 (73.7) 27 (47.4)30 (52.6)
Adenocarcinoma subtypes 0.5 0.67
  Acinar13 (40.6)19 (59.4) 12 (37.5)20 (62.5)
  Micropapillary  3 (27.3)  8 (72.7)   4 (36.4)  7 (63.6)
  Papillary  2 (16.7)10 (83.3)   6 (50.0)  6 (50.0)
  Solid12 (26.1)34 (73.9) 23 (50.0)23 (50.0)
  Mucinous  1 (20.0)  4 (80.0)   3 (60.0)  2 (40.0)
Tumor size 0.82 0.68
  ≤3 cm  9 (27.3)24 (72.7) 16 (48.5)17 (51.5)
  >3 cm20 (30.3)46 (69.7) 29 (43.9)37 (56.1)
EGFR status 0.83 0.019
  Mutation17 (30.4)39 (69.6) 19 (33.9)37 (66.1)
  Wild type14 (28.0)36 (72.0) 29 (58.0)21 (42.0)
ALK status 0.77 0.29
  Fusion gene  4 (23.5)13 (76.5) 10 (58.8)  7 (41.2)
  Non-fusion gene27 (30.3)62 (69.7) 38 (42.7)51 (57.3)
Smoking status 0.36 1.0
  Never smoker19 (26.0)54 (74.0) 33 (45.2)40 (54.8)
  Smoker or ever smoker12 (36.4)21 (63.6) 15 (45.5)18 (54.5)
Clinical stage 0.68 0.28
  I+II16 (32.7)33 (67.3) 22 (44.9)27 (55.1)
  IIIA10 (24.4)31 (75.6) 16 (39.0)25 (61.0)
  IIIB+IV  5 (31.2)11 (68.8) 10 (62.5)  6 (37.5)

[i] CRKL, Crk-like; AXL, AXL receptor tyrosine kinase; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma receptor tyrosine kinase.

Table II.

Association of PFS and hazard ratios with clinicopathological factors in ACs.

Table II.

Association of PFS and hazard ratios with clinicopathological factors in ACs.

Univariate analysisMultivariate analysis


VariablesTotal, nMedian PFS, months95% CIP-valueHazard ratio95% CIP-value
Gender
  Male5011.3   9.3–13.2   0.130
  Female5815.410.3–20.5
Adenocarcinoma subtypes
  Acinar predominant3230.614.3–46.9   0.0520.400.21–0.750.005a
  MicroP predominant1113.010.6–15.4 0.610.28–1.320.210a
  Mucinous predominant  510.5   9.4–11.6 0.960.37–2.520.940a
  Papillary predominant12   7.04.6–9.2 1.160.55–2.430.700a
  Solid predominant4610.5   7.3–13.6
CRKL expression score
  ≤1003113.6   8.3–18.8   0.7900.780.47–1.310.350
  >1007512.5   9.0–15.9
AXL expression score
  ≤1004812.0   6.3–17.7   0.4401.010.61–1.670.980
  >1005813.511.2–15.8
EGFR statusb
  Mutation5616.511.4–21.6   0.053c
  Wild type5011.0   8.8–13.2
  Exon 18 mutation  514.011.4–23.7 0.690.16–2.960.610d
  Exon 19 mutation2727.815.7–40.0 0.520.28–0.960.037d
  Exon 21 mutation2310.0   7.8–12.2 1.330.70–2.550.390d
ALK status
  Fusion gene1711.0   1.5–20.5   0.0571.450.79–2.640.230
  Non-fusion gene9113.6   8.7–18.3
Smoking status
  Never smoker7414.5   9.0–20.0   0.062
  Smoker or ever smoker3410.5   8.2–12.8
Clinical stage (vs. I+II)e
  I+II4927.318.0–36.5<0.0011.971.38–2.83<0.001
  IIIA4210.0   5.2–14.7
  IIIB+IV17   9.7   1.0–15.7

a Comparison with solid predominant among AC subtypes.

b Since the S768I mutation, which is located in exon 20, was found in one patient, this case was not listed in the table. Therefore, only 55 cases are listed in the table.

c Comparison between EGFR mutation and EGFR wild type.

d Comparison with EGFR wild type among EGFR mutation subtypes.

e A total of 108 cases were originally involved in the present study, of which, the results of 2 cases were not incomplete in detecting the expression of AXL and CRKL. Therefore, these 2 cases were excluded from the study. PFS, progression-free survival; CI, confidence interval; CRKL, Crk-like; AXL, AXL receptor tyrosine kinase; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma receptor tyrosine kinase.

Results

Patient characteristics and histopathological features

A total of 108 cases were originally involved in our study; however, the results of 2 cases were not incomplete in detecting the expression of AXL and CRKL. Therefore, 106 cases were analyzed in the present study, whose clinicopathological features are listed in Table I. Out of the 106 patients, AC was slightly more common in females (53.7%; 57/106). The median age was 55 years in females (range, 23–73 years) and 62 years in males (range, 33–79 years). All samples were diagnosed as invasive ACs and 82 (77.4%) of these samples were mixed ACs, which were classified into five histological subtypes based on their predominant components: 32 acinar ACs (30.19%), including 3 samples with cribriform pattern; 12 papillary ACs (11.32%); 5 mucinous carcinomas (4.72%); 11 micropapillary ACs (10.38%); and 46 solid ACs (43.40%). In total, 32.4% of patients did not have a smoking history. PFS comparisons among histological subtypes showed that acinar-predominant AC had a marginally improved chemotherapeutic response than others (P=0.052; Fig. 2A). Overall, 44.4% of patients were diagnosed at early stage (stage I and II), 39.8% of patients were diagnosed at stage IIIA, and 15.7% of patients were diagnosed at stage IIIB and IV. Conventional regimens of chemotherapy-combined use of carboplatin or cisplatin with Taxol®, gemcitabine, navelbine, docetaxel or pemetrexed were administered to 83 patients. Thymopeptide-5 alone was administered to 23 patients.

EGFR mutation and ALK rearrangement in lung ACs

Mutations of the EGFR kinase domain (exons 18–21) were successfully screened and 58 AC samples (53.7%) were positive for EGFR mutations (Table I), Two main types of EGFR mutation conferring TKI sensitivity are deletion in exon 19 and the missense mutation L858R in exon 21 (28). In the present study, 27 and 24 AC samples harbored deletion in exon 19 (range, K745-S753) and the L858R mutation, respectively. Deletion in exon 19 occurred in 12 acinar (12/27; 44.4%), 2 micropapillary (2/27; 7.4%), 3 papillary (3/27; 11.1%) and 10 solid (10/27; 37%) ACs. L858R mutation was found in 12 acinar ACs (12/24; 50%), consisting of one acinar with cribriform pattern, 1 micropapillary (1/24; 4.2%), 1 mucinous (1/24; 4.2%), 1 papillary (1/24; 4.2%) and 9 solid ACs (9/24; 37.5%). Deletion in exon 19 and L858R mutations were mainly harbored in acinar and solid-type ACs. Other less common mutations, such as G719A/S and S768I, were found in 6 patients.

Ventana IHC autostaining is a valid and convenient method for testing ALK fusion protein detection and the validity of Ventana IHC has been verified by FISH (Fig. 1E) (29). In total, 17 ACs (17/106; 16.0%) were determined to be ALK fusion positive by Ventana IHC. In the current study, ACs with ALK rearrangement consisted of 2 micropapillary-predominant (2/11; 18.2%), 1 mucinous-predominant (1/5; 20.0%), 1 papillary-predominant (1/12; 8.3%) and 11 solid-predominant (11/46; 23.9%) tumors, particularly with cytoplasmic mucin. All these tissues stained positive for ALK rearrangement in a uniform pattern, despite the different morphological subtypes (Fig. 1E). In addition, 2/3 ACs with cribriform pattern were ALK rearrangement-positive. EGFR mutation and ALK rearrangement did not coexist in any AC subtype in the present study.

Effect of CRKL and AXL expression with different EGFR and ALK statuses on the prognosis of AC subtypes

Frequency crosstables were used to compare the staining intensity of CRKL and AXL with clinicopathological features (Table I; Fig. 1A-D). CRKL and AXL expression was not significantly associated with clinicopathological features, such as gender, AC subtypes, tumor size, ALK rearrangement and clinical stage (Table I). In total, 81 patients (81/106; 76.4%) experienced elapsed or progression during the follow-up period and 25 patients were censored, since they did not experience progressive disease or were lost to follow-up. The mean clinical follow-up period was 14.9 months (range, 1–48 months). In total, 50 and 58 patients were diagnosed at an early and advanced stage of disease, respectively. It was found that PFS time was not associated with chemotherapeutic regimens (P=0.17; Fig. 2B). However, the prognosis was significantly different between the two groups, with a median PFS time of 24.6 months (95% CI, 15.6–33.6 months) in the early-stage group and 10 months in the advanced group (95% CI, 5.8–14.1 months; HR, 2.83; 95% CI, 1.76–4.5; P<0.0001). The median PFS time of patients with acinar-predominant AC was 30.6 months, which was significantly longer than the micropapillary (HR, 2.9; 95% CI, 1.1–7.6; P=0.03), mucinous (HR, 2.73; 95% CI, 1.1–18.2; P=0.04), papillary (HR, 2.31; 95% CI, 1.1–7.3; P=0.016) and solid-predominant subtypes of AC (HR, 2.16; 95% CI, 1.2–3.5; P=0.009) (Table II). It is known that clinical staging is an important factor that affects the prognosis of patients with lung cancer. Subsequently, the PFS time of different subtypes were stratified by clinical staging and compared; it was found that only acinar AC had a longer PFS time than papillary ACs at an early stage of disease, but prognostic advantage was revealed at an advanced stage of disease (data not shown; P=0.025). The PFS time (16.5 months) of the 56 patients with EGFR mutation was increased compared with the PFS time of patients with wild-type EGFR (11 months; P=0.052). The mutation types were compared with the PFS of patients to elucidate the effect of EGFR mutation types on prognosis and found that they really played roles in prognosis of AC patients (P=0.01; Fig. 2C). It was also found that patients with exon 19 mutation (median PFS time, 27.8 months) had a strikingly improved PFS time compared with patients with L858R mutation (median PFS time, 10 months; HR, 2.5; 95% CI, 1.47–5.86; P=0.003) and wild-type (median PFS time, 11 months; HR, 2.2; 95% CI, 1.29–3.64; P=0.004) (Fig. 2C).

The median PFS time of 17 patients with the ALK fusion gene was 11 months (95% CI, 1.5–20.5 months), whereas the PFS time of the patients with wild-type ALK was 13 months (95% CI, 8.7–18.3 months). The prognosis of patients with wild-type ALK was better than the prognosis of patients with the ALK fusion gene (P=0.057).

No correlation was identified between CRKL and AXL expression and the clinical factors, such as gender, AC subtypes, tumor size, clinical stage, smoking history and ALK status. Despite this finding, the diverse staining resulted in the consideration of the components of ACs. Different staining patterns were correlated with the components of AC. In total, 82 AC lesions (77.4%) were composed of ≥2 variant histological components, and the most frequent combination was solid and acinar patterns (26/106; 24.5%). Other mixtures consisted of solid, papillary, micropapillary and lepidic components of varied proportions. In addition, 46.2 and 24.5% ACs was observed with discrepant expression within components, respectively. In addition, 56 ACs (52.8%) with EGFR mutation had an increased level of AXL expression compared with ACs with wild-type EGFR (P=0.019). In total, 31 and 48 cases were determined with low expression of CRKL and AXL, respectively. A low level of AXL expression was detected in 48/106 ACs (45.3%) and exon 19 deletion was detected in 40.7% (11/27), L858R in 30.4% (7/23) and wild-type EGFR in 58% (29/50) of ACs. PFS comparison revealed that ACs with exon 19 deletion within the group with low AXL expression had a longer PFS time (median PFS time, 28.8 months) than those with the L858R mutation (median PFS time, 9.1 months; HR, 6.04; 95% CI, 6.15–117.7; P<0.0001) and wild-type EGFR (median PFS time, 11 months; HR, 2.88; 95% CI, 1.26–5.38; P=0.012) (Fig. 3A). There was no significant difference in PFS time among the AC subtypes with high AXL expression (P>0.05; Fig. 3B). Among the AC subtypes with low CRKL expression, the median PFS time of patients with exon 19 deletion (median PFS time, 19 months) was not significantly longer than the median PFS time of patients with the L858R mutation (median PFS time, 13 months) and wild-types EGFR (median PFS time, 9.97 months) (P=0.29; Fig. 3C). In contrast, for the 75 ACs with an increased level of CRKL expression, the 20 patients (20/75; 26.7%) with exon 19 deletion had a better PFS time (28.8 months) than the 14 patients (14/75; 18.7%) with the L858R mutation (9.1 months; HR, 2.79; 95% CI, 1.1–7.1; P=0.03) and all 50 patients (50/106; 47.2%) without EGFR mutation (11.3 months; HR, 2.49; 95% CI, 1.33–4.67; P=0.0046) (Fig. 3D).

Discussion

Overall, ~80% of lung ACs are categorized as mixed subtype according to the 2004 WHO classification (2). It has been proposed that a semiquantitative assessment of the percentages of various histological components, such as acinar, papillary, micropapillary, lepidic and solid, should be performed to classify tumors according to the predominant components (30). It is crucial to adopt a practical way to address tumors that are comprised of a complex heterogeneous mixture of histological subtypes, since 70–90% of surgically resected lung tumors are diagnosed as invasive ACs (25). In previous years, multiple independent research groups have classified lung ACs according to the most predominant subtypes (3034). Prominent diverse structures in morphology and heterogeneity in the biology of ACs have been considered in an increasing number of studies following the establishment of the new classification (3,35,36). The present study was commenced subsequent to a review of all the tissue sections, and the diagnoses were renewed based on the new classification. Studies on the topic of micropapillary AC have reported that patients in an early stage of disease have a poor prognosis (32,37). It has recently been shown that micropapillary tumors also have a poor prognosis, similar to that of ACs, with a predominantly solid subtype (38). Patients with papillary or acinar predominance or invasive mucinous AC show similar overall survival (OS). Patients with solid predominance and micropapillary predominance show the worst OS (6). In the present study, 106 AC patients were administrated with conventional chemotherapy, and the PFS times among different regimens were not significantly different (P>0.05). However, the prognosis of different AC histological mixtures was analyzed, and it was found that acinar-predominant AC had a longer PFS time (30.6 months) than other AC subtypes, despite the status of the EGFR, ALK, CRKL and AXL genes. A previous study reports that patients with micropapillary AC have a worse prognosis than patients with mucinous, solid and colloid AC (38). In the present cohort, the clinicopathological findings were analyzed by univariate Kaplan-Meier test and Cox regression analyses stratified with histological subtypes, EGFR and ALK status. The multivariate model showed that AC subtypes and EGFR mutation subtypes were independent factors that affected PFS time in addition to clinical stage. Thereafter, AC subtypes and EGFR status were compared with AXL and CRKL expression. Patients with ACs with exon 19 deletion (PFS time, 27.8 months) demonstrated an improved prognosis compared with patients with ACs with L858R mutation (PFS time, 10 months) and wild-type EGFR (PFS time, 11 months). A previous study investigating 44 patients with lung cancer has reported that the overall response rate to concurrent chemoradiotherapy is significantly increased in the EGFR mutant group compared with the wild-type EGFR group, and local regional relapse occurs less frequently in patients with EGFR mutation compared with patients with wild-type EGFR (39). It is well known that EGFR mutation is most common in ACs in the eastern Asian population, never-smokers and non-mucinous tumors (4042). Lung cancer-associated EGFR mutations are clustered within the tyrosine kinase domain. In-frame exon 19 deletions occur just downstream of a lysine residue at position 745 (K745), which is critical for binding adenosine triphosphate. Absence of a few amino acids located C-terminal to this lysine residue may affect the configuration of the EGFR catalytic site (42). The L858R mutation occurs adjacent to the highly conserved DFG motif in the activation loop region of the kinase (43). Theoretically, these mutations may all result in conformational changes that lead to increased activity and TKI sensitivity (28,44). The actual mechanism of mutant EGFR tumors with target therapy has yet to be elucidated. The present results indicated that activating EGFR mutations, particularly in-frame deletions of exon 19, is more likely to be associated with clinical significance and it is necessary to consider the EGFR status of ACs. The EGFR status of ACs determined the response of the tumors to conventional chemotherapy.

ALK rearrangement results in fusion genes, such as EML4-ALK, KIF5B-ALK and KLC1-ALK (12,45,46). Detection of ALK rearrangement by FISH or RT-PCR is considered to be the standard procedure, but each method possesses limitations. The FISH method, which is based on a break-apart probe, has the limitation that it cannot determine the specific form of translocation, whereas RT-PCR cannot quantify the tumor cells with the ALK fusion gene. IHC using specific antibodies corresponds well with detecting the activating ALK fusion protein, and it may serve as a useful screening method with quantitation and quality outcome (15,16,46,47). The ALK fusion gene defines a distinct molecular subset of NSCLC, in particular AC, which benefits from treatment with ALK-inhibitors. Robust and reliable laboratory tests for predictive biomarkers are critical to select appropriate patients for targeted therapy. Patients with improved performance status and EML4-ALK translocation have an increased overall survival time compared with patients treated with conventional chemotherapy (17,48). There is no significant difference in clinical factors and survival outcome between the patients harboring variant 1 and those harboring non-variant 1 EML4-ALK fusion genes (49). The incidence of 17 ACs (16.0%) possessing ALK rearrangement in the present study was substantially more frequent than that in young male patients in Western countries (5.6%, 20/358) (15). No prognostic advantage of ALK translocation was demonstrated in the present study and the ALK fusion incidence was distributed regardless of histological predilection, which is consistent with other reports of EML4-ALK rearrangement (15,50,51). Improved prognosis was also identified in the patients without ALK rearrangement in the present study. We suspect that this discrepancy with other reports may be caused by the constituents resulting from the ‘acinar’, which is likely to lead to a better outcome than other histological subtypes. Additional stratified analysis is required to illuminate the prognostic difference of AC subtypes.

AXL is a transmembrane receptor tyrosine kinase whose overexpression has been reported in several human cancers. In addition, Gas6-AXL signaling promotes cell proliferation and survival, angiogenesis, and invasion and metastasis through activation of the PI3K-AKT-mTOR and RAS-RAF-MEK-MAPK pathways (52). EGFR-mutant lung cancer models in vitro and in vivo with increased activation of AXL have been shown to possess acquired resistance to erlotinib without T790 M alteration. In lung AC, AXL expression levels are associated with tumor advancement and the survival of patients with adjuvant chemotherapy, thus rendering AXL expression as a reliable biomarker and potential target for treatment of lung AC (53). In the present study, the role of EGFR activating mutation in the prognosis of AC patients was confirmed. Additional stratified pairwise comparisons were performed to elucidate the association between AXL and EGFR activating mutation types. An increased level of AXL expression was more evident in ACs with EGFR mutation. Investigation of the role of AXL in patient prognosis revealed an improved PFS time in patients with low expression of AXL and exon 19 mutation, compared to patients with higher expression of AXL and wild-type EGFR. The AXL/Gas6 system remains an attractive therapeutic target (54), and certain small molecules with AXL inhibitory effects are already under development (55).

CRKL expression is associated with enhanced cancer cell proliferation and invasion (18,19). Overexpression of CRKL is found in NSCLC and is associated with poor tumor differentiation, AC, advanced p-TNM stage, high proliferation index and poor overall survival. In addition, overexpression of CRKL in cell lines promotes cell proliferation by facilitating cell cycle progression (56). In the current study, the CRKL protein level was not demonstrated to be significantly associated with clinical features such as gender, smoking history, clinical staging, EGFR status and ALK status. However, overexpression or amplification of CRKL and activation of AXL is reported associated with the resistance to TKI (9,12,23,57). The original CRKL expression level in AC subtypes with various EGFR mutations remains limited; therefore, the comparison was performed in the present study to investigate the effect of CRKL expression concomitant with EGFR activated mutation on the prognosis of AC patients. Patients with high CRKL expression level and exon 19 mutation had an improved prognosis compared with other patients with a low CRKL expression level and other EGFR status. This indicates that overexpression of CRKL may confer an improved response to conventional chemotherapy in the ACs with exon 19 mutation. To address the diverse expression of AXL and CRKL, the understanding of the exceptional histological and biological heterogeneity of lung carcinoma may be improved if the stem cell theory of cancer development is considered. According to these evolving views, neoplastic cells derive from abnormal progenitors that retain the potential to give rise to a diverse progeny, depending on the large variety of molecular abnormalities affecting the neoplastic clones. Therefore, neoplastic cells produce a large variety of carcinoma subtypes (12,5863). The unique features of these abnormal precursors may determine the phenotype of neoplastic populations, organized in a hierarchy with various properties and degrees of differentiation (64). Numerous studies have focused on the pathological features of cases harboring EGFR mutations to evaluate the predictive significance of morphological characterization, but the available data are partly discordant (65,66). In a recent study in which the histological-genetic correlations of 100 ACs (94% mixed subtype) were analyzed, the strongest molecular correlation was observed between the EGFR mutation and the papillary subtype (30), as previously suggested (33). However, this contrasted with studies claiming that EGFR mutations mainly occur in the non-mucinous BAC and BAC AC mixed histological subtypes (6769). In addition, previous studies investigating the morphological features of ACs that respond and do not respond to TKI therapies confirm that certain histological differences exist (7072). However, there is morphological overlap, and WHO criteria may be considered a confounding factor. Following consideration of these limitations, the use of pure histology as a predictor for targeted EGFR inhibitory therapy in lung ACs is highly controversial. In accordance with the present results, the hypothesis that EGFR mutation does not have a predilection to particular histological subtypes was supported.

In conclusion, due to the incidence of EGFR mutation in the Asian population, it is necessary to consider the EGFR status as an underlying factor in limited therapeutic options. The present findings are considered to contribute to the understanding of CRKL and AXL expression as novel biomarkers and therapeutic targets in lung AC.

Acknowledgements

The present study was supported by the Beijing Foundation for Distinguished Scientists (grant no. 2009D003013000001; Beijing, China) and awards from the Beijing Board of Health (Beijing, China; grant no. QN2009-035).

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November-2016
Volume 12 Issue 5

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Spandidos Publications style
Cai YR, Dong YJ, Wu HB, Yu DP, Zhou LJ, Su D, Zhang L and Chen XJ: Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes. Oncol Lett 12: 3312-3322, 2016
APA
Cai, Y., Dong, Y., Wu, H., Yu, D., Zhou, L., Su, D. ... Chen, X. (2016). Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes. Oncology Letters, 12, 3312-3322. https://doi.org/10.3892/ol.2016.5080
MLA
Cai, Y., Dong, Y., Wu, H., Yu, D., Zhou, L., Su, D., Zhang, L., Chen, X."Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes". Oncology Letters 12.5 (2016): 3312-3322.
Chicago
Cai, Y., Dong, Y., Wu, H., Yu, D., Zhou, L., Su, D., Zhang, L., Chen, X."Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes". Oncology Letters 12, no. 5 (2016): 3312-3322. https://doi.org/10.3892/ol.2016.5080