Lung cancer remains the most prevalent malignant tumor and the leading cause of cancer related death world-widely (1). Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer (2). Advances in the understanding of the molecular biology of lung cancer have made individual therapy into clinical practice for advanced NSCLC. As an example, patients with EGFR mutation or ALK rearrangement showed dramatic response to EGFR or ALK tyrosine kinase inhibitor (TKI) and have been recommended to receive EGFR-TKI or Crizotinib as first line setting (3-5). Thus, precise detection of these driver genes mutation will be urgently needed in clinical practice.
ALK rearrangement can be detected by different methods including immunohistochemistry (IHC), reverse transcriptase polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH). Among them, FISH was approved as a gold standard by the Food and Drug Administration (FDA) with the Vysis ALK Break Apart FISH Probe Kit. ALK rearrangement could also detected by IHC, an auto-mated the Ventana ALK assay kit with D5F3 antibody (Ventana Medical Systems Inc., Tucson, Ariz) coupled with an ultrasensitive amplification and detection system have been verified in ALK protein detection (6,7). Besides that, RT-PCR is a more sensitive and specific method that can allows the detection of even a few molecules of chimeric ALK transcripts. But the accuracy of RT-PCR-based testing ALK rearrangement depends greatly on the RNA quality of specimens. Furthermore, some unknown fusion partners or breakpoint variants may be missed because the primers in the assay are specifically designed to evaluate these known alterations.
As we know, lung cancer is a heterogeneous tumor with diverse molecular and pathological characteristics. For example, several studies have reported the discrepancy of EGFR mutation status between primary tumor and metastatic lesions from the same patient (8,9). However, it remains unclear whether ALK rearrangement between primary and metastatic lesions showed a similar heterogeneous phenomenon as EGFR mutation.
To address this issue, we compare the ALK rearrangement status between primary lesions and paired metastatic lymph nodes in 101 resected patients with adenocarcinoma and EFGR wildtype through the methods of VENTANA IHC and RT-PCR. Discrepancy of ALK rearrangement between IHC and RT-PCR was further confirmed by FISH.
The enrolled criteria included primary pulmonary adenocarcinoma patients with metastatic lymph node who received surgical resection in Tongji University affiliated Shanghai pulmonary hospital, the primary and paired metastatic lymphatic section containing more than 100 tumor cells and EGFR wild type. From September 2013 to May 2014, a total of 101 patients were collected. The histological type was confirmed according to the criteria of ERS/ATS/IASLC multidisciplinary classification of lung adenocarcinoma in our routine practice (10). All patients had signed an informed consent for further molecular analysis.
ALK protein expression
Representative formalin-fixed, paraffin-embedded blocks containing the most tumor cells from primary tumor and paired metastatic lymph node were chosen for this current study by two pathologists. Ten serial tissue sections of each block were prepared to detect ALK rearrangement (two tissue slides for IHC, one for FISH and the rest for RT-PCR). Immunohistochemical staining was performed on a VENTANA BenchMark XT automated slide-processing system at Ventana Medical Systems. Four-µm-thick FFPE tissue sections were deparaffinized with EZ Prep solution and antigen-retrieval with cell conditioning 1 (64 minutes at 100 °C) was performed. Primary antibody (clone D5F3, VMSI) was incubated on tissue sections for 20 minutes. According to the manufacturer’s protocol, OptiView DAB IHC Detection Kit (VMSI) and OptiView Amplification Kit (VMSI) were used. Slides were counterstained with Hematoxylin II and then dehydrated. Tissue slides were protected with coverslips. All immunohistochemical stains were evaluated independently by two pathologists. Specimens were scored positive if strong granular cytoplasmic brown staining in tumor cells (excluding nontumor cells: alveolar macrophages, cells of neural origin, glandular epithelial staining), the absence of strong granular cytoplasmic staining was classified a negative result as described previously (11).
ALK reverse transcriptase-polymerase chain reaction (RT-PCR) detection
The total RNA from tissue sections containing at least 70% tumor cells was extracted and converted to first-strand cDNA according to the manufacturer’s instructions (Amoy Diagnostics, Xiamen, China). This AmoyDx EML4-ALK Fusion Gene Detection kit is designed to qualitatively detect 21 EML4-ALK fusion transcripts in four reaction mixtures (including reference gene beta-actin) with a declared sensitivity of 1% mutant DNA. Real-time PCR was performed on in a 7500 real-time PCR system and analyzed by SDS 2.0 system software (Applied Biosystems, Foster City, CA). As defined by the manufacturer’s instructions, cycle threshold (CT) values ≤30 of the samples detection was interpreted a positive result as described previously (11-13).
ALK fluorescence in situ hybridization (FISH) analysis
When ALK rearrangement was discordant between IHC and RT-PCR, FISH evaluation was further performed according to the guidelines of the LSI ALK Dual Color, Break-apart Rearrangement Probe kit (Abbott Molecular, Abbott Park, Illinois), Briefly, for FISH, tissue sections were baked at 60 °C and deparaffinized, and then pretreated with protease solution at 37 °C for 20 min. A thermoBrite (Abbott Molecular, IL, USA) was used for the denaturation step at 73 °C followed by hybridization at 37 °C overnight for 18 hours. After stringency wash steps, nuclear counterstaining with DAPI was performed. Sections were observed under a ×100 objective with a fluorescence microscope (Leica DM6000, Wetzlar, Germany) equipped with “Applied Imaging 4.0” analysis software (Genetix, England, UK) independently by two experienced pathologists. The positive tumor cells show split signals (at least two signals diameters apart) or a single red signal (deleted green signal) in addition to fused and/or split signals. If the average percentage of positive cells was ≥15% (≥15/100), the sample was considered to be positive.
Differences in demographic characteristics including age, gender, smoking history, histological subtype and TNM stage between ALK+ and ALK− patients were analyzed using pearson’ chi-square test or Fisher’s exact test. Two-sided P values <0.05 was considered statistically significant. Statistical calculations were performed using SPSS17.0 software (SPSS Inc., Chicago, IL, USA).
Clinicopathological features of the enrolled patients
One hundred and one patients were enrolled into this study with a median age of 60 years old (range, 35–78 years). Among them, 50 (49.5%) were females, 61 (60.4%) were non-smokers and 20 patients (20/101, 19.8%) were found to have ALK fusion in either of the paired samples. The patients with ALK fusion were more likely in younger (P<0.001), Nonsmokers (P=0.012), high-stage disease (P=0.021) and predominantly solid tumor growth pattern (P=0.024). The clinicopathological characteristics are summarized in Table 1.
The concordance rate of ALK rearrangement between primary tumor and paired metastatic lymph nodes by immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR)
A total of 101 paired specimens (primary tumor site and paired metastatic lymph nodes) have to be confirmed as ALK rearranged. Of 101 paired specimens detected by IHC and RT-PCR, 81 paired specimens were both IHC and RT-PCR negative, 18 paired specimens were both IHC and RT-PCR positive (as shown in Table 2). Sixty-eight cases showed more than two stations of metastatic lymph nodes. ALK rearrangement was totally concordant between different stations of metastatic lymph nodes in the same patient (Table S1). The concordance rate of ALK rearrangement between primary tumors and paired metastatic lymph nodes was 98.0%.
The discordance of ALK rearrangement between primary tumor and metastatic lymph nodes may derive from the intra-tumoral heterogeneity of primary lesions
Two patients were confirmed with discordance of ALK rearrangement between primary tumors and paired metastatic lymph nodes requiring verification by a third methodology, FISH. One patient (Case #15) was IHC, RT-PCR and FISH positive in primary tumor, triples negative in paired metastatic lymph nodes (Figures 1,2). The other (Case #21) who was IHC and FISH positive in both primary tumor and paired metastatic lymph nodes was RT-PCR positive in primary tumor and negative in paired metastatic lymph nodes (Table S2). By IHC analysis of discordant Cases, We found that most of tumor cells were stained with strong granular cytoplasm in some areas of primary tumor section. In other areas of the same section, some tumor cells were negative in anti-ALK (D5F3) staining. ALK rearrangement showed intra-tumoral heterogeneity in different areas of the same tissue section (Figure 3). For case #21, RT-PCR was enough sensitive to detect the ALK rearrangement in the fewer fraction of tumor cells in metastatic lymph nodes, in which most of the metastatic tumor cells didn’t contain ALK rearrangement by IHC and FISH. Two patients were both female, non-smokers and were diagnosed as adenocarcinoma with predominant solid growth pattern (as shown in Table 3).
This study demonstrates a high concordance rate (98%) in the ALK rearrangement between primary tumors and paired metastatic lymph nodes. Two patients with ALK rearrangement on primary tumor didn’t show ALK gene fusion on paired metastatic lymph nodes. In addition, ALK rearrangement was totally 100% concordant between different stations of metastatic lymph nodes in the same patient by IHC, RT-PCR or FISH.
EML4-ALK rearrangement was reported as a relatively low frequent rearrangement ranging from 1.5% to 6.7% among unselected Caucasian NSCLC patients (14-17). It seems to have higher frequency in Asian population, and was reported as high as 5.1–10% (18-21). Part of the explanation was that in those studies, patients were selected from EGFR wild population. Our previous study has shown ALK rearrangement could be as high as 32.3% among patients who have never smoked, and had adenocarcinoma harboring wild-type EGFR (13). In line with these results, we found the incidence of ALK rearrangement was 19.8% in patients with wild-type EGFR. The subgroup analysis showed that ALK rearrangement was more likely to occur in patients who were younger, never smokers and with adenocarcinoma histology, all of which were consistent with previous studies (22-24).
Intratumor heterogeneity has been described recently and poses a significant challenge to personalized cancer medicine. It was reported that activating EGFR mutations were found to display intratumor heterogeneity and spatial discordance in 6.3–30% of cases in lung cancer (25,26). However, intratumor heterogeneity of ALK rearrangement was rarely evaluated. Rossi et al. firstly reported that the discrepancy of ALK rearrangement in a heavy smoking 54-year-old man with pulmonary adenocarcinoma. This patient showed ALK rearrangement negative on primary lesion while harboring ALK fusion in metastatic peritoneal biopsy specimen 8 month later (27). In the present study, the primary and paired samples were collected simultaneously after surgical resection. Unlike EGFR mutation, our result showed a high concordance between primary tumors and paired metastatic lymph nodes. What’s more, Two patients with discordant ALK rearrangement showed an ALK fusion positive in primary tumor while not in paired metastatic lymph nodes. Recent study reported that ALK rearrangement was discordant among spatially separated tumor areas in the same primary tumor of ALK positive patient by RT-PCR. It therefore seems reasonable to infer that metastatic tumor cells may come from the ALK negative tumor cells in primary lesion because of the genetic intratumoral heterogeneity (28). Different clones and innate tumor heterogeneity of samples may attribute to the discrepancy (29-31).
Previous studies reported that IHC assay was a valuable tool for the screening of patients with ALK rearrangement in clinical practice (7,32,33), IHC with new antibody and modified protocols will extend its serviceable range for detecting ALK rearrangement (34). Although FISH was approved by Food and Drug Administration as a companion diagnostics, FISH required specialized equipment and highly trained and skilled readers for interpretation because of variability between inter-observers. However, surgical samples were only available in minority patients with NSCLC. Several studies investigated the feasibility of cytological sample obtaining from minimally invasive procedures like EBUS-TBNA or CT-guided fine needle biopsy for molecular testing (35-40). A substantial part of patients had insufficient tumor cell number for FISH testing after successful differential diagnosis for NSCLC subtype by IHC marker. RT-PCR is a sensitive method for detecting ALK rearrangement and had an advantage to utility of cytological samples. Thus, RT-PCR is also regarded as an acceptable method to detect ALK rearrangement by Chinese FDA (41,42). Our study also found that FISH, Ventana IHC and RT-PCR have good concordance though one case showed different results. The efficacy of ALK inhibitors detected by RT-PCR need be further evaluated in clinical trials or clinical practice (43).
Several limitations have to be mentioned in this study. Firstly, all the enrolled patients were resected, thus the finding from this study may not represent the whole population. Secondly, most of the patients are still not received the treatment of ALK inhibitor, thus we could not observe the response to ALK inhibitor from the patients who showed a discordant result on primary and metastatic lymph nodes. Thirdly, the samples of this study are still small though enrolling more than 100 patients. Similar results were observed in previous study which showed ALK arrangement was heterogeneous in 67 paired NSCLC specimens between primary tumors and their corresponding metastatic lesions by FISH (44), Thus, large scale study with the data of efficacy to ALK inhibitor is warranted to further support the findings in this study.
In conclusion, we have demonstrated that high concordant rate of ALK rearrangement between primary tumors and paired metastatic lymph nodes through three different detection methods. Though ALK rearrangement was found positive in the primary tumor while not in metastatic lymph nodes in the two discordant patients, primary tumor and metastatic lesions were equally suitable to perform ALK rearrangement testing for therapeutic strategies in patients with advanced NSCLC.
Funding: This work was supported in part by grants from the National Science Foundation of China (No. 81201707), projects of the Science and Technology Commission of Shanghai Municipality (No. 12ZR1426000), Outstanding Young Doctor Program of Shanghai Municipal Commission of Health and Family Planning (No. XYQ2013097) and Shanghai Municipal Commission of Health and Family Planning (No. 201440398) and the Shanghai Science and Technique Committee (No. 134119a3200).
Conflicts of Interest: The authors have no conflicts of interest to declare.
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