Journal of Thoracic Oncology:
Detection of ALK Rearrangement by Immunohistochemistry in Lung Adenocarcinoma and the Identification of a Novel EML4-ALK Variant
To, Ka-Fai MBChB*‡§; Tong, Joanna HM PhD*‡§; Yeung, King SF BSc*‡§; Lung, Raymond WM PhD*‡§; Law, Peggy PY MPhil*‡§; Chau, Shuk Ling MPhil*‡§; Kang, Wei PhD*‡§; Tong, Carol YK MPhil*‡; Chow, Chit PhD*‡; Chan, Anthony WH*; Leung, Linda KS MBBS†‡; Mok, Tony SK MBBS†‡
Departments of *Anatomical and Cellular Pathology, †Clinical Oncology; ‡State Key Laboratory in Oncology in South China; §Li Ka-Shing Institute of Health Sciences, the Chinese University of Hong Kong, Shatin, Hong Kong.
The first two authors contributed equally to this work.
Disclosure: The authors declare no conflict of interest.
Address for correspondence: Ka-Fai To, MBChB, FRCPA, FHKCPath, FHKAM(Path), The Chinese University of Hong Kong, Anatomical and Cellular Pathology, Prince of Wales Hospital, Shatin, Hong Kong. E-mail: firstname.lastname@example.org
Introduction: The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as a potent oncogenic driver in non–small-cell lung cancer, in particular adenocarcinoma (ADC). It defines a unique subgroup of lung ADC, which may be responsive to ALK inhibitors. Detection of ALK rearrangement by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) is considered to be the standard procedure, but each with its own limitation. We evaluated the practical usefulness of immunohistochemistry (IHC) to detect ALK expression as a reliable detection method of ALK rearrangement in lung ADC.
Methods: We tested 373 lung ADCs for ALK rearrangement by IHC and FISH. Multiplex RT-PCR was performed to confirm the fusion variants.
Results: Twenty-two of 373 lung ACs (5.9%) were positive for ALK immunoreactivity. ALK-positive tumor cells demonstrated strong and diffused granular staining in the cytoplasm. All the ALK IHC-positive cases were confirmed to harbor ALK rearrangement, either by FISH, or RT-PCR. Two cases with positive ALK protein expression, but negative for breakapart FISH signal were shown to harbor EML4-ALK variant 1 by RT-PCR. None of the ALK IHC-negative cases were FISH-positive. In addition, we identified a novel EML4-ALK fusion variant (E3:ins53A20), and its potent transformation potential has been confirmed by in vivo tumorigenicity assay.
Conclusion: IHC can effectively detect ALK rearrangement in lung cancer. It might provide a reliable and cost-effective diagnostic approach in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy.
Anaplastic lymphoma kinase (ALK) was first identified as a fusion partner in t(2;5) chromosomal translocation associated with anaplastic large-cell lymphomas.1,2 Increasing evidence support the oncogenic role of ALK in both hematopoietic and nonhematopoietic tumors, including neuroblastoma, inflammatory myofibroblastic tumor, and non–small-cell lung cancer (NSCLC)3–6 by activation mutations or chromosomal translocation. Fusion of ALK with a variety of partner genes results in the expression of oncogenic chimeric proteins that lead to constitutive activation of the ALK kinase domain and the downstream signaling pathways. In lung cancer, a novel gene fusion of ALK and the echinoderm microtubule-associated protein-like 4 (EML4) was identified by in vitro transformation assays.6 Several EML4-ALK variants, resulting from fusion of various EML4 exons to ALK have been reported; all were transforming in vitro.7,8 Subsequent studies confirmed the presence of EML4-ALK fusion in 2% to 7% of NSCLC.8–13 Other uncommon fusion partners for ALK, that is, KIF5B, TFG, and KLC1, have also been reported in NSCLC.14–16
ALK rearrangement has been demonstrated to be a potent oncogenic driver and a promising therapeutic target in NSCLC.6,17,18 It defines a distinct molecular subset of NSCLC, in particular adenocarcinoma (ADC) that can benefit by the treatment of ALK-inhibitors. Development of robust and reliable laboratory tests for predictive biomarkers is essential to select appropriate patients for targeted therapy. A variety of methods have been adopted for the detection of ALK rearrangement, including fluorescent in situ hybridization (FISH), immunohistochemistry (IHC) and reverse transcriptase polymerase chain reaction (RT-PCR). Currently, FISH analysis is the only approved diagnostic test for ALK rearrangement to detect breakapart signals. However, the apparatuses required for FISH analysis are not always readily available in routine diagnostic laboratories. ALK rearrangement frequently involves intrachromosomal inversion. The subtle changes may be difficult to interpret by FISH analysis sometimes, and have led to false-negative results.19,20 IHC has been considered an alternative to FISH, which can detect ALK rearrangements independent of the fusion partners. The initial attempts were not encouraging, owing to a relatively low ALK protein expression in lung cancer with ALK rearrangement. Several studies using different antibodies and amplification strategies have recently been published.15,20–22 The amplified IHC protocols were highly sensitive but less specific. Therefore, a two-tier system, similar to human epidermal growth factor receptor 2, has been proposed for the evaluation of ALK IHC. Given the fact that ALK protein is not ubiquitously expressed in normal lung tissue and ALK-negative lung cancers, it is thus likely that technical issues, rather than intrinsic biology, explain the false-positive results of ALK IHC in recent published reports. Optimal IHC protocol is warranted for the reliable detection. In the current study, we evaluated routine IHC staining method for the detection of ALK protein and compared it with FISH analysis. RT-PCR was performed to detect ALK fusion variants on the cases positive for ALK rearrangement either by IHC, or FISH analysis. We aimed to assess the sensitivity and specificity of ALK IHC in a cohort of Chinese patients with lung ADC.
MATERIALS AND METHODS
Patients diagnosed with lung ADC and the tumor specimens subjected to epidermal growth factor receptor (EGFR) mutational analysis in Prince of Wales Hospital, Hong Kong, between 2007 and 2010 were included in this study. A total of 373 consecutive cases of lung ADC were retrieved from molecular diagnostic unit, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Hong Kong. The histologic diagnosis was confirmed by pathologist (KFT). The patient demographic data and clinicopathologic characteristics were obtained from medical records. The male to female ratio of the abovementioned lung cancer patients was 1.2/1. The median age of the patients was 65 years (range, 27–94 years). On the basis of the 7th tumor, node, metastasis staging system for lung cancer, 154 patients had stage I diseases, 46 patients had stage II, 86 patients had stage III, and 87 patients had stage IV diseases. The study protocol was approved by the Joint Chinese University of Hong Kong-New Territories East Clinical Research Ethics Committee, Hong Kong.
Formalin-fixed paraffin-embedded (FFPE) archive tissues from 315 cases were arranged in tissue array blocks. Hematoxylin and eosin–stained sections were used to define tumor areas, and three representative 1-mm cores were obtained from each case and inserted to a recipient paraffin block, using a tissue arrayer (Beecher Instruments; Silver Spring, MD). For the remaining 58 cases, only small biopsies or pleural fluid cytologic specimens were available. Because the materials were too scanty for tissue microarray (TMA) construction, whole sections were used for these cases
Different amplification and visualization systems for the IHC detection of ALK protein have been evaluated on a TMA comprising 57 lung ADCs. Polymer Refined Detection Kit (Leica Microsystems GmbH, Wetzlar, Germany) gave a slightly stronger staining intensity and was used for the study cases (please refer to Supplemental Digital Content 1, http://links.lww.com/JTO/A410 for the details of the evaluation). Four-micrometer sections taken from each block were deparaffinized, rehydrated, and rinsed in distilled water. Antigen retrieval was done by using pressure cooker with 1-mM ethylenediaminetetraacetic acid (pH 8.0) for 13 minutes. IHC was performed using monoclonal antibody against ALK (1:100, clone 5A4, Abcam, Cambridge, UK) and Polymer Refined Detection Kit (Leica Microsystems GmbH) on a Bond-max fully automated staining system (Leica Microsystems GmbH). All slides were counterstained with hematoxylin.
The cytoplasmic expression of ALK was assessed by assigning a proportion score and an intensity score.23 The proportion score was according to the proportion of tumor cells with positive cytoplasmic staining (0, none; 1, ≤ 10%; 2, 10%–25%; 3, > 25%–50%; 4, > 50%). The intensity score was assigned for the intensity of positive tumor cells (0, none; 1, weak; 2, moderate; 3, strong). Representative images of the staining intensity are shown in Supplementary Figure 1 (Supplemental Digital Content 1, http://links.lww.com/JTO/A410). The cytoplasmic score of ALK was the product of proportion and intensity scores, ranging from 0 (0 × 0) to 12 (4 × 3). The cytoplasmic expression was categorized into negative (score 0), 1+ (score 1–3), 2+ (score 4–6), and 3+ (score 7–12). The slides were assessed blindly by two investigators (JHMT and AWHC) and the inter-rater reliability was determined by kappa statistics. Consensus was established by jointly reviewing the case whenever there was a discrepancy.
Fluorescent In Situ Hybridization
FISH analysis was performed on the FFPE tumor tissues using a breakapart probe specific to the ALK locus (Vysis LSI ALK Dual Color, breakapart rearrangement probe; Abbott Molecular, Abbott Park, IL) according to the manufacturer’s instruction. In brief, 4-µm-thick sections were deparaffinized, dehydrated, immersed with Vysis pretreatment Solution (Abbott Molecular) at 80°C for 15 minutes, and treated with Protease Solution (Abbott Molecular) at 37°C for 20 minutes. Dual probe hybridization was performed using the LSI ALK dual-color probe, which hybridizes to the 2p23 locus with SpectrumOrange and SpectrumGreen on either side of the ALK gene breakpoint. After application of ALK probe mixture, the slides were denatured at 75°C and then incubated at 37°C overnight to allow hybridization. After washing the slides with Washing Buffer I and II (Abbott Molecular), the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ALK FISH was considered positive when more than 15% of 100 or more analyzed cells showed splitting of the fluorescent probes flanking the ALK locus.
Reverse-Transcriptase Polymerase Chain Reaction
Total RNA from frozen tissue was extracted by TRIzol reagent (Life Technologies, Grand Island, NY). For FFPE samples, RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Life Technologies) was used for RNA extraction. First, strand cDNA was obtained from 1 µg of total RNA, using Superscript III reverse transcriptase (Life Technologies). PCR primers specific for all types of known ALK fusion transcripts, including all variants of EML4-ALK, kinesin family member 5B (KIF5B)-ALK, TRK-fused gene (TFG)-ALK, and kinesin light chain 1 (KLC1)-ALK were designed using Primer3 (version 0.4.0; Appliedbiosystems, Foster City, CA).24 The primer sequences are available on request. The amplified PCR products were subjected to direct sequencing, using ABI PRISM 3130xl DNA Sequencer (Applied Biosystems).
Identification of Genomic Breakpoint
Genomic DNA was extracted from frozen tissue, using QIAamp DNA mini kit (QIAGEN, Valencia, CA). PCR was carried out, using a forward primer at intron 3 of EML4 (5′- ATTGCACTGTTGCTTGTTGC-3′) and a reverse primer at intron 19 of ALK (5′- GTACACTGCAGGTGGGTGGT-3′), with the PCR SuperMix High Fidelity (Invitrogen, Grand Island, NY). The genomic breakpoint was confirmed by Sanger sequencing.
Cloning and Transfection
Full-length EML4-ALK new variant (E3;ins53A20) was amplified from tumor tissue by RT-PCR and inserted into pcDNA3.1(+) (Invitrogen) using standard cloning protocol. NIH3T3 cells were maintained in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum. The plasmids were transfected into NIH3T3 cells using FuGENE HD (Roche, Mannheim, Germany). Expression of the chimeric protein was confirmed by Western blot analysis, as described previously, using polyclonal rabbit anti-ALK antibody (ZAL4, 1:5000, Invitrogen).25
In Vivo Tumorigenic Assay
NIH3T3 cells (1 × 106 cells suspended in 0.1-ml phosphate-buffered saline) transfected with EML4-ALK (E3;ins53A20) expression vector or empty vector were injected subcutaneously into the dorsal flank of five 4-week-old male Balb/c nude mice (EML4–ALK-expressing clones on the right and vector control clones on the left). Tumor formation was examined after 3 weeks. The experiment was repeated thrice. The animal handling and all experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.
Statistical analysis of 2 × 2 contingency tables of categorical variables was performed using the χ2 test or Fisher’s exact test, as appropriate. The t test was performed to compare continuous variables between two groups. All statistical analyses were carried out by using statistical program SPSS (version 16.0; SPSS, Armonk, NY). A two-tailed p value of less than 0.05 was regarded as statistically significant.
Detection of ALK Rearrangement by IHC, FISH, and RT-PCR
We studied ALK rearrangement in a cohort of 373 patients with lung ADC, using IHC and FISH. ALK protein expression was detected in 22 of 373 lung ADC patients (5.9%). Twenty of the ALK-positive tumors were identified from the TMA and two were identified on the whole-tissue section. In ALK IHC-positive cases, the tumor cells demonstrated diffused cytoplasmic staining with moderate to strong intensity (Fig. 1). The ALK IHC-positive tumors showed a rather uniform staining pattern, with immunoreactivity in more than 90% tumor cells, and a proportional score of four. The intensity score in those ALK IHC-positive cases were either 2 (moderate), or 3 (strong). Combining the intensity score and proportion score, all 22 cases were categorized as 3+ (Table 1). There was an excellent inter-rater agreement between two investigators when comparing categorized IHC scores: for score 0 versus score 1+ to 3+, κ = 0.911; for score 0 and 1+ versus score 2+ and 3+, κ = 1. No ALK immunoreactivity was observed in normal alveolar or bronchial epithelium, endothelial cells, smooth muscle cells, or stromal cells. The finding was consistent with the biological fact that ALK protein is not expressed in normal lung tissue.6
FISH analysis for ALK rearrangement was successful in 351 cases (94.1%). The success rate was 96.6% (56 of 58) for whole sections and 93.7% (295 of 315) for tissue arrays. Of them, 20 cases demonstrated breakapart signals that indicated chromosomal rearrangements. The FISH-positive cases detected in tissue arrays were further validated using the corresponding whole-tissue sections. A representative case was shown in Figure 2A. Suboptimal tissue fixation, which could result in background autofluorescence, may contribute to the failure in FISH analysis.26 Notably, all 20 FISH-positive cases showed positive ALK protein expression as determined by IHC (Table 1). In two lung ADCs with strong and diffuse ALK immunoreactivity, only fusion signals or close proximity of the red and green signals were observed in tumor cells, despite repeated testing (Fig. 2B). We then repeated FISH analysis using additional tumor blocks. It was again negative for breakapart signals in both cases. These two cases were considered negative for FISH breakapart testing, which was defined by 15% or more split nuclei as indicative of an ALK rearrangement.
In addition to IHC and FISH, RT-PCR has been suggested as a least subjective methodology to detect ALK rearrangements and to determine the specific fusion variants. A multiplex RT-PCR system was designed to capture all reported in-frame fusions of EML4-ALK, KIF5B-ALK, TFG-ALK, and KLC1-ALK. Frozen tumor tissues were available in four of 22 ALK IHC-positive cases, whereas only FFPE tissues were available for the remaining 18 cases. We successfully amplified EML4-ALK fusion transcripts from all four frozen tumors and 14 FFPE tumors. These included 10 cases with EML4-ALK variant 1, five cases with EML4-ALK variant 3b, two cases with EML4-ALK variant 2, and a novel EML4-ALK variant, which has not been reported previously.
Fusion transcript was not detected by RT-PCR in four FFPE tumors (Table 1). All were FISH-positive and IHC-positive for ALK rearrangement. The failure to detect ALK fusion transcript in these FFPE tumor specimens might be because of poor RNA quality, or the presence of fusion variant(s) not assayed for by our RT-PCR system. Notably, in two ALK IHC-positive cases without breakapart FISH signal, fusion transcripts were detected by RT-PCR and confirmed to be EML4-ALK variant 1 by direct sequencing. We therefore, concluded that the FISH result was false-negative.
FISH is the current method of choice in clinical practice. If the sensitivity was calculated using FISH as standard procedure, IHC had a sensitivity of 100%. The two IHC-positive but FISH-negative cases were also proven to harbor EML4-ALK fusion transcripts by RT-PCR, suggesting that IHC is even more sensitive than FISH in detecting ALK translocation. Moreover, all the IHC-positive cases could be confirmed as harboring ALK translocation either by FISH, or RT-PCR, indicating that ALK IHC is highly specific. In summary, we detected ALK protein expression in 22 of 373 lung ADCs (5.9%). All these tumors were confirmed to harbor ALK rearrangement either by FISH breakapart signal or by RT-PCR. False-negative results were found in two cases by FISH. We demonstrated a sensitive IHC-based assay, which showed benefit over FISH analysis.
Clinicopathologic Characteristics of Lung ADC with ALK Rearrangement
Twenty-two ALK-positive patients, comprising 11 men and 11 women, were included for study. None of the patients had exposure to ALK inhibitor, crizotinib. ALK rearrangement was found in eight of 157 smokers (5.1%) as compared with 11 of 176 nonsmokers (6.25%). There was no significant difference between tumor from smokers and nonsmokers with regard to ALK status (p = 0.65). Patients with ALK-positive lung ADCs were significantly younger than those without (p < 0.0001; 52.9 ± 16.1 years versus 64.5 ± 11.5 years). Histologically, the ALK-positive tumors displayed mixed growth patterns, including solid with mucin, papillary, acinia, and lepidic patterns. Signet ring cell component was observed in 10 of 22 ALK-positive tumors (45.5%). Of them, four tumors showed more than 10% signet ring cells. Within the ALK-positive cohort, eight patients (36.4%) had stage I disease, four had stage II (18.2%), three had stage III (13.6%), and seven had stage IV (31.8%) diseases. Among the 373 cases, 159 cases (42.6%) harbored EGFR mutation and 214 cases (57.4%) were EGFR wild type (wt). No EGFR mutation was identified in ALK-positive tumors (p < 0.0001). A total of 192 cases were negative for both EGFR and ALK (designated wt/wt). There was no significant difference in tumor size, positive lymph node, distant metastasis, and pathologic stage between the ALK-positive and ALK-negative groups (Table 2).
The follow-up data were available in 364 patients. At the time of evaluation, 211 patients (58%) were still alive, whereas 153 (42%) had died. The median follow-up time was 52.0 months. There was no significant difference in overall survival (OS) times between the ALK-positive and ALK-negative groups (median OS 55.8 ± 4.2 and 50.3 ± 4.1 months, respectively, p = 0.255; Supplementary Figure 2, Supplemental Digital Content 1, http://links.lww.com/JTO/A410). When further stratifying the ALK-negative group, the median OS was 66.3 ± 4.7 months for EGFR-positive patients and 46.2 ± 5.3 months for wt/wt (double negative: ALK-negative/EGFR-negative) patients. Although there was a trend toward longer OS time in ALK-positive patients compared with wt/wt patients, the difference was not statistically significant (p = 0.076; Supplementary Fig. 3, Supplemental Digital Content 1, http://links.lww.com/JTO/A410). In patients with stage IV diseases, median OS was 19.9 ± 8.1 months for ALK-positive group, compared with 25.1 ± 3.3 months for EGFR-positive group (p = 0.958), and 16.1 ± 3.0 months for wt/wt group (p = 0.253; Supplementary Figure 4, Supplemental Digital Content 1, http://links.lww.com/JTO/A410). We defined recurrence and metastasis as disease progression events for early-stage patients (stage I and II). Therefore, the median time to progression was 52.9 ± 16.6 months for early-stage ALK-positive patients, compared with 39.7 ± 9.6 months for EGFR-positive patients (p = 0.775), and 36.2 ± 11.7 months for wt/wt patients (p = 0.248; Supplementary Figure 5, Supplemental Digital Content 1, http://links.lww.com/JTO/A410).
Identification of a Novel EML4-ALK Variant
A novel EML4-ALK fusion variant was detected in a lung ADC with ALK rearrangement. The tumor showed strong ALK protein expression and was also positive for ALK breakapart signal by FISH analysis. RT-PCR, using the primer set designed for EML4-ALK variant 5 (fusion between EML4 exon 2 and ALK exon 20), yielded a PCR band larger than the expected product size. Direct sequencing of the PCR product revealed a fusion transcript, resulting from the connection of EML4 exon 3 to a position 53 base pair (bp) upstream of ALK exon 20 (Fig. 3). PCR direct sequencing of genomic DNA also revealed that EML4 was disrupted at a position 2241 bp (Chr 2, NT 022184.15 21307898) downstream of exon 3 and was ligated to a position 60 bp (Chr 2, NT 022184.15, 8268341) upstream of exon 20 of ALK. The novel variant was designated E3;ins53A20 provisionally. The full-length sequence has been submitted to GenBank (accession number JQ828841). Because the frozen tumor tissue of this case was available, we amplified the full-length cDNA of variant E3;ins53A20 from tumor and cloned into pcDNA3.1(+) expression vector. The transforming potential of variant E3;ins53A20 was investigated by in vivo tumorigenicity assay. NIH 3T3 mouse embryo fibroblast cells were transfected with variant E3;ins53A20 expression vectors and injected subcutaneously into nude mice. Only the variant E3;ins53A20-expressing clones formed tumors but not the mock-transfected clones (p = 0.0026; Fig. 3), confirming the potent transforming ability of variant E3;ins53A20.
To date, multiple ALK fusion genes have been identified in human cancer, including the fusion of ALK with NPM,1 TFG,14 CLTC,27 ATIC,28 CARS,29 MSN,30 TPM3,31 EML4,17 KIF5B,15 KLC1,16 and C2orf44.32 In lung cancer, the primary ALK fusion partners were reported to be EML4, followed by KIF5, TFG, and KLC1. The fusion point of ALK is conserved among most of the chimeric proteins, resulting in the fusion of the entire intracellular kinase domain of ALK to the corresponding partner. In this study, we reported the detection of a novel EML4-ALK variant (E3;ins53A20) in a lung ADC, with strong ALK protein expression and breakapart FISH signals. The novel variant involved an inframe fusion between EML4 exon 3 and 53 bp upstream of ALK exon 20, comprising the coiled-coil domain of EML4, and the juxtamembrane intracellular region of ALK, including the entire tyrosine kinase domain. The EML4 portion of this novel variant comprised only the basic coiled-coil domain, which is always preserved in all reported EML4-ALK fusion variants. Its constitutive expression in lung is believed to play an essential role in the dimerization and activation of downstream oncogenic effects of EML4-ALK isoforms.6 The potent transformation potential of variant E3;ins53A20 has been confirmed by in vivo tumorigenicity assay. It is likely to play an important role in the tumorigenesis of lung cancer. The reported EML4-ALK fusion genes comprised variable truncations of EML4 (occurring at exons 2, 6, 13, 14, 15, 18, and 20), and the kinase domain of ALK and all demonstrated gain of function properties. Identification of E3;ins53A20 as an alternative EML4-ALK variant further consolidates the role of ALK signaling in lung cancers.
After the recent discovery of ALK rearrangement in lung cancer, ALK inhibition has been emerging as a promising targeted therapy for this subset of patients. Identification of appropriate patient population is the key to the overall success of such targeted therapy. A variety of methods can be used for the detection of ALK rearrangements, including FISH, IHC, and RT-PCR. Each method, inevitably, has its own advantages and disadvantages. FISH has been adopted as the method of choice for patient selection in clinical trials. The breakapart probe allows detection of rearrangements, independent of the fusion partners or specific breakpoint. However, FISH requires specialized laboratory techniques, expert interpretation, relatively high costs, and a longer turnaround time. The current criteria, for a positive FISH result, that has been set for clinical trial is that more than 15% of the tumor cells must show split red and green signals, separated by at least two signal diameters, or a single red signal.18,33
An alternative to FISH is IHC. Similar to the FISH assay, IHC can detect ALK independent of the fusion partner. However, researchers found it difficult to reliably detect EML4-ALK protein expression in NSCLC. The detection threshold of IHC depends on the tissue preparation, affinity of the antibody, the sensitivity of detection system used, the scoring system, and experience of the scorer. Unlike the strong expression of ALK protein in anaplastic large-cell lymphoma, which can be readily detected by IHC, ALK antibodies seem to give variable results in lung cancer. The early studies, using commercial ALK antibodies and standard protocol, showed that IHC was specific but not sensitive for the detection of lung cancer with ALK rearrangement.12,34 The low sensitivity may be attributed to the low level of EML4 transcriptional activity or to instability of EML4-ALK in cells.6 A variety of signal amplification technologies have been developed to improve the sensitivity of ALK protein detection in lung cancer, including tyramide amplification19 and intercalation of an antibody-enhanced polymer.15 Using the highly sensitive detection methods in combination with high affinity antibodies, for example, 5A4 and D5F3, IHC can effectively detect ALK fusion protein in lung ADCs with high sensitivity and specificity.20,34,35 Multiplex RT-PCR system has been the usual screening strategy applied for ALK gene rearrangements,6,7,36,37 and has been considered the least subjective. The presence of fusion transcripts as detected by RT-PCR provides direct evidence of chromosomal translocation. However, the requirement of high-quality RNA makes it difficult to implement in a routine clinical diagnostic laboratory, using FFPE samples. Furthermore, RT-PCR can only detect fusion transcripts with known fusion partners.
Given that IHC is a routine methodology in most pathology laboratories to detect a protein of interest, it is desirable to establish a sensitive and accurate detection method for ALK fusion protein based on IHC. In this study, we reported the immunohistochemical detection of ALK protein expression in 22 of 373 lung ADCs. The staining was considered specific as all ALK IHC-positive cases were confirmed to harbor ALK rearrangement either by FISH or by RT-PCR analysis. Two-tier systems for the evaluation of ALK status with initial IHC screening followed by FISH analysis of IHC score 2+, or both 1+ and 2+ cases have been proposed.21,22 However, we did not observe a background or nonspecific staining using our staining protocol, consistent with the biological fact that ALK protein is not constitutively expressed in normal lung tissue. The positive cases showed diffuse and strong immunoreactivity that were scored as 3+ according to the combined intensity and proportion scoring criteria. The strong immunoreactivity in ALK-positive cases would be extremely helpful in avoiding interobserver variation in the assessment of ALK IHC status.
It has been reported that some of the ALK IHC-positive tumors that had been confirmed by RT-PCR showed no breakapart FISH signals.19,34 In our cases series, two cases demonstrated false-negative FISH results, despite repeat testing. The EML4-ALK chimeric proteins are the result of inversions within the short arm of chromosome 2, involving 2p21 and 2p23, approximately 12 megabases apart. Because the EML4 and ALK loci are mapped relatively close on chromosome 2p, the subtle changes in fluorescent signal, caused by intrachromosomal inversion in some positive cases, might be difficult to interpret and might lead to false-negative results. The limited probe separation in such cases reduces the sensitivity of FISH assay. Peled et al.38 reported a crizotinib-sensitive NSCLC with complex ALK rearrangement that was negative by FISH analysis. Next-generation sequencing analysis of the tumor DNA revealed multiple breakpoints and complex rearrangement at genomic level, which was not detected by breakapart FISH assay. Nevertheless, high concordant rates between FISH and IHC have been reported, using high affinity antibodies and sensitive visualization systems.
In conclusion, we report that IHC is a sensitive and specific detection method to detect ALK rearrangement in lung cancer. IHC would be served as an effective and rapid detection method in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. In addition, we reported a novel oncogenic EML4-ALK variant (E3;ins53A20). Our series demonstrated that some ALK IHC-positive but FISH-negative lung cancers did harbor the translocation events as confirmed by RT-PCR. Thus, this subgroup of patients should also benefit from ALK inhibitory therapy. Further clinical trials are required to address the predictive value of ALK IHC in these patients.
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Lung adenocarcinoma; Anaplastic lymphoma kinase; Fusion transcript; Echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase; Immunohistochemistry; Fluorescence in situ hybridization; Immunohistochemistry
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