Journal of Thoracic Oncology:
Bright-Field Dual-Color Chromogenic In Situ Hybridization for Diagnosing Echinoderm Microtubule-Associated Protein-Like 4-Anaplastic Lymphoma Kinase-Positive Lung Adenocarcinomas
Yoshida, Akihiko MD*†; Tsuta, Koji MD, PhD*; Nitta, Hiroaki PhD‡; Hatanaka, Yutaka PhD§; Asamura, Hisao MD∥; Sekine, Ikuo MD, PhD¶; Grogan, Thomas M. MD‡; Fukayama, Masashi MD, PhD†; Shibata, Tatsuhiro MD, PhD#; Furuta, Koh MD, PhD*; Kohno, Takashi PhD**; Tsuda, Hitoshi MD, PhD*
*Department of Pathology and Clinical Laboratories, National Cancer Center Hospital; †Department of Pathology, The University of Tokyo, Tokyo, Japan; ‡Medical Innovation, Ventana Medical Systems, Inc, Tucson, Arizona; §Department of Surgical Pathology, Hokkaido University Hospital, Sapporo, Japan; and ∥Thoracic Surgery Division, ¶Thoracic Oncology Division, #Division of Cancer Genomics, and **Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan.
Disclosure: H.N. and T.M.G. are employed by Ventana Medical Systems, Inc.
Address for correspondence: Koji Tsuta, MD, PhD, Department of Pathology and Clinical Laboratories, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. E-mail: firstname.lastname@example.org
Introduction: A subset of lung cancers harbors an EML4-ALK (echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase) gene fusion, and detecting this subset may hold therapeutic implications. Many prior studies used fluorescence in situ hybridization (FISH) analysis for this detection, but FISH may have disadvantages including signal decay and dark-field examination that may obscure tissue architecture. In this study, we explored the potential of the ALK-break-apart chromogenic in situ hybridization (CISH) method to detect ALK-rearranged lung cancer.
Methods: We examined 15 lung adenocarcinomas with reverse-transcriptase polymerase chain reaction-proven EML4-ALK fusion transcripts and 30 ALK-negative cases. One hundred tumor cells were evaluated by CISH and FISH for each case, and a detailed signal profile was recorded and compared.
Results: CISH preserved tissue architecture and cytomorphology considerably and facilitated the signal evaluation using a routine light microscope. Positive rearrangement signals (splits or isolated 3′ signals) were identified in 13 to 78% (mean ± SD, 41% ± 19%) of tumor cells in the ALK-positive cohort and in 0 to 15% (mean ± SD, 6% ± 4%) of cells in the ALK-negative cohort. The two groups were best separated by a cutoff value of 20%, with a sensitivity of 93% and a specificity of 100%. The only false-negative tumor having only 13% CISH-positive cells displayed predominantly (76%) isolated 5′ signals unaccompanied by 3′ signals. FISH showed largely similar signal profiles, and the results were completely concordant with CISH.
Conclusions: We have successfully introduced CISH for diagnosing EML4-ALK-positive lung adenocarcinoma. This method allows simultaneous visualization of genetics and tumor cytomorphology and facilitates the molecular evaluation and could be applicable in clinical practice to detect lung cancer that may be responsive to ALK inhibitors.
The recent discovery of a fusion gene that joins the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) in a subset (1–5%) of non-small cell lung carcinomas (NSCLCs) has added a novel molecular subtype to the classification scheme for pulmonary neoplasms.1 The EML4-ALK fusion seems to be formed as the result of a small inversion within the short arm of chromosome 2, and the encoded protein, a chimera comprising the N-terminal portion of EML4 and the intracellular catalytic domain of ALK, is dimerized, leading to constitutive activation.2 To date, a number of fusion variants have been identified, and KIF5B was discovered to be another fusion partner of ALK.3 The importance of recognizing this molecular subtype was highlighted by an international phase I/II clinical trial in which the ALK inhibitor crizotinib (PF02341066) yielded encouraging overall response and disease control rates in a cohort of patients with ALK-rearranged NSCLCs.4 Therefore, an accurate and practical assay is urgently needed to detect this molecular subset of lung cancer.
Currently, the methods available for detecting ALK rearrangement are reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH). ALK immunohistochemistry (IHC), which previously yielded low sensitivity,3,5 has recently been modified to yield high detection rates approaching those of RT-PCR and FISH.3,6 RT-PCR is a single direct test to detect EML4-ALK; however, it generally requires good quality RNA and a multiplex system7 because of the wide variations in fusion types and the rare presence of ALK fusion partners other than EML4. Many prior studies thus favored FISH analysis with the ALK break-apart probe as a genetic confirmation,4,8,9 because it is easily applied to formalin-fixed paraffin-embedded tissue, and it covers multiple ALK fusion variants. Nevertheless, FISH is not without limitations, including a requirement for highly specialized equipment, cumbersome manipulation of the fluorescent microscope, inevitable signal decay after storage, and dark-field examination that may obscure tissue architecture and cytomorphology.6 The latter feature is particularly undesirable in testing for lung cancers, because they often assume complex morphologies10 intimately admixed with nonneoplastic cells, and differentiating tumor cells from the nonneoplastic elements may be difficult without architectural/cytoplasmic information.
Chromogenic in situ hybridization (CISH), which was developed to overcome the aforementioned disadvantages of FISH, has been used in the diagnostic pathology of other organ systems with excellent concordance with FISH.11–13 When applied to formalin-fixed paraffin-embedded materials and examined under the routine bright-field microscope, CISH enables detection of specific genetic alterations while preserving tumor architecture and cytomorphology. In this study, we explore the potential of CISH in diagnosing ALK-positive NSCLCs in correlation with the results of multiplex RT-PCR for EML4-ALK and KIF5B-ALK, ALK-break-apart FISH, and a sensitive ALK IHC newly developed at our laboratory.
Additionally, we aim to provide a detailed description of the hybridization signal pattern in the tested cases. In most prior FISH studies, the test results were simply recorded as either positive or negative in relationship to the preset cutoff value4–6,8,14; however, this value varied among laboratories including 5%,14 15%,5,8 and 50%,15 as did the definition of FISH-“positive” cells.4,5,8,14 The number and type of cells to be counted were not mentioned in many reports.5–8,15 ALK-rearranged NSCLCs tend to show complex in situ signal patterns on break-apart probes; therefore, formulating practical diagnostic criteria requires more than just setting a cutoff value, and studies that evaluate actual signal profiles should make reliable interpretation possible. Recently, Camidge et al.,9 inspired by Perner et al.,16 specifically described FISH signal patterns in ALK-positive NSCLCs and took an important step toward formulating objective criteria for the in situ assessment. Unfortunately, their study lacked RT-PCR confirmation or IHC correlation, and the number of ALK-positive cases was understandably small (13 cases), considering the rarity of this entity in the general population. Herein, we add the detailed in situ signal profile of a further 15 cases of RT-PCR-proven ALK-positive tumors, and these data should help us better understand the spectrum of signal patterns and to develop appropriate diagnostic criteria.
MATERIALS AND METHODS
This study was approved by the institutional review board of the National Cancer Center, Tokyo, Japan. Two cohorts of primary lung adenocarcinomas were retrieved from the National Cancer Center archive; all the tumors were surgically resected, and the histological diagnosis was confirmed according to the latest World Health Organization Classification.10 The first cohort (ALK-positive study group) consisted of 15 cases of lung adenocarcinoma (P1–P15), previously confirmed by multiplex RT-PCR (see method later) to harbor EML4-ALK chimeric transcripts. The second cohort (ALK-negative control group) consisted of 30 cases of lung adenocarcinoma (N1–N30), previously shown by multiplex RT-PCR to lack the EML4-ALK and KIF5B-ALK fusion genes. Tissue microarray was constructed using duplicate 2.0 mm tissue cores sampled from two different representative areas of each tumor (Azumaya, Tokyo).
Reverse-Transcriptase Polymerase Chain Reaction
Fresh-frozen tumor tissues from each tumor were powdered using CP02 (Covaris, Woburn, MA) and sonicated using a Covaris S2 (Covaris). Total RNA was extracted using a mirVana RNA Isolation Kit (Ambion, Foster City, CA), and complementary DNA was synthesized using MMTV reverse transcription (Transcriptor First Strand cDNA Synthesis Kit, Roche Diagnostics, Switzerland). For amplification of the ALK fusion genes, a mixture of primers covering potential breakpoints of fusion transcripts (EML4-ALK and KIF5B-ALK) were used as reported previously.17 The multiplex PCR conditions were 95°C for 60 seconds, followed by 50 cycles at 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 60 seconds.
Chromogenic In Situ Hybridization
The ALK break-apart CISH assay was performed on a BenchMark XT (Ventana, Tucson, AZ) automated slide processing system as described previously.18 Briefly, a custom-designed ALK break-apart probe set, based on a previous publication,16 which hybridizes with the neighboring centromeric (5′ probe labeled with digoxigenin) and telomeric (3′ probe labeled with 2,4 dinitrophenyl) sequence of the ALK gene, was cohybridized after pretreatment. The 5′ ALK probe signal was visualized with alkaline phosphatase (AP)-based fast blue detection; the AP was subsequently inactivated with hybridization buffer before the second AP detection step. The 3′ ALK probe signal was visualized with AP-based fast red detection (Figure 1). Tissue sections were counterstained lightly with diluted hematoxylin II, and nonneoplastic lung tissue was used as a negative control. One hundred nonoverlapping tumor cells with hybridization signals were examined for each case with a light microscope (Olympus BX41, Olympus, Tokyo, Japan) under a 60× objective lens without oil immersion, and a detailed signal pattern was recorded for each cell. Cells lacking any hybridization signal were not evaluated. The signal in each cell was categorized into one of the following seven patterns: (1) fused 3′/5′ only; (2) fused 3′/5′ and both isolated 3′ and 5′ (split); (3) both isolated 3′ and 5′ (split) only; (4) fused 3′/5′ and isolated 5′; (5) fused 3′/5′ and isolated 3′; (6) isolated 5′ only; and (7) isolated 3′ only (Figure 2). A fused 3′/5′ signal looked purple or black due to colocalization of red (3′) and blue (5′) signals. A split signal was defined by 5′ and 3′ probes observed at a distance more than 1 time the signal size, and signals separated by less than that was regarded as fused signals and counted as such. The CISH-positive cells were defined as having any split signals or any isolated 3′ (red) signals. In addition, we evaluated separate sets of 50 and 20 tumor cells in a similar manner to determine how the number of assessed cells affects the test performance. To verify that a lone 3′ (red) signal in ALK-rearranged lung cancer should be considered equivalent to a true split,4,19,20 we also evaluated the test performance when limiting the CISH positivity to split signals and excluding the lone 3′ signals.
Fluorescence In Situ Hybridization
FISH used a commercially available break-apart probe for the ALK gene (Vysis LSI ALK Dual Color, Abbott Molecular, Abbott Park, IL) in accordance with the manufacturer's instructions. The 5′ ALK signal was labeled with SpectrumGreen (green), and the 3′ ALK signal was labeled with SpectrumOrange (orange) in this probe design. One hundred nonoverlapping tumor cells with hybridization signals were examined for each case with a fluorescent microscope (Olympus BX50, Olympus, Tokyo, Japan) under a 100× objective lens with oil immersion, and a detailed signal pattern was recorded for each cell. Cells lacking any hybridization signal were not evaluated. The signals were categorized into seven types as in CISH assay. A split signal was defined by 5′ and 3′ probes observed at a distance more than 1 time the signal size, and signals separated by less than that was regarded as fused signals and counted as such. The FISH-positive cells were defined as having any split signals or any isolated 3′ (orange) signals.
Four-micrometer-thick sections were deparaffinized, and heat-induced epitope retrieval was performed with Targeted Retrieval Solution (pH 9.0, Dako, Carpinteria, CA). The slides were treated with 3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity, followed by washing in deionized water for 2 to 3 minutes. The slides were then incubated with primary antibodies against ALK protein (1:40, 5A4; Abcam, Cambridge, UK) for 1 hour at room temperature, and immunoreactions were detected using the Envision-FLEX and LINKER (Dako). The reactions were visualized with 3,3′-diaminobenzidine, followed by counterstaining with hematoxylin. Cytoplasmic staining was regarded as positive; as staining was diffuse in all the tested cases, it was graded only by intensity as weak (1+), moderate (2+), and strong (3+). Appropriate positive and negative controls were used.
Chromogenic In Situ Hybridization
The distinction between neoplastic and nonneoplastic elements was easily made by well-preserved tissue architecture and cytoplasmic characteristics. Detailed hybridization signal profiles are provided in Table 1 and summarized in Figure 3A, and representative preparations are illustrated in Figure 4. In situ signals were of variable types, including fused, split, and isolated patterns, in all the cases examined. Signal gain (up to eight copies) was common and was observed with varying degrees in all cases. For ALK-positive tumors, the proportion of cells with positive CISH signals was in the range of 13 to 78% (mean ± standard deviation [SD], 41% ± 19%), and the stochastic “pseudo-positive” signals for the ALK-negative group were 0 to 15% (mean ± SD, 6% ± 4%). The two cohorts were best separated, albeit narrowly, when the cutoff was set at 20%, with a sensitivity of 93% and a specificity of 100%. The only false-negative case (P4) harbored 13% of CISH-positive cells, and a large number (76%) of tumor cells displayed isolated 5′ (blue) signals without accompanying 3′ (red) signals (Figure 5A). Among the EML4-ALK-positive cases, the proportion of CISH-positive cells did not seem to correlate with clinicopathological parameters, such as age, sex, stage, and tobacco exposure. When isolated 3′ signals (such as those seen in Figures 2E, G) were excluded from the definition of CISH positivity, the test sensitivity decreased to 47%, and eight EML4-ALK-positive cancers were wrongly classified as negative by CISH. The test results based on counting 50 cells were generally in accordance with those based on 100 cells, with the sensitivity and specificity retained at 93% and 100%, respectively. However, the specificity was reduced to 93% when only 20 tumor cells were evaluated (Table 2).
Fluorescence In Situ Hybridization
Detailed hybridization signal profiles are provided in Table 3 and summarized in Figure 3B. For ALK-positive tumors, the proportion of cells with positive FISH signals was in the range of 3 to 72% (mean ± SD, 41% ± 17%), and the stochastic “pseudo-positive” signals for the control group were 1 to 10% (mean ± SD, 5% ± 3%). The two cohorts were best separated, albeit narrowly, when the cutoff was set at approximately 15 to 20%, with a sensitivity of 93% and a specificity of 100%. The only false-negative case (P4) harbored 3% of FISH-positive cells, and a large number (75%) of tumor cells displayed isolated 5′ (green) signals without accompanying 3′ (orange) signals (Figure 5B). Among the EML4-ALK positive cases, the proportion of FISH-positive cells did not seem to correlate with clinicopathological parameters, including age, sex, stage, and tobacco exposure.
Correlation between CISH and FISH
The results of CISH and FISH are compared in Table 4. The test sensitivity and specificity were completely concordant between the two assays. The proposed cutoff value that best separates two cohorts was also similar, approximately 20% in both modalities. Moreover, the two techniques provided a largely similar signal profile in each case. For example, cases showing predominantly isolated 3′ signals with CISH showed a similar pattern with FISH (cases P8, P11, and P12). Similarly, case P4 showed predominantly isolated 5′ signals with both CISH and FISH; this case was false negative for ALK rearrangement by both modalities. Differences in signal patterns were identified in a few cases; this likely reflects the differences in probe design. For example, P14 showed a significant number of split signals with CISH but predominantly isolated 3′ signals with FISH.
Immunohistochemical results are summarized in Tables 1 and 3. All the 15 cases in the EML4-ALK-positive group were positively labeled for ALK (Figure 6). The staining intensity was strong in six cases, moderate in eight cases, and weak in one case. The staining pattern was diffuse and cytoplasmic, and virtually all the cells in the cores were labeled. No significant intratumoral staining heterogeneity was observed; although cells with abundant intracytoplasmic mucus tended to exhibit weaker staining than those without, perhaps because cytoplasmic ALK protein is diminished by a large volume of mucus material. All the 30 cases in the control group were immunonegative. There was no apparent correlation between the proportion of CISH- or FISH-positive cells and the intensity of immunostaining.
This study has produced data suggesting CISH as a promising method to detect ALK rearrangement in EML4-ALK-positive lung adenocarcinomas. The previously reported advantages of CISH over FISH11–13 were confirmed as follows: (1) the morphological indicators of tumor cells were adequately preserved, which greatly facilitated the overall evaluation and (2) CISH signals were easily observed under a routine light microscope without oil immersion. The high (93%) sensitivity and perfect (100%) specificity of CISH are totally concordant with those of FISH and indicates its large potential for future clinical applications.
The difference in the proportion of CISH/FISH-positive cells between the two groups was relatively modest (41% ± 19% versus 6% ± 4% in CISH; 41% ± 17% versus 5% ± 3% in FISH). The results generally agree with the FISH analysis by Camidge et al.,9 who showed a range of 22 to 87% for ALK-positive tumors and up to 11% in ALK-negative tissues. The inherent difficulty associated with in situ hybridization analysis for EML4-ALK-positive lung cancer was previously emphasized6,15,17 and may have resulted partly from this narrow separation. These data highlight the importance of a quantitative approach in the CISH/FISH analysis for diagnosing EML4-ALK-positive NSCLC cases. Determining an appropriate cutoff value is imperative for accurate assessment, and this figure may vary among laboratories (20% in this study and 15% in others) depending on actual test settings. Although the cutoff value was within the range of 10 to 20% in many studies, it is essential that each laboratory performs a validation assay with an RT-PCR-confirmed set of EML4-ALK-positive cases.
Limiting the number of examined cells to 20 reduced the test accuracy, and our data suggested that at least 50 tumor cells should be evaluated to accurately detect ALK status. Sixty was also proposed to be a viable cell number for accurate evaluation.9 We are uncertain whether diagnosis is facilitated if more than 100 cells are examined, especially when dealing with a tumor that shows rearrangement signals in a range on the borderline between positives and negatives (10–20%). In such cases, we suggest that rather than performing exhaustive repeated counting, it is more practical to follow a corroborative approach that takes other data into consideration. In particular, the immunohistochemical method we have developed and described herein showed complete concordance with RT-PCR results and may supplement CISH/FISH analysis in difficult cases. The clinicopathological characteristics of EML4-ALK-positive NSCLCs have also been well documented5,21 and may assist diagnosis in such cases.
The relatively low rate of rearrangement-positive cells in EML4-ALK-positive NSCLCs has been a subject of debate.9,16 Perner et al. first observed the focal (50–100%) nature of the FISH-positive cells in the majority of EML4-ALK-positive NSCLCs and hypothesized that EML4-ALK fusion represents an acquired event developed at a late stage in tumorigenesis, not an oncogenic molecular drive. Camidge et al., in contrast, ascribed this low positivity rate (22% at lowest in their series) primarily to a technical artifact because the intrachromosomal proximity (∼12 Mb) of the EML4 and ALK genes produces only a narrow split that is difficult to resolve by contemporary in situ technology. In addition to confirming the low rate of positive cells (13–78% in CISH and 3–72% in FISH), we demonstrated diffuse ALK immunoreactivity even in cases harboring only a low percentage of in situ hybridization-positive cells. The intensity of immunolabeling seemed unrelated to the proportion of CISH-positive cells. Although a previous study showed expression of wild-type ALK in an EML4-ALK-positive tumor,1 the diffuse cytoplasmic immunoreactivity in this study seems incompatible with wild-type ALK expression because wild-type ALK is expected to show membranous subcellular localization.2 Our IHC data, therefore, suggest diffuse distribution of chimeric ALK protein in EML4-ALK-positive tumors and indicate that the apparently low rate of rearrangement-positive cells by in situ hybridization is a technical artifact, not authentic intratumoral heterogeneity.
We confirmed the widely held view that isolated 3′ hybridization signals should be regarded as positive for ALK rearrangement in the diagnosis of NSCLCs.4,9,19 Disregarding these signals would have misclassified more than half of the EML4-ALK-positive cases in our CISH study. The molecular basis for the loss of the 5′ signal is unclear, but it is believed to represent a deletion spanning a fusion locus.19 Notably, the first reported EML4-ALK-positive lung cancer did not harbor a reciprocal ALK-EML4 gene,1 which seems inconsistent with a proposed simple inversion mechanism and may indicate a coexisting deletion near the fusion point; the in situ data were not provided for that case. Deletion associated with translocation has been described in other tumors such as chronic myelogenous leukemia22 and ERG-rearranged prostate cancer,23 where such a deletion implicates an aggressive clinical course. The biological significance of the loss of 5′ signals in EML4-ALK-positive lung cancer is yet to be determined. In our study, there was no evident behavioral difference between the three cases (P8, P11, and P12) that showed predominant 5′ loss and the remaining 12 ALK-positive cases.
Because the 5′ hybridization signal represents the noncatalytic domain of ALK kinase, isolated 5′ signals without accompanying 3′ signals seem to be irrelevant with respect to EML4-ALK activity and ALK overexpression.9 Lone 5′ signals have thus been disregarded and have not been deemed positive in any prior studies including a clinical trial.4 Notably, the only false-negative case (P4) in our series harbored a large number (76% in CISH and 75% in FISH) of tumor cells with isolated 5′ signals unaccompanied by 3′ signals. This tumor, which harbored a typical variant 1 chimeric EML4-ALK transcript and was strongly immunopositive for ALK, occurred in a young patient with light smoking exposure, and it had a predominantly acinar histology and a focal signet-ring cell component, characteristic of ALK-rearranged adenocarcinoma.5,21 If lone 5′ signals should be considered in isolation as positive evidence of rearrangement without concurrent 3′ signals, then the proportion of CISH-positive cells for the case P4 would be 89% and that for the entire ALK-positive group would range from 28 to 89%. The proportion of CISH-positive cells for the ALK-negative group would range from 1 to 20%, and the two groups would be differentiated from each other with a new cutoff at approximately 25%. Nevertheless, because the molecular basis of the isolated 5′ signal is unknown, and because one ALK-negative case (N9) showed moderate number (23/100) of cells with lone 5′ signals in FISH, we are reluctant to immediately incorporate such an ad hoc approach into routine diagnostics. Further study is needed to elucidate the mechanism of lone 5′ signals and to determine their value in CISH/FISH for diagnosing ALK-positive NSCLCs. We recommend, for the time being, that cases with a predominantly lone 5′ profile should be treated with caution and that the clinical, histological, and immunohistochemical findings should be carefully correlated. We recently encountered another FISH-false-negative case (not included in this series) displaying a similar profile predominated by isolated 5′ signals, and we suspect that this may be a recurring pattern of CISH/FISH false negativity in ALK-rearranged lung cancer.
In conclusion, we have successfully introduced CISH for diagnosing EML4-ALK-positive lung adenocarcinoma. This novel method allows simultaneous visualization of genetics and tumor cytomorphology and markedly facilitates the molecular evaluation. The cutoff value for positivity was best set at ∼20% in our series, but each laboratory should perform a validation series for the clinical application of the in situ technology. ALK IHC may supplement the CISH/FISH analysis for difficult cases. Although the proportion of rearrangement-positive cells may be low in EML4-ALK-positive tumors, the invariable diffuse ALK immunoreactivity of tumor cells suggested that the apparent low rate represents a technical artifact and not true intratumoral genetic heterogeneity. We confirmed that a lone 3′ signal should be treated as equivalent to a split signal and regarded as positive rearrangement. Predominantly isolated 5′ signal may be a recognizable pattern in CISH/FISH false negativity, and this atypical profile warrants further investigation.
Supported, in part, by Grant-in-Aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan (H.T.).
The authors thank Dr. H. Mano for providing us information about the sequence of primers and multiplex PCR condition. They also thank Mses. Sachiko Miura, Chizu Kina, Karin Yokozawa, and Mr. Susumu Wakai for superb technical assistance.
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