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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0000000000000068
Original Articles

Detection of Rearrangements and Transcriptional Up-Regulation of ALK in FFPE Lung Cancer Specimens Using a Novel, Sensitive, Quantitative Reverse Transcription Polymerase Chain Reaction Assay

Gruber, Kim MSc*; Horn, Heike PhD*; Kalla, Jörg MD*; Fritz, Peter MD*; Rosenwald, Andreas MD; Kohlhäufl, Martin MD; Friedel, Godehard MD; Schwab, Matthias MD§; Ott, German MD*; Kalla, Claudia PhD*

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Author Information

*Department of Clinical Pathology, Robert-Bosch-Krankenhaus, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Germany; Institute of Pathology, University of Würzburg, Würzburg, Germany; Center for Pulmonology and Thoracic Surgery, Klinik Schillerhöhe, Stuttgart-Gerlingen, Germany; and § Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and Department of Clinical Pharmacology, University Hospital, Tübingen, Germany.

Drs Ott and Kalla are senior coauthors of this article.

Current address of Dr. Kalla: Institute of Pathology, Schwarzwald-Baar-Klinikum, Villingen-Schwenningen, Germany.

Disclosure: The study was supported by the Robert Bosch Foundation, Stuttgart, Germany (to KG, HH, JK, MS, GO, and CK) and by the Federal Ministry for Education and Research (BMBF, Berlin, Germany) grant # 03 IS 2061C (to MS). The other authors declare no conflict of interest.

Address for correspondence: Claudia Kalla, PhD, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany. E-mail: claudia.kalla@ikp-stuttgart.de

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Abstract

Introduction: The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK.

Methods: We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry.

Results: qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding.

Conclusions: Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

Recently, considerable progress has been made in understanding the molecular mechanisms of lung cancer pathogenesis. This especially pertains to the development of novel treatment strategies. In particular, the identification of aberrantly activated tyrosine kinases (e.g., epidermal growth factor receptor [EGFR]) in subsets of non–small-cell lung cancer (NSCLC) together with the development of specific kinase inhibitors, has proven to be a successful approach to target individual tumors.

A subset of NSCLC harbors rearrangements of the anaplastic lymphoma receptor tyrosine kinase (ALK) gene, often with the echinoderm microtubule associated protein like 4 (EML4) gene because of a small inversion within chromosome 2p,1,2 and more rarely with other fusion partners such as KIF5B3 and TFG.1 These rearrangements lead to the expression of a chimeric tyrosine kinase in which the ALK kinase domain is fused to the N-terminal part of the fusion partner. The tyrosine kinase inhibitor crizotinib has shown dramatic clinical responses in patients and transgenic mice with ALK-rearranged tumors.46 The prevalence of ALK rearrangements, however, is low, ranging from 3% to 6% in adenocarcinomas.7,8 Therefore, efficient patient selection may be a crucial issue in clinical practice. In the search for predictive characteristics, we found that parameters such as younger age, never/light smoking history, and adenocarcinoma histology with signet-ring cells were found to be associated with EML4-ALK gene fusions.9,10 However, fusion genes were also observed in older patients with smoking history.9 Therefore, selection of patients by clinical and structural characteristics is inappropriate, and molecular screening would clearly be desirable to identify the subset of ALK-positive tumors.

Currently, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) are used to detect ALK rearrangements.11 Although each method has its specific strengths, there is no perfect method for the detection of ALK gene alterations especially in formalin-fixed, paraffin-embedded (FFPE) tissue samples in routine pathology practice. FISH is the gold standard technique that, however, requires specialized technical equipment and expertise and is a low-throughput approach. Immunostaining of the ALK protein detects ALK rearrangements reliably in cases with substantial protein expression; however, the interpretation of the result is often challenging because of a weak and variable immunoreactivity. RT-PCR amplification of specific ALK fusion transcripts is very sensitive and can identify the fusion partner, but is rather complex because of the large number of fusion variants and cannot detect rearrangements with hitherto unknown partners. Very recently, three assays were published that detect unbalanced ALK transcript expression. All three methods showed excellent sensitivity and specificity for the detection of ALK rearrangements, but are not adapted to poor-quality RNA characteristic of FFPE specimens12 or are based on exon array or NanoString’s (Seattle, WA) nCounter technology, which is not available in most pathology laboratories.13,14 Therefore, those new approaches are difficult to exploit in daily diagnostic practice.

The aim of the present study, therefore, was to develop a robust diagnostic assay for the detection of ALK alterations in FFPE samples based on quantitative RT-PCR (qRT-PCR). This approach was applied to a large cohort of routine NSCLC specimens to determine (1) the reliability of the technique to detect ALK rearrangements in comparison with FISH and IHC and (2) the prevalence of transcriptional up-regulation of ALK in NSCLC.

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MATERIALS AND METHODS

Specimen Collection

In this study we included FFPE tissue biopsies or resection specimens from 652 patients diagnosed with NSCLC that had been classified according to the World Health Organization classification for lung tumors.15 Basic medical records of the patients were reviewed to obtain clinical information (age, sex, smoking status). The study was approved by the Ethics Committee of the University Hospital, Eberhard-Karls-Universität Tübingen (Germany), and all sampling was performed according to respective protocols. FFPE samples from one anaplastic large-cell lymphoma, the NSCLC cell line H2228 (CRL-5935; ATCC, Rockville, MD), and the rhabdomyosarcoma cell line RH30 (DSMZ, Braunschweig, Germany) were used as positive controls for the expression of nucleophosmin (NPM)-ALK EML4-ALK, and full-length ALK transcripts, respectively.

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RNA and DNA Preparation

Total RNA and genomic DNA were isolated from FFPE tissue samples by using the AllPrep DNA/RNA FFPE Kit as described by the manufacturer (Qiagen, Hilden, Germany). This extraction method was optimized by the manufacturer to reverse formaldehyde modification without further RNA degradation and turned out to be the most efficient one for isolating nucleic acids of sufficient quality for PCR amplification when tested in our laboratory. Total RNA and genomic DNA were isolated from cell lines by using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions.

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Quantitative RT-PCR

Total RNA (1 μg for resection specimens, large biopsies, or control samples; 0.1–1 μg for small biopsies) was reverse transcribed using random hexamers and the SuperScript-First Strand Synthesis System for RT-PCR (Life Technologies, Darmstadt, Germany). Primers for qRT-PCR targeting the 5` and the 3` portions of the ALK transcript (exons 4/5 and 24/25) were designed based on published sequence data (NM_004304). As reference gene, phosphoglycerate kinase 1 (PGK1) was used, which had been proven to represent a suitable internal control for normalizing NSCLC samples (data not shown). Primer sequences are shown in Supplementary Table 1 (Supplemental Digital Content 1, http://links.lww.com/JTO/A511). qRT-PCR was carried out in an ABI PRISM 7700 Sequence Detector by using the SYBR Green PCR kit (Life Technologies). Complementary DNA (50 ng for NSCLC samples, 10 ng for cell lines) was subjected to qRT-PCR reactions containing 750 nM primer in a total volume of 25 μl. The cycling conditions were as follows: two initial incubations of 2 minutes at 50°C and 10 minutes at 95°C, then 40 cycles of 30 seconds at 95°C, and 1 minute at 60°C. Each sample was analyzed at least in duplicate (analysis was repeated in rare cases with discordant data). For data analysis, the threshold fluorescence was set at 0.045. NSCLC samples were excluded from the analysis if PGK1 amplification resulted in a fluorescence threshold (Ct) value above 31. For ALK, we defined Ct=40 as our limit of detection, and Ct values beyond this limit were defined as negative and set to 40 for further calculations. ALK expression was calculated by applying ΔΔCt: For each sample, ALK expression was normalized to PGK1 and calculated relative to the average value in cell line RH30, which was arbitrarily defined as 1. The cutoff value for altered ALK expression was 0.3 for both amplicons, corresponding to the mean value of ALK in 50 NSCLC cases with wild-type ALK (as confirmed by FISH) plus 3 SDs.

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DNA Sequence Analysis

Direct sequencing of PCR products was performed by cycle sequencing with ABI PRISM BigDye Terminator chemistry followed by electrophoresis on an ABI-3100 automated sequencer (Life Technologies). ALK primer sequences are shown in Supplementary Table 1 (Supplemental Digital Content 1, http://links.lww.com/JTO/A511). EGFR sequence analysis was performed as reported.16,17

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Fluorescence In Situ Hybridization

FISH was done on 4-μm thick FFPE tissue sections according to published protocols.18 Experiments were evaluated using an epifluorescence microscope (Leica DMRA, Wetzlar, Germany). Images were captured using an Isis workstation (Version 5.4; Metasystems, Altlussheim, Germany). ALK rearrangements were analyzed using a breakapart probe specific for the ALK locus (ZytoLight SPEC ALK/EML4 TriCheckTM Probe; Zytomed Systems, Berlin, Germany). Signal evaluation was performed on at least 100 nuclei. Cases were regarded as FISH-positive if (1) separated green and orange signals (translocation) or (2) single orange signals (translocation and deletion of the ALK 5` portion) were identified in at least 15% of tumor cells analyzed.

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Immunohistochemistry

IHC was performed on 2-μm thick FFPE tissue sections using the Bond Automated Immunohistochemistry & In-Situ Hybridisation System (Menarini Diagnostics, Berlin, Germany). After heat-induced epitope retrieval with 1 mM EDTA (pH 9.0) rabbit monoclonal antihuman ALK antibody (1:100; D5F3; New England Biolabs, Frankfurt, Germany) was incubated for 30 minutes, followed by washing and detection according to the manufacturer’s protocol. ALK IHC was scored as follows: 0, no staining; 1+, faint or weak; 2+, moderate; 3+, strong staining intensity in at least 10% tumor cells. IHC, FISH, and qRT-PCR evaluations were performed in a blinded fashion.

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Statistical Analysis

For all calculations, Analyse-it software (Leeds, United Kingdom) for Microsoft Excel was used.

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RESULTS

Establishment of the ALK qRT-PCR Assay

The ALK rearrangement leads to the overexpression of the 3` portion of ALK encoding the kinase domain (exons 20–29) whereas the 5` portion (exons 1–19) remains unexpressed. On the basis of this we constructed a qRT-PCR assay consisting of two amplicons targeting the 5` and the 3` portions of the ALK transcript separately (Fig. 1). Small amplicons (66–83 base pairs in size) were designed to overcome the technical problems associated with the use of fragmented FFPE RNA to arrive at efficient amplification and detection of ALK cDNA. Furthermore, we used an RNA isolation method that had been optimized by the manufacturer to reverse formaldehyde modification without further RNA degradation.

Figure 1
Figure 1
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The amplification efficiency and theoretical sensitivity of the ALK qRT-PCT assay was validated by fourfold dilution series of RH30 cDNA in ddH2O (Supplementary Figure 1 [Supplemental Digital Content 2, http://links.lww.com/JTO/A512] shows representative standard curves). For both amplicons the Ct values correlated with the ALK mRNA input over at least three orders of magnitude (linear regression: p= 0.0001).

The reliability of the ALK assay was assessed by studying a series of FFPE samples with known ALK status as determined by FISH: one t(2;5) positive lymphoma and two NSCLC cases (#10, #17) with ALK translocations as well as 10 NSCLC cases with wild-type ALK (#35 to #44). For the expression of EML4-ALK and full-length ALK transcripts, cell lines H2228 and RH30 were used as positive controls, respectively. Unbalanced ALK transcript expression was seen in all ALK translocated cases, all of them showing a significant expression of the 3` exons whereas the 5` exons were not expressed. In all wild-type cases, neither 5` nor 3` exons were expressed (Fig. 1).

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Screening for ALK Rearrangements by qRT-PCR Assay and Correlation with FISH

To validate the ALK qRT-PCR assay in a larger series of FFPE diagnostic specimens, bronchoscopic biopsies or resection specimens (irrespective of size) from 652 NSCLC patients were screened for ALK rearrangements. In all cases, the tumor cell content was at least 10%. Five hundred twenty-three cases (80%) were interpretable, that is, with RNA of sufficient quality obtained as indicated by efficient amplification of the control gene PGK1.

In 24 of 523 tumors (4.6%) an expression of the ALK 3` portion with values above the cutoff level of 0.3 was observed (median value: 1.76, range, 0.32–7.10) whereas the 5` portion was not expressed (Table 1, Fig. 1B). Six tumors (1.1%) showed a significant expression of the full-length ALK transcript (Table 1, Fig. 1B). In those cases, the expression of the 3` part ranged from 0.42 to 0.96 (median: 0.61) and of the 5` part from 0.66 to 1.85 (median: 1.12). ALK was not expressed or at only very low levels (medium value: 0.05; range, 0.001–0.29) in 493 samples (94.3%; Fig. 1B).

Table 1
Table 1
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To relate ALK expression to ALK gene rearrangements, 198 samples were subjected to FISH analysis (tests performed in a blinded fashion). ALK breakapart FISH was interpretable in 92% (182 of 198) tumors and in 22 of the 24 tumors with unbalanced ALK expression. Of the latter, all 19 cases with 3` expression values greater than 0.4 were confirmed to be ALK rearranged. Of note, in one case (# 8) FISH analysis revealed a translocation accompanied by a partial deletion of the 3` part of ALK as indicated by separated green and very small orange signals. The critical question whether the crucial target region was still present in this case was answered by qRT-PCR demonstrating a significant expression of the 3` ALK portion (value 1.30). Three cases had expression levels between 0.3 and 0.4 but showed a wild-type genomic constellation by FISH (Table 1). According to these observations, a cutoff value of 0.4 was considered a relevant threshold for qRT-PCR rather than the mathematical cutoff value of 0.3 originally applied. Of 154 qRT-PCR–negative tumors with interpretable FISH, 152 were confirmed negative by FISH, whereas two cases with an unbalanced, yet low expression of the 3` portion (# 20: 0.22; # 21: 0.15) were found positive on FISH. The six tumors with expression of the full-length transcript were all negative for genomic ALK rearrangements (Fig. 2). In four of them, additional ALK gene copies (3–4 gene copies, occasionally polysomic nuclei) were detected in more than 25% of the tumor cells. A comparable genomic constellation, however, was observed in about one third of the 152 FISH-negative cases without detectable expression of ALK. Our qRT-PCR assay, thus, accurately typed 97% (177 of 182) of tumors analyzed by FISH in parallel and strongly suggested rearrangements of ALK in three tumors with insufficient or ambiguous FISH analysis (data not shown). In addition, qRT-PCR was able to identify cases with full-length ALK overexpression not detectable by FISH.

Figure 2
Figure 2
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Influence of Sample Size and Tumor Cell Content on ALK Expression

Positive qRT-PCR results were obtained for resection specimens and for bronchoscopic biopsies, with the smallest specimens being 0.1 cm2 in size (Table 1). The relative expression of the 3` ALK portion was independent of the sample size. The tumor cell content of cases with significant ALK transcript expression ranged from 10% to 90%. Although high expression values (3` portion expression >1.0) were only observed in samples with a tumor cell content of more than 50%, the expression did not strictly correlate with tumor content (Pearson’s correlation, p= 0.21), and ALK mRNA was reliably detectable in four cases with 10% to 30% tumor cells (expression values 0.49–0.86).

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ALK Protein Expression in ALK-Rearranged versus ALK–Wild-Type Tumors

ALK protein expression was assessed in 250 samples by immunostaining. The D5F3 antibody detected ALK protein overexpression in all 21 samples harboring an ALK rearrangement verified by FISH (Table 1, Fig. 2). Four ALK-rearranged adenocarcinomas showed strong staining of the tumor cells for the protein, and another 10 cases were moderately positive. Seven cases altogether showed weak and often focal staining. Expression levels of the 3` portion of the ALK transcript did not necessarily correlate with the intensity of the immunohistochemical staining (Pearson’s correlation, p= 0.20; Supplementary Figure 2 [Supplemental Digital Content 3, http://links.lww.com/JTO/A513]). Of five samples with up-regulated full-length transcription analyzed, weak ALK protein expression was detected in four samples (#22, #23, #25, #27), whereas ALK protein was not detectable in one case (#24) (Table 1, Fig. 2). Of importance, 224 ALK wild-type tumors showed no staining with the ALK antibody.

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Mutation Analysis of the Kinase Region

In neuroblastoma, expression of full-length ALK transcripts is frequently associated with mutations in the kinase domain.19 Therefore, we examined this region (exons 20–29) for mutations in all six cases with full-length ALK expression. In case #25, a heterozygous nucleotide substitution was detected (3659C>A), which results in the substitution of serine by tyrosine within the central part of the kinase domain (S1220Y) (Fig. 3). This nucleotide change was also present in DNA extracted from tumor-free lung tissue from the patient, and was thus of germline origin; the expression of ALK transcripts, however, was restricted to tumor cells (Fig. 3).

Figure 3
Figure 3
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Clinico-Pathological Characteristics of ALK-Positive Lung Cancers

Patients with ALK rearrangements had a median age of 59 years; about half of them (48%) were 60 years old or younger. Two patients were aged 75 years or older. The majority of patients with ALK-rearranged tumors were women (74%). We did not observe any prevalence of the ALK translocation in nonsmoking versus smoking women (8 versus 7 patients; Table 1), whereas four of five male patients with proven ALK translocation had been nonsmokers. In contrast, three of six patients with tumors expressing the full-length transcript were male smokers. The median age of patients with ALK-expressing tumors was 59 years (range, 42–82) (Table 1).

In our series, ALK rearrangement did not correlate with tumor grading nor with TNM status. ALK translocations were not restricted to advanced cancers, but were also detected in pT1 and low-grade tumors (Table 1). The presence of a signet-ring cell component was observed in roughly one third of ALK-rearranged and ALK-expressing tumors (39% and 33%, respectively). There was no clear prevalence of a specific histological subtype among ALK-positive tumors. 34% were classified as predominantly solid and 31% as glandular adenocarcinomas; 24% had a mucinous differentiation (Table 1). None of the 22 ALK-rearranged or full-length ALK–expressing tumors analyzed harbored coexisting EGFR mutations (data not shown).

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DISCUSSION

Rearrangement of ALK results in the expression of the 3` part of the gene fused with EML4 or alternative partners, whereas the ALK 5` portion remains unexpressed. Taking advantage of this feature, we have developed a diagnostic test that reliably detects ALK rearrangement in FFPE tissues by means of recognizing an unbalanced ALK transcript expression independent of the fusion partner. Most notably, the assay also provides information about the expression of intact ALK transcripts. Recent reports have shown the principal potential of such an approach. All these approaches, however, are of limited utility in routine diagnostics using FFPE material. The qRT-PCR assay developed by Wang and colleagues12 detected rearrangements in frozen tissue samples with excellent sensitivity, but had not been adapted to FFPE specimens. Two alternative technologies based on exon array analyses or NanoString’s nCounter technology were highly sensitive for the detection of ALK fusion transcripts, but are not readily available in most pathology laboratories.13,14 In contrast, our approach is based on the qRT-PCR technique that is wellestablished in many routine laboratories. To cope with the poor quality of RNA isolated from routine FFPE specimens we took advantage of (1) an RNA isolation method that was optimized to reverse formaldehyde modification and (2) small RT-PCR amplicons to allow for the use of fragmented nucleic acids for efficient amplification of ALK cDNA.

When applied to a large series of 652 routine NSCLC specimens, the qRT-PCR assay was interpretable in 80% of cases, which is about the range reported for ALK breakapart FISH (60%–90%20,21; 92% in this study), and showed a high sensitivity and specificity for the detection of ALK rearrangements. Compared with FISH, the qRT-PCR accurately typed 97% of the tumors (19 ALK-rearranged and 158 nonrearranged cases) and also strongly suggested rearrangements in three tumors with insufficient or ambiguous FISH results. Three tumors with low qRT-PCR expression (between 0.3 and 0.4) were negative by FISH and IHC. The risk of providing false-positive results would thus be minimized by selecting a cutoff value of 0.4. However, in two FISH-positive cases an unbalanced expression of the 3` portion of ALK was noted, which, however, was below the cutoff level. The number of tumor cells (40%–50%) in those cases, obviously, was not a limiting factor, because unbalanced ALK expression was reliably detected in four specimens with as few as 10% to 30% tumor cells. The low expression encountered might rather be caused by a non–EML4-ALK rearrangement as reported in one case that was positive by FISH and IHC but negative by RT-PCR.20 Whether a low number of fusion transcripts impedes treatment with ALK kinase inhibitors and whether, consequently, its quantification should be part of the diagnostic procedure, warrants further investigation.

Currently, FISH using breakapart probes is the gold standard technique for the diagnosis of ALK rearrangements in lung adenocarcinomas. FISH, however, is a low-throughput approach that requires specialized technical equipment and expertise, especially with respect to the complex signal constellation arising from intrachromosomal inversions combined with deletions in the setting of polysomy typical of lung cancer. qRT-PCR, in contrast, is an inexpensive and rapid technique with easy-to-use protocols. By using a 96-well or 384-well detection system this assay becomes relatively high-throughput, which makes the rapid screening of larger patient cohorts efficient. In addition, small biopsy fragments with low tumor cell content will suffice for the diagnosis: ALK expression was reliably detected in resection specimens and in small biopsies with as few as 10% to 30% tumor cells. Furthermore, using the extraction method used here, it will be possible to isolate simultaneously both RNA and DNA from the same specimens, which opens up the possibility of combining PCR-based molecular testing with DNA mutation analysis and optimizing tissue processing.

Several authors have recommended IHC testing as a prescreening tool.7,21,22 In our study, ALK protein expression was restricted to tumors that harbored ALK rearrangements or overexpressed full-length ALK. This raises the possibility of diagnosing ALK-positive tumors using routine IHC. However, several limitations of the IHC approach have to be considered, such as: (1) Tissue staining for ALK is often weak and focal (in this study in one third of rearranged cases), which requires confirmatory FISH or RT-PCR; (2) Although not observed in this study, false-negative results have been reported for ALK-rearranged cases using IHC23; and (3) the use of ALK antibodies raised against the C-terminal portion of the protein cannot discriminate between the expression of a fusion protein or the full-length ALK protein. This, again, requires ALK protein expressing cases to be re-examined by qRT-PCR (or FISH).

In lung adenocarcinoma, ALK is generally activated by the expression of chimeric proteins containing the ALK kinase domain, whereas in other ALK-positive neoplasms (e.g., neuroblastoma, sarcoma) ALK activation is caused by overexpression of wild-type or mutated transcripts (see ref. 24 for review). Only recently, oncogenic point mutations of ALK have been detected in lung cancer.25–27 In our series of NSCLC, up-regulated expression of nonrearranged transcripts was observed in six cases altogether (1.1%). In four of five tumors analyzed, up-regulated transcription was associated with detectable amounts of ALK protein. In one of those cases, an amino acid substitution within the kinase domain (S1220Y) was identified. The replacement of a serine by a tyrosine residue might interfere with the kinase activity and with the binding of a kinase inhibitor as demonstrated for other mutations in this region.26,28,29 Although this mutation was of germline origin, its expression was restricted to tumor cells. To date, this nucleotide exchange has not been reported (http://www.ncbi.nlm.nih.gov/SNP/), which argues against a polymorphic character but rather points to a genetic predisposition for NSCLC comparable with familial and sporadic cases of neuroblastoma carrying ALK germline mutations.30,31 In our study, DNA sequence analysis focused on the kinase domain, because the majority of activating mutations in neuroblastoma affects this region.32 Oncogenic ALK mutations detected in lung cancer, however, are distributed in different protein domains.25–27 Thus, it is possible that mutations exist outside the kinase domain in the five other cases expressing full-length ALK.

Three lines of evidence indicate that the identification of up-regulated full-length ALK transcripts in lung adenocarcinoma might be of therapeutic relevance: (1) Several ALK inhibitors (crizotinib, CEP-14083, TAE684, CH5424802) inhibit the growth of neuroblastoma cells overexpressing wild-type or mutated full-length ALK in vitro and in xenograft models.33–36 Crizotinib is currently being tested for the treatment of neuroblastoma (NCT00939770, ClinicalTrials.gov); (2) Ectopic overexpression of wild-type or mutated full-length ALK confers a tumorigenic phenotype to NSCLC cells and xenograft tumors, and treatment with different ALK inhibitors (WHI-P154, NVP-TAE684) resulted in the regression of tumors and suppression of metastasis26; (3) Several reports describe NSCLC tumors that were IHC positive but FISH negative,7,9,37–41 which is indicative of up-regulated ALK expression without gene rearrangement. Of pivotal interest, one of those patients was treated with crizotinib and showed a dramatic therapy response.40 To what extent treatment response of lung adenocarcinomas expressing full-length ALK might be dependent on certain mutations or requires a critical threshold of expression (as shown for neuroblastoma)34 will have to be determined in future studies.

The clinical and structural features of the ALK-rearranged cases in our series confirms previous observations that ALK rearrangement tends to be associated with younger age, female sex, and the presence of a signet-ring cell component.9,10,20,22,42 Preanalytical selection of patients using these parameters, however, would have missed as many as 74% of patients with detectable ALK rearrangements, which underlines the necessity for molecular screening. In contrast to other reports,10,13,22,43 an association with advanced disease or smoking behavior of the predominantly female patients was not observed in our series. Thus, our observations are well in line with the proposed guidelines for the selection of lung cancer patients for ALK tyrosine kinase inhibitors, which recommends molecular ALK testing in all patients with advanced-stage adenocarcinoma, regardless of age, sex, or smoking history.44 Most notably, full-length ALK expression was preferentially found in male patients, most of them smokers.

In conclusion, the qRT-PCR approach presented here reliably detects ALK-rearranged tumors independently of the fusion partner and also identifies cases with full-length transcript expression of the gene not detectable by FISH. Thus, qRT-PCR seems to be a sensitive, easy-to-perform, and high-throughput technique suitable for the routine diagnosis of ALK activation not only in lung cancer, but also in other tumor entities where rearrangements with alternative fusion partners (t(2;5)+ anaplastic large-cell lymphoma, ALK+ diffuse large B-cell lymphoma, ALK+ inflammatory myofibroblastic tumors), or transcriptional up-regulation of ALK (neuroblastoma, sarcomas) are prevalent.

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ACKNOWLEDGMENTS

We thank Katja Bräutigam and Daniela Rauh for expert technical assistance, Elisabeth Schroeder-Lüttgen for excellent documentation of clinico-pathological data, and Annette Staiger for inspiring discussions.

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Non–small-cell lung cancer; Anaplastic lymphoma receptor tyrosine kinase; Translocation and overexpression; Quantitative expression analysis; Routine diagnosis

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