Cancer Letters 362 (2015) 116–121
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Novel ALK fusion partners in lung cancer Aglaya G. Iyevleva a,b, Grigory A. Raskin c, Vladislav I. Tiurin a, Anna P. Sokolenko a,b, Natalia V. Mitiushkina a, Svetlana N. Aleksakhina a, Aigul R. Garifullina a, Tatiana N. Strelkova a, Valery O. Merkulov a, Alexandr O. Ivantsov a, Ekatherina Sh. Kuligina a, Kazimir M. Pozharisski a,b, Alexandr V. Togo a, Evgeny N. Imyanitov a,b,d,* a
Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, St.-Petersburg 197758, Russia Department of Medical Genetics, St.-Petersburg Pediatric Medical University, St.-Petersburg 194100, Russia c Department of Morphology, Russian Research Centre for Radiology and Surgical Technologies, St.-Petersburg 197758, Russia d Department of Oncology, I.I. Mechnikov North-Western Medical University, St.-Petersburg 191015, Russia b
A R T I C L E
I N F O
Article history: Received 15 February 2015 Received in revised form 18 March 2015 Accepted 18 March 2015 Keywords: Lung cancer ALK Translocation Rearrangement Fusion partner PCR
A B S T R A C T
Detection of ALK rearrangements in patients with non-small cell lung cancer (NSCLC) presents a significant technical challenge due to the existence of multiple translocation partners and break-points. To improve the performance of PCR-based tests, we utilized the combination of 2 assays, i.e. the variant-specific PCR for the 5 most common ALK rearrangements and the test for unbalanced 5′/3′-end ALK expression. Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ALKex20 variants and 3 tumors with novel translocation partners. 386 (96.5%) out of 400 EGFR mutation-negative NSCLCs were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK translocation; 49 of these samples (6 ALK+, 43 ALK−) were further evaluated by FISH, and there were no instances of disagreement. Among the 14 (3.5%) “discordant” tumors, 5 demonstrated ALK translocation by the first but not by the second PCR assay, and 9 had unbalanced ALK expression in the absence of known ALK fusion variants. 5 samples from the latter group were subjected to FISH, and the presence of translocation was confirmed in 2 cases. Next generation sequencing analysis of these 2 samples identified novel translocation partners, DCTN1 and SQSTM1; furthermore, the DCTN1/ALK fusion was also found in another NSCLC sample with unbalanced 5′/3′-end ALK expression, indicating a recurrent nature of this translocation. We conclude that the combination of 2 different PCR tests is a viable approach for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners. © 2015 Published by Elsevier Ireland Ltd.
Introduction Detection of ALK translocations has recently become a mandatory part of clinical examination of patients with advanced nonsmall cell lung cancer (NSCLC). The significance of ALK testing is supported by the availability of several highly efficient therapeutic ALK inhibitors, which almost always provide remarkable benefit to the patients with ALK-driven NSCLC [1–5]. Unfortunately, ALK rearrangements may involve distinct break-points and multiple fusion partners, therefore routine ALK testing presents a significant technical challenge. Fluorescent in situ hybridization (FISH) breakapart assay is considered to be a “gold standard” for the evaluation of ALK status. It relies on a spatial separation of 5′- and 3′-portions
* Corresponding author. Tel.: +7 812 4399528; fax: +7 812 5968947. E-mail address: [email protected] (E.N. Imyanitov). http://dx.doi.org/10.1016/j.canlet.2015.03.028 0304-3835/© 2015 Published by Elsevier Ireland Ltd.
of ALK gene upon rearrangement, and produces characteristic split ALK-specific signals in case of the translocation. Being apparently the most reliable approach to ALK testing for the time being, FISH assay has a number of critical disadvantages. FISH requires significant time input of an extensively trained personnel and cannot be subjected to a reasonable automation; furthermore, it demonstrates relatively high failure rates at least in some sample series and may provide poorly interpretable results in a noticeable fraction of NSCLC cases. In addition, FISH always relies on the purchase of commercial kits, which are highly expensive [2,6–11]. There are several alternative technical approaches pretending to substitute FISH or serve as a prescreening test. The development of highly sensitive ALK diagnostic antibodies offered an opportunity to detect ALK-driven tumors by a standard immunohistochemical (IHC) method. The principle of IHC is based on the fact that activating ALK rearrangements are accompanied by significant overexpression of the catalytic portion of this tyrosine kinase. IHC is generally capable to produce highly reliable results when
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performed in reference laboratories, however its interlaboratory reproducibility and performance in heterogenous lung cancer tissue collections remains to be evaluated [8,9,11–14]. Use of PCR-based assays is also a common approach for ALK testing. Conventional PCR may have significant advantages as compared to FISH and IHC. First of all, while FISH and IHC detect rather indirect signs of the presence of ALK translocation, PCR usually reveals the exact variant of the rearrangement and therefore provides definite evidence for the presence of ALK fusion. Furthermore, allele-specific PCR is highly sensitive, i.e. it can detect single ALK-driven NSCLC cells in the presence of excess of normal tissues. In addition, PCR ALK-analysis utilizes the same technical platform as other kinds of molecular NSCLC diagnosis, i.e. it can be performed in parallel or after EGFR testing using the same pool of nucleic acids [2,15]. A number of commercial PCR kits for detection of ALK rearrangements have been developed recently (e.g., ALK RGQ RT-PCR Kit (Qiagen); EML4-ALK Fusion Gene Detection Kit (AmoyDx); EML4 ALK Gene Fusion, PCR (Quest Diagnostics), etc.). Conventional PCR test-systems are designed to detect the most common individual types of ALK translocations. Given that the number of already known ALK fusion variants exceeds a dozen, multiple PCR assays or sophisticated multiplexing of PCR reactions is required for comprehensive ALK testing. There is a critical disadvantage of the variant-specific PCR testing as compared to FISH or IHC, i.e. its inability to detect yet unknown, novel types of ALK rearrangements. This limitation can be overcome by an elegant approach, which relies on the PCR-based comparison of expression of 5′- and 3′-portions of the ALK transcript. It is assumed that ALK translocation usually results in accelerated production of RNA fragments specific to catalytic portion of the gene, therefore ALKdriven NSCLCs can be distinguished from other tumors on the basis of unbalanced expression of distinct parts of ALK message [16,17]. Test for unbalanced expression appears to combine many advantages of PCR and FISH, however its actual performance in routine diagnostic setting has not been evaluated yet. We present here the results of the use of the PCR test for unbalanced 5′/3′-end ALK expression for detection of ALK rearrangements. Furthermore, we report the identification of novel ALK fusion partners in NSCLC. Materials and methods This study included patients with NSCLC, who were forwarded to molecular diagnostics by their physicians in the years 2012–2014. All tissue specimens were subjected to the manual dissection of cancer cells; isolation of nucleic acids and cDNA synthesis were performed as described in Ref. 15. The consecutive patients (n = 895) were initially screened for EGFR mutations using the standard protocol [18]. Tumors carrying EGFR alterations (n = 152) or those where EGFR analysis failed (n = 3) were excluded from further study. The remaining 685 samples were considered for the ALK testing. cDNA of sufficient quality was obtained in 597/685 (87.2%) samples (Supplementary Fig. S1). Further analysis included 400 randomly selected cDNA specimens and involved a combination of the variant-specific PCR and the test for unbalanced 5′/3′end ALK expression. Variant-specific assays were performed separately for the 5 most common EML4/ALK fusions (E13;A20 (also known as variant 1), E20;A20 (also known as variant 2), E6a/b;A20 (also known as variant 3a/b), E18;A20 and E2;A20). Tumors showing unbalanced 5′/3′-end ALK expression but negative for the above listed ALK variants were further tested for 10 rare types of ALK rearrangements involving EML4, KIF5B, TFG and KLC1 (E15;A20, E14;A20, E14;ins11A20, E13;ins69A20, E2;ins117A20, K15;A20, K17;A20, K24;A20, T3;A20, KLC1ex9;A20). Test for unbalanced 5′/3′-end ALK expression was done as described by Wang et al. [16]. Briefly, each sample was amplified simultaneously in three PCR reactions, which contained primers and probes for ALK exons 9–11 (5′-fragment), ALK exons 22–23 (3′-fragment) and SDHA (genereferee), respectively (Supplementary Table S1). ALK3′/ALK5′ ratio was calculated as 1.8−[(CtALK22/23-CtSDHA)−(CtALK9/11-CtSDHA)]. 60 NSCLC samples were also subjected to the FISH analysis using ALK Split Signal DNA probe (DAKO) according to the manufacturer’s instructions. Search for novel ALK partners was performed by the RACE (rapid amplification of cDNA ends) method using SMARTer RACE cDNA Amplification Kit (Clontech) with ALK-specific primer as described in Ref. 19. 5′-RACE PCR products were processed for the next generation sequencing (NGS) with Nextera DNA Sample Prep Kit (Illumina)
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and sequenced on the Illumina MiSeq instrument using 100 base paired-end reads. Those genes, which demonstrated maximal depth of coverage upon NGS run, were considered as potential ALK fusion partners. NGS-identified ALK translocations were validated by conventional Sanger sequencing.
Results 400 NSCLC were analyzed by PCR both for unbalanced 5′/3′end ALK expression and for the presence of five most common individual translocations (Fig. 1). 386 (96.5%) tumors were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK rearrangement. Among the 14 (3.5%) “discordant” tumors, 5 demonstrated ALK translocation by the variant-specific PCR but were negative upon the assay for unbalanced 5′/3′-end ALK expression. The remaining 9 samples showed evidence for translocation by the unbalanced expression test, but did not contain any of common ALK fusion variants; they were further subjected to the analysis of 10 rare translocation types, however these variant-specific PCR tests were negative as well (Table 1). Among 35 tumors with 5′/3′-end ALK imbalance, 16 had high level of ALK 3′-portion in the absence of the ALK 5′-transcript (high ALK 3′ expression defined as CtALK3′ − CtSDHA < 5), while 19 showed a clearly unbalanced expression of two ALK fragments, with ALK3′/ ALK5′ ratio varying from 12.1 to 891.4. 31 samples were positive upon variant-specific PCR assay; this group included 14 EML4ex13/ ALKex20 (V.1), 12 EML4ex6/ALKex20 (V.3), 3 EML4ex18/ALKex20 and 2 EML4ex20/ALKex20 (V.2) translocations. 60 tumor specimens were further analyzed by FISH, and the interpretable results were obtained for 55 (91.7%) cases. The latter panel included 6 samples positive both by the test for unbalanced 5′/3′-end ALK expression and for the assay for individual translocations, 5 cases with clearly unbalanced expression but negative results of variant-specific PCR, 1 tumor with the normal 5′/3′transcript ratio but detectable individual rearrangement, and 43 specimens negative for both PCR assays. None of samples from the latter category turned out to be positive by FISH; therefore, the combined PCR test has sufficient reliability to exclude ALK translocation-negative cases from further consideration. Similarly, all 6 cases positive for both PCR assays produced concordant results upon FISH analysis. FISH confirmed the presence of translocation in 2 out of 5 samples with unbalanced expression but lack of known translocation variant; importantly, novel ALK translocations were subsequently identified in both of these samples. Finally, FISH validated the existence of ALK fusion in the sample, which had detectable EML4ex6/ALKex20 variant by PCR but showed balanced 5′/3′-transcript ratio (Table 2). Two FISH-positive cases with clearly unbalanced ALK levels were further analyzed by 5′-RACE technique coupled with the next generation sequencing. This approach resulted in identification of two new ALK fusion partners – DCTN1 and SQSTM1 (Fig. 2). The remaining 4 cases demonstrating unbalanced ALK expression but negative for known frequent and rare fusion types were tested for the novel translocations involving DCTN1 and SQSTM1. One of the four tumors also contained the DCTN1/ALK transcript, therefore, the DCTN1/ ALK may represent not a unique, but a recurrent type of ALK rearrangement.
Table 1 Comparison of the variant-specific PCR and the test for unbalanced 5′/3′-end ALK expression. Sample groups
Unbalanced ALK+
Unbalanced ALK−
Total
Variant-specific RT-PCR+ Variant-specific RT-PCR− Total
26 9 35
5 360 365
31 369 400
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Fig. 1. Detection of ALK translocation by the test for unbalanced 5′/3′-end ALK expression (upper left) followed by the variant identification by allele-specific PCR (lower left) and validation by Sanger sequencing (lower right). An example of the expression analysis of the control (wild-type ALK) sample is presented at the upper right of the figure.
Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ ALKex20 variants and 3 tumors with novel translocation partners
Table 2 Comparison of break-apart FISH and PCR assays. Sample groups Unbalanced ALK+ and variant-specific RT-PCR+ (n = 6) Unbalanced ALK+ and variant-specific RT-PCR− (n = 5) Unbalanced ALK− and variant-specific RT-PCR+ (n = 1) Unbalanced ALK− and variant-specific RT-PCR− (n = 43)
FISH+ 6 (100%)
FISH−
(Table 3 and Supplementary Table S2). ALK translocations were more prevalent in young-onset NSCLC, while their occurrence in patients aged above 70 years was low. Activating ALK alterations were clearly associated with the non-smoking status and female gender. When the studied patients were grouped into corresponding 4 categories, i.e. male non-smokers, female non-smokers, male smokers and female non-smokers, it became apparent that the observed gender differences were almost entirely attributed to the distinct smoking attitude, not to the gender per se (Table 3).
0 (0%)
Discussion 2* (40%) 1 (100%) 0 (0%)
* NGS analysis revealed novel translocations in both these samples.
3 (60%) 0 (0%) 43 (100%)
Current standards for molecular diagnosis of NSCLC include testing for EGFR mutations and ALK rearrangements [20]. EGFR testing requires isolation of DNA, while ALK analysis is being performed using FISH- and/or IHC-based visualization of tissue sections. The requirement of two independent diagnostic platforms complicates
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Fig. 2. Sanger sequencing of cDNA samples bearing novel ALK translocations.
the logistics of NSCLC management; furthermore, in many instances the amount of tumor tissue obtained from a tiny biopsy is not sufficient for performing multiple parallel tests. The described above approach utilizes the same pool of nucleic acids for both EGFR and ALK testing that may be considered as a major advantage of the method. PCR-based ALK analysis has been repeatedly questioned for its reliability, therefore only a few ALK testing guidelines allow the use of this category of assays [21]. Here we demonstrate that the combination of the variant-specific PCR and the test for unbalanced 5′/3′-end expression provides fairly satisfactory results. Indeed, the above two PCR assays demonstrate concordant results in most instances (386/400 (96.5%)), and the status of these “PCR-concordant” samples is always in good agreement with the results of FISH evaluation (Table 3). In the present study only 14/400 (3.5%) samples showed discordance between the above PCR tests and thus required further evaluation. There are two potential major reasons of discordance. Low content of the tumor cells in the analyzed tissue specimen appears to be the main cause of false-negative results of the test for unbalanced 5′/3′-end expression. On the other hand, failure to identify the translocation variant in the presence of an elevated amount of 3′-end specific ALK message is likely to indicate the involvement of a yet unknown ALK fusion.
Given that neither FISH nor IHC is capable to identify the ALK translocation variant, it is not surprising that novel ALK rearrangements are rarely revealed upon routine diagnostics or clinical trials. This study has identified two novel NSCLC ALK fusions (DCTN1/ ALK and SQSTM1/ALK), and the DCTN1/ALK rearrangement was shown to be recurrent. DCTN1 (dynactin 1) encodes for a subunit of a macromolecular complex dynactin, which is involved in various aspects of intercellular transport and organelle movements [22–25]. Mutations in DCTN1 cause hereditary neurodegenerative disorders [26,27]. DCTN1 has been described as an ALK fusion partner in inflammatory myofibroblastic tumors [28] and Spitz tumors [29], but not yet in NSCLC. Similar to all previously reported ALK fusion partners in NSCLC (EML4, KIF5B, KLC1, TFG, TPR, HIP1, STRN), DCTN1 contains the coiled coil region, which facilitates constitutive ALK dimerization. SQSTM1 (sequestosome 1) encodes for ubiquitin-binding protein that mediates activation of the NF-kB pathway and is involved in oxidative stress response and autophagy [30]. SQSTM1 mutations are associated with Paget bone disease [31]. SQSTM1/ALK fusion was previously identified in large B-cell lymphoma [32]. Data on the clinical features of ALK-rearranged tumors are in good agreement with previous findings [33,34]. It is essential to acknowledge that the observed increase of the frequency of ALK
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Table 3 ALK translocations in non-small cell lung carcinomas. ALK translocations
Age Mean age (age range) 20–40 41–50 51–60 61–70 >/=71 ND Gender Males Females Smoking status Non-smokers Smokers Unknown Gender and smoking Male non-smokers Male smokers Males with unknown smoking status Female non-smokers Female smokers Females with unknown smoking status Histology Adenocarcinoma Adenosquamous Squamous Large cell Other ND Total
All
Present*
Absent
57.1 (39–75) 2 (22.2%) 5 (12.2%) 15 (9.3%) 10 (7.6%) 1 (1.9%) 1 (20.0%)
60.2 (20–88) 7 (77.8%) 36 (87.8%) 146 (90.7%) 122 (92.4%) 51 (98.1%) 4 (80.0%)
59.9 (20–88) 9 (100%) 41 (100%) 161 (100%) 132 (100%) 52 (100%) 5 (100%)
10 (3.5%) 24 (20.7%)
274 (96.5%) 92 (79.3%)
284 (100%) 116 (100%)
27 (22.3%) 1 (0.6%) 6 (5.1%)
94 (77.7%) 160 (99.4%) 112 (94.9%)
121 (100%) 161 (100%) 118 (100%)
8 (19.0%) 0 (0%) 2 (2.1%)
34 (81.0%) 147 (100%) 93 (97.9%)
42 (100%) 147 (100%) 95 (100%)
19 (24.1%) 1 (7.1%) 4 (17.4%)
60 (75.9%) 13 (92.9%) 19 (82.6%)
79 (100%) 14 (100%) 23 (100%)
32 (9.0%) 0 (0%) 1 (4.8%) 1 (16.7%) 0 (0%) 0 (0%) 34 (8.5%)
325 (91.0%) 6 (100%) 20 (95.2%) 5 (83.3%) 7 (100%) 3 (100%) 366 (91.5%)
357 (100%) 6 (100%) 21 (100%) 6 (100%) 7 (100%) 3 (100%) 400 (100%)
* Cases positive by RT-PCR and/or FISH, including tumors with newly discovered fusion variants.
translocations in women is entirely attributed to the high proportion of non-smokers in female lung cancer patients, but not to the genuine gender differences. In conclusion, the combination of variant-specific PCR and PCR test for unbalanced 5′/3′-end gene expression provides a viable alternative for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners. Acknowledgements This work has been supported by the Russian Federation for Basic Research (grants 13-04-01786 and 14-04-92110) and the Dynasty Foundation (contract 18/13). Conflict of interest There are no conflicts of interest in the studies reported in the paper. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.03.028. References [1] E.L. Kwak, Y.J. Bang, D.R. Camidge, A.T. Shaw, B. Solomon, R.G. Maki, et al., Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer, N. Engl. J. Med. 363 (2010) 1693–1703.
[2] E. Iwama, I. Okamoto, T. Harada, K. Takayama, Y. Nakanishi, Development of anaplastic lymphoma kinase (ALK) inhibitors and molecular diagnosis in ALK rearrangement-positive lung cancer, Onco Targets Ther. 7 (2014) 375–385. [3] H. Kim, H.S. Shim, L. Kim, T.J. Kim, K.Y. Kwon, G.K. Lee, et al., Guideline recommendations for testing of ALK gene rearrangement in lung cancer: a proposal of the Korean Cardiopulmonary Pathology Study Group, Korean J. Pathol. 48 (2014) 1–9. [4] K. McKeage, Alectinib: a review of its use in advanced ALK-rearranged non-small cell lung cancer, Drugs (2014) doi:10.1007/s40265-014-0329-y in press. [5] A.T. Shaw, J.A. Engelman, Ceritinib in ALK-rearranged non-small-cell lung cancer, N. Engl. J. Med. 370 (2014) 2537–2539. [6] E. Thunnissen, L. Bubendorf, M. Dietel, G. Elmberger, K. Kerr, F. Lopez-Rios, et al., EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations, Virchows Arch. 461 (2012) 245–257. [7] N.I. Lindeman, P.T. Cagle, M.B. Beasley, D.A. Chitale, S. Dacic, G. Giaccone, et al., Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology, J. Mol. Diagn. 15 (2013) 415–453. [8] A. Marchetti, A. Ardizzoni, M. Papotti, L. Crinò, G. Rossi, C. Gridelli, et al., Recommendations for the analysis of ALK gene rearrangements in non-small-cell lung cancer: a consensus of the Italian Association of Medical Oncology and the Italian Society of Pathology and Cytopathology, J. Thorac. Oncol. 8 (2013) 352–358. [9] W. Cooper, S. Fox, S. O’Toole, A. Morey, G. Frances, N. Pavlakis, et al., National Working Group Meeting on ALK diagnostics in lung cancer, Asia Pac. J. Clin. Oncol. 10 (Suppl. 2) (2014) 11–17. [10] R.E. Shackelford, M. Vora, K. Mayhall, J. Cotelingam, ALK-rearrangements and testing methods in non-small cell lung cancer: a review, Genes Cancer 5 (2014) 1–14. [11] L. Tembuyser, V. Tack, K. Zwaenepoel, P. Pauwels, K. Miller, L. Bubendorf, et al., The relevance of external quality assessment for molecular testing for ALK positive non-small cell lung cancer: results from two pilot rounds show room for optimization, PLoS ONE 9 (2014) e112159. [12] M. von Laffert, A. Warth, R. Penzel, P. Schirmacher, D. Jonigk, H. Kreipe, et al., Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): results of a multi-centre ALK-testing, Lung Cancer 81 (2013) 200–206. [13] F. Cabillic, A. Gros, F. Dugay, H. Begueret, L. Mesturoux, D.C. Chiforeanu, et al., Parallel FISH and immunohistochemical studies of ALK status in 3244 nonsmall-cell lung cancers reveal major discordances, J. Thorac. Oncol. 9 (2014) 295–306. [14] J.C. Cutz, K.J. Craddock, E. Torlakovic, G. Brandao, R.F. Carter, G. Bigras, et al., Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer, J. Thorac. Oncol. 9 (2014) 1255–1263. [15] N.V. Mitiushkina, A.G. Iyevleva, A.N. Poltoratskiy, A.O. Ivantsov, A.V. Togo, I.S. Polyakov, et al., Detection of EGFR mutations and EML4-ALK rearrangements in lung adenocarcinomas using archived cytological slides, Cancer Cytopathol. 121 (2013) 370–376. [16] R. Wang, Y. Pan, C. Li, H. Hu, Y. Zhang, H. Li, et al., The use of quantitative real-time reverse transcriptase PCR for 5′ and 3′ portions of ALK transcripts to detect ALK rearrangements in lung cancers, Clin. Cancer Res. 18 (2012) 4725–4732. [17] K. Gruber, H. Horn, J. Kalla, P. Fritz, A. Rosenwald, M. Kohlhäufl, et al., 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, J. Thorac. Oncol. 9 (2014) 307–315. [18] V.M. Moiseyenko, S.A. Procenko, E.V. Levchenko, A.S. Barchuk, F.V. Moiseyenko, A.G. Iyevleva, et al., High efficacy of first-line gefitinib in non-Asian patients with EGFR-mutated lung adenocarcinoma, Onkologie 33 (2010) 231–238. [19] Y. Togashi, M. Soda, S. Sakata, E. Sugawara, S. Hatano, R. Asaka, et al., KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffinembedded tissue only, PLoS ONE 7 (2012) e31323. [20] C. Gridelli, F. de Marinis, F. Cappuzzo, M. Di Maio, F.R. Hirsch, T. Mok, et al., Treatment of advanced non-small-cell lung cancer with epidermal growth factor receptor (EGFR) mutation or ALK gene rearrangement: results of an international expert panel meeting of the Italian Association of Thoracic Oncology, Clin. Lung Cancer 15 (2014) 173–181. [21] Y. Murakami, T. Mitsudomi, Y. Yatabe, A screening method for the ALK fusion gene in NSCLC, Front. Oncol. 2 (2012) 24. [22] T.A. Schroer, Dynactin, Annu. Rev. Cell Dev. Biol. 20 (2004) 759–779. [23] H. Kim, S.C. Ling, G.C. Rogers, C. Kural, P.R. Selvin, S.L. Rogers, et al., Microtubule binding by dynactin is required for microtubule organization but not cargo transport, J. Cell Biol. 176 (2007) 641–651. [24] J.E. Lazarus, A.J. Moughamian, M.K. Tokito, E.L. Holzbaur, Dynactin subunit p150(Glued) is a neuron-specific anti-catastrophe factor, PLoS Biol. 11 (2013) e1001611. [25] A.J. Moughamian, G.E. Osborn, J.E. Lazarus, S. Maday, E.L. Holzbaur, Ordered recruitment of dynactin to the microtubule plus-end is required for efficient initiation of retrograde axonal transport, J. Neurosci. 33 (2013) 13190–13203. [26] M.J. Farrer, M.M. Hulihan, J.M. Kachergus, J.C. Dächsel, A.J. Stoessl, L.L. Grantier, et al., DCTN1 mutations in perry syndrome, Nat. Genet. 41 (2009) 163–165. [27] P. Caroppo, I. Le Ber, F. Clot, S. Rivaud-Péchoux, A. Camuzat, A. De Septenville, et al., DCTN1 mutation analysis in families with progressive supranuclear palsy-like phenotypes, JAMA Neurol. 71 (2014) 208–215.
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[28] C. Murga-Zamalloa, M.S. Lim, ALK-driven tumors and targeted therapy: focus on crizotinib, Pharmgenomics Pers. Med. 7 (2014) 87–94. [29] K.J. Busam, H. Kutzner, L. Cerroni, T. Wiesner, Clinical and pathologic findings of Spitz nevi and atypical Spitz tumors with ALK fusions, Am. J. Surg. Pathol. 38 (2014) 925–933. [30] M. Komatsu, S. Kageyama, Y. Ichimura, p62/SQSTM1/A170: physiology and pathology, Pharmacol. Res. 66 (2012) 457–462. [31] N. Laurin, J.P. Brown, J. Morissette, V. Raymond, Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone, Am. J. Hum. Genet. 70 (2002) 1582–1588.
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[32] K. Takeuchi, M. Soda, Y. Togashi, Y. Ota, Y. Sekiguchi, S. Hatano, et al., Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma, Haematologica 96 (2011) 464–467. [33] J. Vidal, S. Clavé, S. de Muga, I. González, L. Pijuan, J. Gimeno, et al., Assessment of ALK status by FISH on 1000 Spanish non-small cell lung cancer patients, J. Thorac. Oncol. 9 (2014) 1816–1820. [34] J. Wang, Y. Cai, Y. Dong, J. Nong, L. Zhou, G. Liu, et al., Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR, PLoS ONE 9 (2014) e101551.
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Novel ALK fusion partners in lung cancer Aglaya G. Iyevleva a,b, Grigory A. Raskin c, Vladislav I. Tiurin a, Anna P. Sokolenko a,b, Natalia V. Mitiushkina a, Svetlana N. Aleksakhina a, Aigul R. Garifullina a, Tatiana N. Strelkova a, Valery O. Merkulov a, Alexandr O. Ivantsov a, Ekatherina Sh. Kuligina a, Kazimir M. Pozharisski a,b, Alexandr V. Togo a, Evgeny N. Imyanitov a,b,d,* a
Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, St.-Petersburg 197758, Russia Department of Medical Genetics, St.-Petersburg Pediatric Medical University, St.-Petersburg 194100, Russia c Department of Morphology, Russian Research Centre for Radiology and Surgical Technologies, St.-Petersburg 197758, Russia d Department of Oncology, I.I. Mechnikov North-Western Medical University, St.-Petersburg 191015, Russia b
A R T I C L E
I N F O
Article history: Received 15 February 2015 Received in revised form 18 March 2015 Accepted 18 March 2015 Keywords: Lung cancer ALK Translocation Rearrangement Fusion partner PCR
A B S T R A C T
Detection of ALK rearrangements in patients with non-small cell lung cancer (NSCLC) presents a significant technical challenge due to the existence of multiple translocation partners and break-points. To improve the performance of PCR-based tests, we utilized the combination of 2 assays, i.e. the variant-specific PCR for the 5 most common ALK rearrangements and the test for unbalanced 5′/3′-end ALK expression. Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ALKex20 variants and 3 tumors with novel translocation partners. 386 (96.5%) out of 400 EGFR mutation-negative NSCLCs were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK translocation; 49 of these samples (6 ALK+, 43 ALK−) were further evaluated by FISH, and there were no instances of disagreement. Among the 14 (3.5%) “discordant” tumors, 5 demonstrated ALK translocation by the first but not by the second PCR assay, and 9 had unbalanced ALK expression in the absence of known ALK fusion variants. 5 samples from the latter group were subjected to FISH, and the presence of translocation was confirmed in 2 cases. Next generation sequencing analysis of these 2 samples identified novel translocation partners, DCTN1 and SQSTM1; furthermore, the DCTN1/ALK fusion was also found in another NSCLC sample with unbalanced 5′/3′-end ALK expression, indicating a recurrent nature of this translocation. We conclude that the combination of 2 different PCR tests is a viable approach for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners. © 2015 Published by Elsevier Ireland Ltd.
Introduction Detection of ALK translocations has recently become a mandatory part of clinical examination of patients with advanced nonsmall cell lung cancer (NSCLC). The significance of ALK testing is supported by the availability of several highly efficient therapeutic ALK inhibitors, which almost always provide remarkable benefit to the patients with ALK-driven NSCLC [1–5]. Unfortunately, ALK rearrangements may involve distinct break-points and multiple fusion partners, therefore routine ALK testing presents a significant technical challenge. Fluorescent in situ hybridization (FISH) breakapart assay is considered to be a “gold standard” for the evaluation of ALK status. It relies on a spatial separation of 5′- and 3′-portions
* Corresponding author. Tel.: +7 812 4399528; fax: +7 812 5968947. E-mail address: [email protected] (E.N. Imyanitov). http://dx.doi.org/10.1016/j.canlet.2015.03.028 0304-3835/© 2015 Published by Elsevier Ireland Ltd.
of ALK gene upon rearrangement, and produces characteristic split ALK-specific signals in case of the translocation. Being apparently the most reliable approach to ALK testing for the time being, FISH assay has a number of critical disadvantages. FISH requires significant time input of an extensively trained personnel and cannot be subjected to a reasonable automation; furthermore, it demonstrates relatively high failure rates at least in some sample series and may provide poorly interpretable results in a noticeable fraction of NSCLC cases. In addition, FISH always relies on the purchase of commercial kits, which are highly expensive [2,6–11]. There are several alternative technical approaches pretending to substitute FISH or serve as a prescreening test. The development of highly sensitive ALK diagnostic antibodies offered an opportunity to detect ALK-driven tumors by a standard immunohistochemical (IHC) method. The principle of IHC is based on the fact that activating ALK rearrangements are accompanied by significant overexpression of the catalytic portion of this tyrosine kinase. IHC is generally capable to produce highly reliable results when
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performed in reference laboratories, however its interlaboratory reproducibility and performance in heterogenous lung cancer tissue collections remains to be evaluated [8,9,11–14]. Use of PCR-based assays is also a common approach for ALK testing. Conventional PCR may have significant advantages as compared to FISH and IHC. First of all, while FISH and IHC detect rather indirect signs of the presence of ALK translocation, PCR usually reveals the exact variant of the rearrangement and therefore provides definite evidence for the presence of ALK fusion. Furthermore, allele-specific PCR is highly sensitive, i.e. it can detect single ALK-driven NSCLC cells in the presence of excess of normal tissues. In addition, PCR ALK-analysis utilizes the same technical platform as other kinds of molecular NSCLC diagnosis, i.e. it can be performed in parallel or after EGFR testing using the same pool of nucleic acids [2,15]. A number of commercial PCR kits for detection of ALK rearrangements have been developed recently (e.g., ALK RGQ RT-PCR Kit (Qiagen); EML4-ALK Fusion Gene Detection Kit (AmoyDx); EML4 ALK Gene Fusion, PCR (Quest Diagnostics), etc.). Conventional PCR test-systems are designed to detect the most common individual types of ALK translocations. Given that the number of already known ALK fusion variants exceeds a dozen, multiple PCR assays or sophisticated multiplexing of PCR reactions is required for comprehensive ALK testing. There is a critical disadvantage of the variant-specific PCR testing as compared to FISH or IHC, i.e. its inability to detect yet unknown, novel types of ALK rearrangements. This limitation can be overcome by an elegant approach, which relies on the PCR-based comparison of expression of 5′- and 3′-portions of the ALK transcript. It is assumed that ALK translocation usually results in accelerated production of RNA fragments specific to catalytic portion of the gene, therefore ALKdriven NSCLCs can be distinguished from other tumors on the basis of unbalanced expression of distinct parts of ALK message [16,17]. Test for unbalanced expression appears to combine many advantages of PCR and FISH, however its actual performance in routine diagnostic setting has not been evaluated yet. We present here the results of the use of the PCR test for unbalanced 5′/3′-end ALK expression for detection of ALK rearrangements. Furthermore, we report the identification of novel ALK fusion partners in NSCLC. Materials and methods This study included patients with NSCLC, who were forwarded to molecular diagnostics by their physicians in the years 2012–2014. All tissue specimens were subjected to the manual dissection of cancer cells; isolation of nucleic acids and cDNA synthesis were performed as described in Ref. 15. The consecutive patients (n = 895) were initially screened for EGFR mutations using the standard protocol [18]. Tumors carrying EGFR alterations (n = 152) or those where EGFR analysis failed (n = 3) were excluded from further study. The remaining 685 samples were considered for the ALK testing. cDNA of sufficient quality was obtained in 597/685 (87.2%) samples (Supplementary Fig. S1). Further analysis included 400 randomly selected cDNA specimens and involved a combination of the variant-specific PCR and the test for unbalanced 5′/3′end ALK expression. Variant-specific assays were performed separately for the 5 most common EML4/ALK fusions (E13;A20 (also known as variant 1), E20;A20 (also known as variant 2), E6a/b;A20 (also known as variant 3a/b), E18;A20 and E2;A20). Tumors showing unbalanced 5′/3′-end ALK expression but negative for the above listed ALK variants were further tested for 10 rare types of ALK rearrangements involving EML4, KIF5B, TFG and KLC1 (E15;A20, E14;A20, E14;ins11A20, E13;ins69A20, E2;ins117A20, K15;A20, K17;A20, K24;A20, T3;A20, KLC1ex9;A20). Test for unbalanced 5′/3′-end ALK expression was done as described by Wang et al. [16]. Briefly, each sample was amplified simultaneously in three PCR reactions, which contained primers and probes for ALK exons 9–11 (5′-fragment), ALK exons 22–23 (3′-fragment) and SDHA (genereferee), respectively (Supplementary Table S1). ALK3′/ALK5′ ratio was calculated as 1.8−[(CtALK22/23-CtSDHA)−(CtALK9/11-CtSDHA)]. 60 NSCLC samples were also subjected to the FISH analysis using ALK Split Signal DNA probe (DAKO) according to the manufacturer’s instructions. Search for novel ALK partners was performed by the RACE (rapid amplification of cDNA ends) method using SMARTer RACE cDNA Amplification Kit (Clontech) with ALK-specific primer as described in Ref. 19. 5′-RACE PCR products were processed for the next generation sequencing (NGS) with Nextera DNA Sample Prep Kit (Illumina)
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and sequenced on the Illumina MiSeq instrument using 100 base paired-end reads. Those genes, which demonstrated maximal depth of coverage upon NGS run, were considered as potential ALK fusion partners. NGS-identified ALK translocations were validated by conventional Sanger sequencing.
Results 400 NSCLC were analyzed by PCR both for unbalanced 5′/3′end ALK expression and for the presence of five most common individual translocations (Fig. 1). 386 (96.5%) tumors were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK rearrangement. Among the 14 (3.5%) “discordant” tumors, 5 demonstrated ALK translocation by the variant-specific PCR but were negative upon the assay for unbalanced 5′/3′-end ALK expression. The remaining 9 samples showed evidence for translocation by the unbalanced expression test, but did not contain any of common ALK fusion variants; they were further subjected to the analysis of 10 rare translocation types, however these variant-specific PCR tests were negative as well (Table 1). Among 35 tumors with 5′/3′-end ALK imbalance, 16 had high level of ALK 3′-portion in the absence of the ALK 5′-transcript (high ALK 3′ expression defined as CtALK3′ − CtSDHA < 5), while 19 showed a clearly unbalanced expression of two ALK fragments, with ALK3′/ ALK5′ ratio varying from 12.1 to 891.4. 31 samples were positive upon variant-specific PCR assay; this group included 14 EML4ex13/ ALKex20 (V.1), 12 EML4ex6/ALKex20 (V.3), 3 EML4ex18/ALKex20 and 2 EML4ex20/ALKex20 (V.2) translocations. 60 tumor specimens were further analyzed by FISH, and the interpretable results were obtained for 55 (91.7%) cases. The latter panel included 6 samples positive both by the test for unbalanced 5′/3′-end ALK expression and for the assay for individual translocations, 5 cases with clearly unbalanced expression but negative results of variant-specific PCR, 1 tumor with the normal 5′/3′transcript ratio but detectable individual rearrangement, and 43 specimens negative for both PCR assays. None of samples from the latter category turned out to be positive by FISH; therefore, the combined PCR test has sufficient reliability to exclude ALK translocation-negative cases from further consideration. Similarly, all 6 cases positive for both PCR assays produced concordant results upon FISH analysis. FISH confirmed the presence of translocation in 2 out of 5 samples with unbalanced expression but lack of known translocation variant; importantly, novel ALK translocations were subsequently identified in both of these samples. Finally, FISH validated the existence of ALK fusion in the sample, which had detectable EML4ex6/ALKex20 variant by PCR but showed balanced 5′/3′-transcript ratio (Table 2). Two FISH-positive cases with clearly unbalanced ALK levels were further analyzed by 5′-RACE technique coupled with the next generation sequencing. This approach resulted in identification of two new ALK fusion partners – DCTN1 and SQSTM1 (Fig. 2). The remaining 4 cases demonstrating unbalanced ALK expression but negative for known frequent and rare fusion types were tested for the novel translocations involving DCTN1 and SQSTM1. One of the four tumors also contained the DCTN1/ALK transcript, therefore, the DCTN1/ ALK may represent not a unique, but a recurrent type of ALK rearrangement.
Table 1 Comparison of the variant-specific PCR and the test for unbalanced 5′/3′-end ALK expression. Sample groups
Unbalanced ALK+
Unbalanced ALK−
Total
Variant-specific RT-PCR+ Variant-specific RT-PCR− Total
26 9 35
5 360 365
31 369 400
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Fig. 1. Detection of ALK translocation by the test for unbalanced 5′/3′-end ALK expression (upper left) followed by the variant identification by allele-specific PCR (lower left) and validation by Sanger sequencing (lower right). An example of the expression analysis of the control (wild-type ALK) sample is presented at the upper right of the figure.
Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ ALKex20 variants and 3 tumors with novel translocation partners
Table 2 Comparison of break-apart FISH and PCR assays. Sample groups Unbalanced ALK+ and variant-specific RT-PCR+ (n = 6) Unbalanced ALK+ and variant-specific RT-PCR− (n = 5) Unbalanced ALK− and variant-specific RT-PCR+ (n = 1) Unbalanced ALK− and variant-specific RT-PCR− (n = 43)
FISH+ 6 (100%)
FISH−
(Table 3 and Supplementary Table S2). ALK translocations were more prevalent in young-onset NSCLC, while their occurrence in patients aged above 70 years was low. Activating ALK alterations were clearly associated with the non-smoking status and female gender. When the studied patients were grouped into corresponding 4 categories, i.e. male non-smokers, female non-smokers, male smokers and female non-smokers, it became apparent that the observed gender differences were almost entirely attributed to the distinct smoking attitude, not to the gender per se (Table 3).
0 (0%)
Discussion 2* (40%) 1 (100%) 0 (0%)
* NGS analysis revealed novel translocations in both these samples.
3 (60%) 0 (0%) 43 (100%)
Current standards for molecular diagnosis of NSCLC include testing for EGFR mutations and ALK rearrangements [20]. EGFR testing requires isolation of DNA, while ALK analysis is being performed using FISH- and/or IHC-based visualization of tissue sections. The requirement of two independent diagnostic platforms complicates
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Fig. 2. Sanger sequencing of cDNA samples bearing novel ALK translocations.
the logistics of NSCLC management; furthermore, in many instances the amount of tumor tissue obtained from a tiny biopsy is not sufficient for performing multiple parallel tests. The described above approach utilizes the same pool of nucleic acids for both EGFR and ALK testing that may be considered as a major advantage of the method. PCR-based ALK analysis has been repeatedly questioned for its reliability, therefore only a few ALK testing guidelines allow the use of this category of assays [21]. Here we demonstrate that the combination of the variant-specific PCR and the test for unbalanced 5′/3′-end expression provides fairly satisfactory results. Indeed, the above two PCR assays demonstrate concordant results in most instances (386/400 (96.5%)), and the status of these “PCR-concordant” samples is always in good agreement with the results of FISH evaluation (Table 3). In the present study only 14/400 (3.5%) samples showed discordance between the above PCR tests and thus required further evaluation. There are two potential major reasons of discordance. Low content of the tumor cells in the analyzed tissue specimen appears to be the main cause of false-negative results of the test for unbalanced 5′/3′-end expression. On the other hand, failure to identify the translocation variant in the presence of an elevated amount of 3′-end specific ALK message is likely to indicate the involvement of a yet unknown ALK fusion.
Given that neither FISH nor IHC is capable to identify the ALK translocation variant, it is not surprising that novel ALK rearrangements are rarely revealed upon routine diagnostics or clinical trials. This study has identified two novel NSCLC ALK fusions (DCTN1/ ALK and SQSTM1/ALK), and the DCTN1/ALK rearrangement was shown to be recurrent. DCTN1 (dynactin 1) encodes for a subunit of a macromolecular complex dynactin, which is involved in various aspects of intercellular transport and organelle movements [22–25]. Mutations in DCTN1 cause hereditary neurodegenerative disorders [26,27]. DCTN1 has been described as an ALK fusion partner in inflammatory myofibroblastic tumors [28] and Spitz tumors [29], but not yet in NSCLC. Similar to all previously reported ALK fusion partners in NSCLC (EML4, KIF5B, KLC1, TFG, TPR, HIP1, STRN), DCTN1 contains the coiled coil region, which facilitates constitutive ALK dimerization. SQSTM1 (sequestosome 1) encodes for ubiquitin-binding protein that mediates activation of the NF-kB pathway and is involved in oxidative stress response and autophagy [30]. SQSTM1 mutations are associated with Paget bone disease [31]. SQSTM1/ALK fusion was previously identified in large B-cell lymphoma [32]. Data on the clinical features of ALK-rearranged tumors are in good agreement with previous findings [33,34]. It is essential to acknowledge that the observed increase of the frequency of ALK
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Table 3 ALK translocations in non-small cell lung carcinomas. ALK translocations
Age Mean age (age range) 20–40 41–50 51–60 61–70 >/=71 ND Gender Males Females Smoking status Non-smokers Smokers Unknown Gender and smoking Male non-smokers Male smokers Males with unknown smoking status Female non-smokers Female smokers Females with unknown smoking status Histology Adenocarcinoma Adenosquamous Squamous Large cell Other ND Total
All
Present*
Absent
57.1 (39–75) 2 (22.2%) 5 (12.2%) 15 (9.3%) 10 (7.6%) 1 (1.9%) 1 (20.0%)
60.2 (20–88) 7 (77.8%) 36 (87.8%) 146 (90.7%) 122 (92.4%) 51 (98.1%) 4 (80.0%)
59.9 (20–88) 9 (100%) 41 (100%) 161 (100%) 132 (100%) 52 (100%) 5 (100%)
10 (3.5%) 24 (20.7%)
274 (96.5%) 92 (79.3%)
284 (100%) 116 (100%)
27 (22.3%) 1 (0.6%) 6 (5.1%)
94 (77.7%) 160 (99.4%) 112 (94.9%)
121 (100%) 161 (100%) 118 (100%)
8 (19.0%) 0 (0%) 2 (2.1%)
34 (81.0%) 147 (100%) 93 (97.9%)
42 (100%) 147 (100%) 95 (100%)
19 (24.1%) 1 (7.1%) 4 (17.4%)
60 (75.9%) 13 (92.9%) 19 (82.6%)
79 (100%) 14 (100%) 23 (100%)
32 (9.0%) 0 (0%) 1 (4.8%) 1 (16.7%) 0 (0%) 0 (0%) 34 (8.5%)
325 (91.0%) 6 (100%) 20 (95.2%) 5 (83.3%) 7 (100%) 3 (100%) 366 (91.5%)
357 (100%) 6 (100%) 21 (100%) 6 (100%) 7 (100%) 3 (100%) 400 (100%)
* Cases positive by RT-PCR and/or FISH, including tumors with newly discovered fusion variants.
translocations in women is entirely attributed to the high proportion of non-smokers in female lung cancer patients, but not to the genuine gender differences. In conclusion, the combination of variant-specific PCR and PCR test for unbalanced 5′/3′-end gene expression provides a viable alternative for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners. Acknowledgements This work has been supported by the Russian Federation for Basic Research (grants 13-04-01786 and 14-04-92110) and the Dynasty Foundation (contract 18/13). Conflict of interest There are no conflicts of interest in the studies reported in the paper. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.03.028. References [1] E.L. Kwak, Y.J. Bang, D.R. Camidge, A.T. Shaw, B. Solomon, R.G. Maki, et al., Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer, N. Engl. J. Med. 363 (2010) 1693–1703.
[2] E. Iwama, I. Okamoto, T. Harada, K. Takayama, Y. Nakanishi, Development of anaplastic lymphoma kinase (ALK) inhibitors and molecular diagnosis in ALK rearrangement-positive lung cancer, Onco Targets Ther. 7 (2014) 375–385. [3] H. Kim, H.S. Shim, L. Kim, T.J. Kim, K.Y. Kwon, G.K. Lee, et al., Guideline recommendations for testing of ALK gene rearrangement in lung cancer: a proposal of the Korean Cardiopulmonary Pathology Study Group, Korean J. Pathol. 48 (2014) 1–9. [4] K. McKeage, Alectinib: a review of its use in advanced ALK-rearranged non-small cell lung cancer, Drugs (2014) doi:10.1007/s40265-014-0329-y in press. [5] A.T. Shaw, J.A. Engelman, Ceritinib in ALK-rearranged non-small-cell lung cancer, N. Engl. J. Med. 370 (2014) 2537–2539. [6] E. Thunnissen, L. Bubendorf, M. Dietel, G. Elmberger, K. Kerr, F. Lopez-Rios, et al., EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations, Virchows Arch. 461 (2012) 245–257. [7] N.I. Lindeman, P.T. Cagle, M.B. Beasley, D.A. Chitale, S. Dacic, G. Giaccone, et al., Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology, J. Mol. Diagn. 15 (2013) 415–453. [8] A. Marchetti, A. Ardizzoni, M. Papotti, L. Crinò, G. Rossi, C. Gridelli, et al., Recommendations for the analysis of ALK gene rearrangements in non-small-cell lung cancer: a consensus of the Italian Association of Medical Oncology and the Italian Society of Pathology and Cytopathology, J. Thorac. Oncol. 8 (2013) 352–358. [9] W. Cooper, S. Fox, S. O’Toole, A. Morey, G. Frances, N. Pavlakis, et al., National Working Group Meeting on ALK diagnostics in lung cancer, Asia Pac. J. Clin. Oncol. 10 (Suppl. 2) (2014) 11–17. [10] R.E. Shackelford, M. Vora, K. Mayhall, J. Cotelingam, ALK-rearrangements and testing methods in non-small cell lung cancer: a review, Genes Cancer 5 (2014) 1–14. [11] L. Tembuyser, V. Tack, K. Zwaenepoel, P. Pauwels, K. Miller, L. Bubendorf, et al., The relevance of external quality assessment for molecular testing for ALK positive non-small cell lung cancer: results from two pilot rounds show room for optimization, PLoS ONE 9 (2014) e112159. [12] M. von Laffert, A. Warth, R. Penzel, P. Schirmacher, D. Jonigk, H. Kreipe, et al., Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): results of a multi-centre ALK-testing, Lung Cancer 81 (2013) 200–206. [13] F. Cabillic, A. Gros, F. Dugay, H. Begueret, L. Mesturoux, D.C. Chiforeanu, et al., Parallel FISH and immunohistochemical studies of ALK status in 3244 nonsmall-cell lung cancers reveal major discordances, J. Thorac. Oncol. 9 (2014) 295–306. [14] J.C. Cutz, K.J. Craddock, E. Torlakovic, G. Brandao, R.F. Carter, G. Bigras, et al., Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer, J. Thorac. Oncol. 9 (2014) 1255–1263. [15] N.V. Mitiushkina, A.G. Iyevleva, A.N. Poltoratskiy, A.O. Ivantsov, A.V. Togo, I.S. Polyakov, et al., Detection of EGFR mutations and EML4-ALK rearrangements in lung adenocarcinomas using archived cytological slides, Cancer Cytopathol. 121 (2013) 370–376. [16] R. Wang, Y. Pan, C. Li, H. Hu, Y. Zhang, H. Li, et al., The use of quantitative real-time reverse transcriptase PCR for 5′ and 3′ portions of ALK transcripts to detect ALK rearrangements in lung cancers, Clin. Cancer Res. 18 (2012) 4725–4732. [17] K. Gruber, H. Horn, J. Kalla, P. Fritz, A. Rosenwald, M. Kohlhäufl, et al., 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, J. Thorac. Oncol. 9 (2014) 307–315. [18] V.M. Moiseyenko, S.A. Procenko, E.V. Levchenko, A.S. Barchuk, F.V. Moiseyenko, A.G. Iyevleva, et al., High efficacy of first-line gefitinib in non-Asian patients with EGFR-mutated lung adenocarcinoma, Onkologie 33 (2010) 231–238. [19] Y. Togashi, M. Soda, S. Sakata, E. Sugawara, S. Hatano, R. Asaka, et al., KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffinembedded tissue only, PLoS ONE 7 (2012) e31323. [20] C. Gridelli, F. de Marinis, F. Cappuzzo, M. Di Maio, F.R. Hirsch, T. Mok, et al., Treatment of advanced non-small-cell lung cancer with epidermal growth factor receptor (EGFR) mutation or ALK gene rearrangement: results of an international expert panel meeting of the Italian Association of Thoracic Oncology, Clin. Lung Cancer 15 (2014) 173–181. [21] Y. Murakami, T. Mitsudomi, Y. Yatabe, A screening method for the ALK fusion gene in NSCLC, Front. Oncol. 2 (2012) 24. [22] T.A. Schroer, Dynactin, Annu. Rev. Cell Dev. Biol. 20 (2004) 759–779. [23] H. Kim, S.C. Ling, G.C. Rogers, C. Kural, P.R. Selvin, S.L. Rogers, et al., Microtubule binding by dynactin is required for microtubule organization but not cargo transport, J. Cell Biol. 176 (2007) 641–651. [24] J.E. Lazarus, A.J. Moughamian, M.K. Tokito, E.L. Holzbaur, Dynactin subunit p150(Glued) is a neuron-specific anti-catastrophe factor, PLoS Biol. 11 (2013) e1001611. [25] A.J. Moughamian, G.E. Osborn, J.E. Lazarus, S. Maday, E.L. Holzbaur, Ordered recruitment of dynactin to the microtubule plus-end is required for efficient initiation of retrograde axonal transport, J. Neurosci. 33 (2013) 13190–13203. [26] M.J. Farrer, M.M. Hulihan, J.M. Kachergus, J.C. Dächsel, A.J. Stoessl, L.L. Grantier, et al., DCTN1 mutations in perry syndrome, Nat. Genet. 41 (2009) 163–165. [27] P. Caroppo, I. Le Ber, F. Clot, S. Rivaud-Péchoux, A. Camuzat, A. De Septenville, et al., DCTN1 mutation analysis in families with progressive supranuclear palsy-like phenotypes, JAMA Neurol. 71 (2014) 208–215.
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[28] C. Murga-Zamalloa, M.S. Lim, ALK-driven tumors and targeted therapy: focus on crizotinib, Pharmgenomics Pers. Med. 7 (2014) 87–94. [29] K.J. Busam, H. Kutzner, L. Cerroni, T. Wiesner, Clinical and pathologic findings of Spitz nevi and atypical Spitz tumors with ALK fusions, Am. J. Surg. Pathol. 38 (2014) 925–933. [30] M. Komatsu, S. Kageyama, Y. Ichimura, p62/SQSTM1/A170: physiology and pathology, Pharmacol. Res. 66 (2012) 457–462. [31] N. Laurin, J.P. Brown, J. Morissette, V. Raymond, Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone, Am. J. Hum. Genet. 70 (2002) 1582–1588.
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[32] K. Takeuchi, M. Soda, Y. Togashi, Y. Ota, Y. Sekiguchi, S. Hatano, et al., Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma, Haematologica 96 (2011) 464–467. [33] J. Vidal, S. Clavé, S. de Muga, I. González, L. Pijuan, J. Gimeno, et al., Assessment of ALK status by FISH on 1000 Spanish non-small cell lung cancer patients, J. Thorac. Oncol. 9 (2014) 1816–1820. [34] J. Wang, Y. Cai, Y. Dong, J. Nong, L. Zhou, G. Liu, et al., Clinical characteristics and outcomes of patients with primary lung adenocarcinoma harboring ALK rearrangements detected by FISH, IHC, and RT-PCR, PLoS ONE 9 (2014) e101551.
