- Email: [email protected]
RET fusion gene analysis in a selected series of cytological specimens of EGFR, KRAS and EML4-ALK wild-type adenocarcinomas of the lung
Lung Cancer 81 (2013) 377–381
Contents lists available at SciVerse ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
KIF5B/RET fusion gene analysis in a selected series of cytological specimens of EGFR, KRAS and EML4-ALK wild-type adenocarcinomas of the lung Nicla Borrelli a,1 , Riccardo Giannini a,∗,1 , Agnese Proietti b , Greta Alì b , Serena Pelliccioni b , Cristina Niccoli b , Marco Lucchi c , Franca Melfi c , Alfredo Mussi d , Fulvio Basolo a,b , Gabriella Fontanini a,b a
Department of Surgical, Medical, Molecular, and Critical Area Pathology, Division of Pathological Anatomy, University of Pisa, Pisa, Italy Unit of Pathological Anatomy, Azienda Ospedaliera Universitaria Pisana, AOUP, Pisa, Italy c Unit of Thoracic Surgery, Azienda Ospedaliera Universitaria Pisana, AOUP, Pisa, Italy d Department of Surgical, Medical, Molecular, and Critical Area Pathology, Division of Thoracic Surgery, University of Pisa, Pisa, Italy b
a r t i c l e
i n f o
Article history: Received 5 April 2013 Received in revised form 27 June 2013 Accepted 30 June 2013 Keywords: Adenocarcinoma Solid Lung KIF5B/RET rearrangement Cytology Reverse transcriptase-polymerase chain reaction Fluorescent in situ hybridization
a b s t r a c t A new RET fusion gene has been recently described in a subset of non-small cell lung cancer (NSCLC) identified by specific clinico-pathologic characteristics. This transforming gene arise from the fusion of KIF5B and the RET proto-oncogene, and it is mutually exclusive with EGFR, KRAS and EML4/ALK alterations. For this reason it could represent a putative target for specific inhibitory drugs and its evaluation could be necessary in the future daily molecular characterization of NSCLCs. One of the major challenge in diagnostic molecular pathology is to optimize genotyping tests with the minimally invasive techniques used to acquire diagnostic tumor tissue or cells. This is a significant relevant issue for approximately 60% of NSCLC patients presenting with unresectable disease, where the only pathologic materials available for diagnostic use are small biopsy or cytological specimens. Thus, the aim of this study was to verify the possibility to use RNA purified from cytological specimens to perform KIF5B/RET gene fusion expression analysis. Accordingly, we looked for the presence of the rearrangement in formalin fixed paraffin embedded tissues (FFPETs) and cytological specimens (CSs) of a selected series of “triple-marker” negative adenocarcinomas. The tests conducted revealed the presence of 1 positive patient for variant 1 of KIF5B/RET among the 49 analyzed. The presence of this fusion transcript was found in both FFPET and CS of the same patient demonstrating that the RNA obtained from minimally invasive techniques is perfectly suitable for this kind of tests. The presence of the rearrangement was also confirmed by FISH analysis. In conclusion, our findings confirm that the performance of cytology-based molecular testing for KIF5B/RET rearrangements is at least as effective as histology-based analysis, both with regard to the success rate for nucleic acid isolation and the ability to detect gene alterations. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is the leading cause of cancer deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for 80% of these cases [1–3]. Approximately 60% of patients with NSCLC present with unresectable stage IIIB or IV disease, in which the only pathologic materials available for diagnostic and therapeutic use may be small
∗ Corresponding author at: Department of Surgery, Medical, Molecular, and Critical Area Pathology, Division of Pathological Anatomy, University of Pisa, Via Roma 57, 56126 Pisa, Italy. Tel.: +39 50 993277; fax: +39 50 992481. E-mail address: [email protected] (R. Giannini). 1 These authors contributed equally to this work. 0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2013.06.026
biopsy or cytological specimens [4]. Moreover, as a consequence of an increased understanding of the molecular mechanisms underlying disease and treatment, a rapid increase in the accessibility and use of molecular testing has been observed. Furthermore, significant progress has been made with “tailored therapies” based on molecular markers [2,5,6]. Initially, the success of such therapies was largely due to the discovery that the epidermal growth factor receptor (EGFR) and KRAS mutations, which are largely confined to adenocarcinomas, are predictive of responsiveness and resistance, respectively, to the EGFR tyrosine kinase inhibitors, erlotinib and gefitinib [7,8]. Recently, the EML4/ALK translocation was identified as a predictive biomarker in patients with NSCLC. This translocation leads to the oncogenic constitutive activation of ALK and is predictive of a patient’s responsiveness to crizotinib, an inhibitor
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of MET and ALK [9]. In addition, by integrating whole-genome and transcriptome sequencing, Ju et al. were able to identify a novel transforming gene resulting from the fusion of KIF5B and the RET proto-oncogene. The fusion of KIF5B and the RET proto-oncogene is the result of a pericentric inversion on chromosome 10, which leads to the formation of the KIF5B/RET fusion oncogene [10]. Because such rearrangements do not always occur in the same location, multiple KIF5B/RET variants can be generated. Thus far, at least 13 variants have been reported, involving KIF5B introns 15, 16, 23 and 24 and RET introns 7 and 11 [11,12]. KIF5B/RET rearrangements have been detected in 1–2% of “triple-marker” negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild-type patients [10–18], although the clinical significance of this rearrangement is not fully clear [11,13,19]. To date, several phase I or II clinical trials have been launched to investigate the effects of specific drugs in patients whose tumors have KIF5B/RET rearrangements (NCT01390818, NCT01529593, NCT01049776, NCT01639508). The increased diagnostic demands of NSCLC dictate that the amount of material obtained for pathological evaluation and molecular analysis should be maximized to reduce the need for additional invasive sampling procedures. While the requirements for tissue quantity have increased, sample sizes have decreased. Thus, optimizing the use of the available tumor material has become a major challenge. Under normal conditions, cytology samples and biopsies can be used for mutational analysis, provided that the sample contains a sufficient quantity of vital tumor cells and that the extracted nucleic acid is of sufficient quality [20,21]. The aim of the present study was to verify the possibility of using RNA purified from cytological specimens to perform molecular tests, such as gene fusion expression analysis. In detail, we investigated the expression of KIF5B/RET fusion gene variants in triple-negative (EGFR and KRAS wild-type and EML4/ALK-negative) lung cancer patients. Moreover, the KIFB5/RET gene rearrangement status was investigated using newly established FISH analysis on resection specimens.
using both cytological and histological samples and were classified according to the recent indications of the International Association for the Study of Lung Cancer/American Thoracic Society/European Society working group. Diagnoses were confirmed by at least two pathologists (G.F., A.P., G.A.) after reviewing the cytological and histological samples from each patient. In detail, we analyzed 39 smears of aspirative and 10 smears of exfoliative cytology. These samples were used to assess the presence of the fusion gene transcript. Histological and cytological samples were obtained in agreement with protocols approved by the institutional review board, and informed consent for genetic analysis was obtained from all patients. 2.2. RNA purification After demounting Papanicolaou-stained slides and performing a standard deparaffinization of 10 m FFPET sections, cells or tissues were enriched by manual microdissection. Then, RNA was extracted and purified using the Qiagen RNeasy FFPE kit (Qiagen), as described by the manufacturer. RNA was quantified by measuring the absorbance at 260 nm, and the purity of the RNA was assessed according to A260/280 ratios using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The RNA integrity and yield were also evaluated using the RNA 6000 Pico Chip kit with the Agilent 2100 bioanalyzer (Agilent Technologies) by calculating the RNA Integrity Number (RIN). 2.3. Reverse transcription Total RNA was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas). Briefly, 600 ng of total RNA in a maximum volume of 22 L was added to each 40-L reaction and incubated according to the manufacturer’s protocol.
2. Materials and methods
2.4. KIF5B/RET real-time PCR
2.1. Patients and samples
cDNA was subjected to real-time PCR amplification using the Taq PCR Master Mix (Qiagen). Specific primer sets for each of the analyzed KIF5B-RET variants, covering almost 90% of the fusion variants (COSF1233, COSF1231, COSF1254, COSF1256, COSF123740) [22], were designed using Primer3 software (Table 1), and 4 independent PCR reactions were performed. In addition, the gene encoding beta actin (-actin) was amplified to estimate the efficiency of cDNA synthesis. PCRs were performed in triplicate, with each 25-L reaction containing 4 L cDNA, 12.5 L of Taq PCR Master Mix (Qiagen) and 0.5 L of each primer (20 M) and water to
In total, 49 patients (15 female and 34 male) with a mean age of 66.2 years (ranging from 37 to 80) were selected from our database of patients who had undergone the molecular diagnostic routine in the last two years. The inclusion criteria required adenocarcinoma histology and a “triple-marker” negative genotype (i.e., EGFR and KRAS wild-type and EML4/ALK-negative) and the availability both of surgical formalin-fixed, paraffin-embedded tissues (FFPETs) and of cytological specimens (CSs). All of the patients were diagnosed Table 1 Varianta
Exonb
Genec
Primer sequence (5 –3 )d
Product lengthe (bp)
1
15 12
KIF5B RET
AGG AAA TGA CCA ACC ACC AG GTA CCC TGC TCT GCC TTT CA
224
2
16 12
KIF5B RET
TGC AAG CAG TTA GAA AGC ACA TCC AAA TCC GCC TTC TCC TA
165
3
22 12
KIF5B RET
GAC AGT GGC AAA AGA ACT TCA G TCC AAA TTC GCC TTC TCC TA
151
4
15 11
KIF5B RET
AGG AAA TGA CCA ACC ACC AG CGG AAG AGG AGT AGC TGA CC
189
KIF5B/RET. a Rearrangement variants. b Involved exons. c Involved gene symbols. d RT-PCR primer sequences. e Amplicons products length in base pairs (bp).
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Fig. 1. Schematic representation of the KIF5B/RET solid poorly differentiated adenocarcinomas rearranged case. A, Hematoxylin and eosin-stained (10×) section. B, Bronchial brushing, Papanicolau stain. A1, B1 real time RT-PCR amplification and amplicon melt analysis; A2, B2 amplicon electrophoresis; A3, B3 direct sequencing of the produced amplicons showing the fusion region (bolded box). A1, A2, A3 and B1, B2, B3 represent analysis on the RNA from FFPE and cytological specimen respectively.
the final volume. Then, a Rotor-Gene Q (Qiagen) thermal cycler was used under the following conditions: pre-denaturation at 95 ◦ C for 15 min, followed by denaturation at 95 ◦ C for 20 s and 57 ◦ C for 30 s. Amplicon size and specificity were assessed and visualized using the Agilent DNA 1000 Kit with an Agilent 2100 bioanalyzer (Agilent Technologies).
2.5. Sanger sequencing Sequencing analysis was performed in all amplified samples to confirm that the amplicon corresponded to the fusion transcript. PCR products were sequenced in both directions using the same primers as in real-time PCR and the BigDye
Terminator kit with an ABI 3100 DNA Sequencer (Applied Biosystems).
2.6. RET FISH testing Unstained 3–5 m sections of formalin-fixed paraffin embedded tumor tissue were subjected to dual-color FISH analysis using the KBI-60007 Tissue Digestion Kit I in combination with POSEIDONTM Repeat-FreeTM fluorescently labeled DNA probes (Kreatech Diagnostic). One hundred non-overlapping cells with hybridization signals were examined for each case with a fluorescence microscope (BX61, Olympus) at a 63× magnification (oil immersion objective). A split or break was defined as a green/red or
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yellow fusion signal splitting into separate red and green signals. Only signals that are more than one signal diameter apart from one another are counted as a break. Two co-localized green/red or yellow fusion signals identify the normal chromosome. To compile the frequency of the positive cells for RET rearrangements, 15% of the cells carrying split red and green or single red (RET 5 region) signals was considered to be a positive result [12,18]. 3. Results 3.1. Histological patterns Fourteen cases (28%) demonstrated a solid pattern; 14 (28%) had an acinar pattern; 9 (19%) had a lepidic pattern; 3 (6%) were mucinous adenocarcinomas; and 9 (19%) were unclassified adenocarcinomas. 3.2. RNA purification and cDNA synthesis All purified total RNA samples showed A260/280 ratios ranging from 1.8 to 2. The quantity of RNA purified from aspirative, exfoliative and FFPET samples eluted in 25 l of RNase free water was sufficient to reverse transcribe 600 ng of total RNA in all cases, with an average concentration of 114.8 ng/l (range 27.3–295.7 ng/l). In addition, beta actin gene amplification was successful in all of the analyzed samples, confirming the efficiency of cDNA synthesis (data not shown). 3.3. KIF5B/RET rearrangements One solid, poorly differentiated adenocarcinoma sample resulted in a positive PCR reaction when using the variant 1 primer set, i.e., the set designed for the fusion between KIF5B exon 15 and RET exon 12 (Fig. 1). The fusion amplicon was found in the cytological aspirative specimen as well as in its corresponding FFPE tissue. The reaction specificity was verified by Agilent electrophoresis, confirming the correct amplicon length (Fig. 1). PCR performed using the primer sets designed to detect variants 1, 2, 3 and 4 of the KIF5B/RET rearrangements were negative for all other samples. 3.4. Amplicon characterization The gene fusion between the KIF5B exon 15 and RET exon 12 was further confirmed by the direct sequencing of the produced amplicons. Sequences from the FFPET and cytological specimens showed the same fusion point between 2 two genes involved (Fig. 1). 3.5. Patterns of KIF5/RET in FISH Normal signals (fused 3 –5 RET) are frequently observed in lung cancer cells, and the typical KIF5/RET fusion is visualized as distinct red and green signals separated within the same cell. To compile the frequency of positive cells for RET rearrangements, 15% of the cells carrying split red and green or single red signals was considered to be a positive result. Positive cases at sequencing were confirmed by FISH testing on a FFPET sample (Fig. 2), as the cytological samples were used for the molecular analysis. 4. Discussion In the era of personalized medicine, tailored treatment based on gene alterations and molecular marker expression has become standard practice. To appropriately assign such treatments, the strategic use of small samples derived from non-surgical procedures is required to obtain adequate amounts of well-preserved
Fig. 2. FISH analysis showing cells with RET translocation with one normal (yellow) signal, and two pairs of separated signals (red and green).
material for histological diagnosis, subtyping and molecular profiling. In lung adenocarcinomas, the use of specific EGFR tyrosinekinase inhibitors (TKIs) depends on the EGFR mutational status, and KRAS mutations are well known to predict a lack of response to treatment [7,23–27]. A minority of lung tumors have been investigated for a small inversion within chromosome 2p, which gives rise to the transforming fusion gene EML4-ALK and is present in approximately 5% of lung adenocarcinomas [9,28]. Tumors with EML4-ALK translocations seem to be mutually exclusive of EGFR and KRAS mutations and have a low frequency of p53 mutations. Another ALK translocation involving KIF5B-ALK fusion has been identified in lung adenocarcinomas; however, currently, insufficient data exist to define its specific histological nature. Patients carrying this fusion oncoprotein show a decreased sensitivity to EGFR TKIs [29], an increased sensitivity to ALK TKIs [30] and an increased sensitivity to HSP90 inhibitors [29]. Recently, the KIF5B/RET fusion oncogene was discovered, although its clinical significance remains unclear. Even after Tackeuchi et al. demonstrated that vandetanib inhibited the proliferation of Ba/F3 cells expressing KIF5B/RET [12], data regarding the effect of kinase inhibitors with anti-RET activity on KIF5B/RETpositive patients are not yet available [31–35]. Consequently, several phase I or II clinical trials are currently investigating the effects of specific drugs in patients whose tumors possess this rearrangement (NCT01390818, NCT01529593, NCT01049776, NCT01639508). Accordingly, we looked for the presence of the KIF5B/RET fusion oncogene in our selected series of “triple-marker” negative adenocarcinomas. The tests conducted on resected FFPE tissues revealed the presence of 1 positive patient for variant 1 of KIF5B/RET among the 49 analyzed, thus supporting the overall low frequency of this rearrangement [10–13]. The presence of this fusion transcript was also found in an aspirative cytological specimen of the same patient and was confirmed by FISH analysis. The RET fusion tumor histology presented as a predominantly solid, poorly differentiated subtype, in agreement with a previous report [36]. These data confirm that the performance of cytology-based molecular testing is at least as effective as histology-based analysis, both with regard to the success rates for nucleic acid isolation and the ability to detect gene alterations. Although virtually all prior studies in this field have led to similar conclusions [37], the suitability of cytological material for routine molecular testing remains under discussion. Furthermore, the data collected support the appropriateness of our KIF5B/RET analysis as a source of nucleic
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acid. Our next goal should be establishing a more extensive analysis to identify additional variants generated by KIF5B and RET fusion. Conflicts of interest statement The authors declare that they have no conflicts of interest. References [1] Weir HK, Thun MJ, Hankey BF, Ries LA, Howe HL, Wingo PA, et al. Annual report to the nation on the status of cancer, 1975–2000, featuring the uses of surveillance data for cancer prevention and control. J Natl Cancer Inst 2003;95:1276–99. [2] Scagliotti GV, Parikh P, von Pawel J, Biesma B, Vansteenkiste J, Manegold C, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-smallcell lung cancer. J Clin Oncol 2008;26:3543–51. [3] Chuman Y, Bergman A, Ueno T, Saito S, Sakaguchi K, Alaiya AA, et al. a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas. FEBS Lett 1999;462:129–34. [4] Travis WDCT, Corrin B. Hystological typing of lung and pleural tumours. Berlin: Springer Verlag; 1999. [5] Smouse JH, Cibas ES, Janne PA, Joshi VA, Zou KH, Lindeman NI. EGFR mutations are detected comparably in cytologic and surgical pathology specimens of nonsmall cell lung cancer. Cancer 2009;117:67–72. [6] Boldrini L, Ali G, Gisfredi S, Ursino S, Baldini E, Melfi F, et al. Epidermal growth factor receptor and K-RAS mutations in 411 lung adenocarcinoma: a population-based prospective study. Oncol Rep 2009;22:683–91. [7] Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500. [8] Fukuoka M, Wu YL, Thongprasert S, Sunpaweravong P, Leong SS, Sriuranpong V, et al. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS). J Clin Oncol 2011;29:2866–74. [9] Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561–6. [10] Ju YS, Lee WC, Shin JY, Lee S, Bleazard T, Won JK, et al. A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing. Genome Res 2012;22:436–45. [11] Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, Nammo T, et al. KIF5BRET fusions in lung adenocarcinoma. Nat Med 2012;18:375–7. [12] Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med 2012;18:378–81. [13] Lipson D, Capelletti M, Yelensky R, Otto G, Parker A, Jarosz M, et al. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nat Med 2012;18:382–4. [14] Li F, Feng Y, Fang R, Fang Z, Xia J, Han X, et al. Identification of RET gene fusion by exon array analyses in “pan-negative” lung cancer from never smokers. Cell Res 2012;22:928–31. [15] Cai W, Su C, Li X, Fan L, Zheng L, Fei K, et al. KIF5B-RET fusions in Chinese patients with non-small cell lung cancer. Cancer 2013;119:1486–94. [16] Suehara Y, Arcila M, Wang L, Hasanovic A, Ang D, Ito T, et al. Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions. Clin Cancer Res 2012;18:6599–608. [17] Yokota K, Sasaki H, Okuda K, Shimizu S, Shitara M, Hikosaka Y, et al. KIF5B/RET fusion gene in surgically-treated adenocarcinoma of the lung. Oncol Rep 2012;28:1187–92. [18] Sasaki H, Shimizu S, Tani Y, Maekawa M, Okuda K, Yokota K, et al. RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer. Cancer Med 2012;1:68–75.
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Contents lists available at SciVerse ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
KIF5B/RET fusion gene analysis in a selected series of cytological specimens of EGFR, KRAS and EML4-ALK wild-type adenocarcinomas of the lung Nicla Borrelli a,1 , Riccardo Giannini a,∗,1 , Agnese Proietti b , Greta Alì b , Serena Pelliccioni b , Cristina Niccoli b , Marco Lucchi c , Franca Melfi c , Alfredo Mussi d , Fulvio Basolo a,b , Gabriella Fontanini a,b a
Department of Surgical, Medical, Molecular, and Critical Area Pathology, Division of Pathological Anatomy, University of Pisa, Pisa, Italy Unit of Pathological Anatomy, Azienda Ospedaliera Universitaria Pisana, AOUP, Pisa, Italy c Unit of Thoracic Surgery, Azienda Ospedaliera Universitaria Pisana, AOUP, Pisa, Italy d Department of Surgical, Medical, Molecular, and Critical Area Pathology, Division of Thoracic Surgery, University of Pisa, Pisa, Italy b
a r t i c l e
i n f o
Article history: Received 5 April 2013 Received in revised form 27 June 2013 Accepted 30 June 2013 Keywords: Adenocarcinoma Solid Lung KIF5B/RET rearrangement Cytology Reverse transcriptase-polymerase chain reaction Fluorescent in situ hybridization
a b s t r a c t A new RET fusion gene has been recently described in a subset of non-small cell lung cancer (NSCLC) identified by specific clinico-pathologic characteristics. This transforming gene arise from the fusion of KIF5B and the RET proto-oncogene, and it is mutually exclusive with EGFR, KRAS and EML4/ALK alterations. For this reason it could represent a putative target for specific inhibitory drugs and its evaluation could be necessary in the future daily molecular characterization of NSCLCs. One of the major challenge in diagnostic molecular pathology is to optimize genotyping tests with the minimally invasive techniques used to acquire diagnostic tumor tissue or cells. This is a significant relevant issue for approximately 60% of NSCLC patients presenting with unresectable disease, where the only pathologic materials available for diagnostic use are small biopsy or cytological specimens. Thus, the aim of this study was to verify the possibility to use RNA purified from cytological specimens to perform KIF5B/RET gene fusion expression analysis. Accordingly, we looked for the presence of the rearrangement in formalin fixed paraffin embedded tissues (FFPETs) and cytological specimens (CSs) of a selected series of “triple-marker” negative adenocarcinomas. The tests conducted revealed the presence of 1 positive patient for variant 1 of KIF5B/RET among the 49 analyzed. The presence of this fusion transcript was found in both FFPET and CS of the same patient demonstrating that the RNA obtained from minimally invasive techniques is perfectly suitable for this kind of tests. The presence of the rearrangement was also confirmed by FISH analysis. In conclusion, our findings confirm that the performance of cytology-based molecular testing for KIF5B/RET rearrangements is at least as effective as histology-based analysis, both with regard to the success rate for nucleic acid isolation and the ability to detect gene alterations. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is the leading cause of cancer deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for 80% of these cases [1–3]. Approximately 60% of patients with NSCLC present with unresectable stage IIIB or IV disease, in which the only pathologic materials available for diagnostic and therapeutic use may be small
∗ Corresponding author at: Department of Surgery, Medical, Molecular, and Critical Area Pathology, Division of Pathological Anatomy, University of Pisa, Via Roma 57, 56126 Pisa, Italy. Tel.: +39 50 993277; fax: +39 50 992481. E-mail address: [email protected] (R. Giannini). 1 These authors contributed equally to this work. 0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2013.06.026
biopsy or cytological specimens [4]. Moreover, as a consequence of an increased understanding of the molecular mechanisms underlying disease and treatment, a rapid increase in the accessibility and use of molecular testing has been observed. Furthermore, significant progress has been made with “tailored therapies” based on molecular markers [2,5,6]. Initially, the success of such therapies was largely due to the discovery that the epidermal growth factor receptor (EGFR) and KRAS mutations, which are largely confined to adenocarcinomas, are predictive of responsiveness and resistance, respectively, to the EGFR tyrosine kinase inhibitors, erlotinib and gefitinib [7,8]. Recently, the EML4/ALK translocation was identified as a predictive biomarker in patients with NSCLC. This translocation leads to the oncogenic constitutive activation of ALK and is predictive of a patient’s responsiveness to crizotinib, an inhibitor
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of MET and ALK [9]. In addition, by integrating whole-genome and transcriptome sequencing, Ju et al. were able to identify a novel transforming gene resulting from the fusion of KIF5B and the RET proto-oncogene. The fusion of KIF5B and the RET proto-oncogene is the result of a pericentric inversion on chromosome 10, which leads to the formation of the KIF5B/RET fusion oncogene [10]. Because such rearrangements do not always occur in the same location, multiple KIF5B/RET variants can be generated. Thus far, at least 13 variants have been reported, involving KIF5B introns 15, 16, 23 and 24 and RET introns 7 and 11 [11,12]. KIF5B/RET rearrangements have been detected in 1–2% of “triple-marker” negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild-type patients [10–18], although the clinical significance of this rearrangement is not fully clear [11,13,19]. To date, several phase I or II clinical trials have been launched to investigate the effects of specific drugs in patients whose tumors have KIF5B/RET rearrangements (NCT01390818, NCT01529593, NCT01049776, NCT01639508). The increased diagnostic demands of NSCLC dictate that the amount of material obtained for pathological evaluation and molecular analysis should be maximized to reduce the need for additional invasive sampling procedures. While the requirements for tissue quantity have increased, sample sizes have decreased. Thus, optimizing the use of the available tumor material has become a major challenge. Under normal conditions, cytology samples and biopsies can be used for mutational analysis, provided that the sample contains a sufficient quantity of vital tumor cells and that the extracted nucleic acid is of sufficient quality [20,21]. The aim of the present study was to verify the possibility of using RNA purified from cytological specimens to perform molecular tests, such as gene fusion expression analysis. In detail, we investigated the expression of KIF5B/RET fusion gene variants in triple-negative (EGFR and KRAS wild-type and EML4/ALK-negative) lung cancer patients. Moreover, the KIFB5/RET gene rearrangement status was investigated using newly established FISH analysis on resection specimens.
using both cytological and histological samples and were classified according to the recent indications of the International Association for the Study of Lung Cancer/American Thoracic Society/European Society working group. Diagnoses were confirmed by at least two pathologists (G.F., A.P., G.A.) after reviewing the cytological and histological samples from each patient. In detail, we analyzed 39 smears of aspirative and 10 smears of exfoliative cytology. These samples were used to assess the presence of the fusion gene transcript. Histological and cytological samples were obtained in agreement with protocols approved by the institutional review board, and informed consent for genetic analysis was obtained from all patients. 2.2. RNA purification After demounting Papanicolaou-stained slides and performing a standard deparaffinization of 10 m FFPET sections, cells or tissues were enriched by manual microdissection. Then, RNA was extracted and purified using the Qiagen RNeasy FFPE kit (Qiagen), as described by the manufacturer. RNA was quantified by measuring the absorbance at 260 nm, and the purity of the RNA was assessed according to A260/280 ratios using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The RNA integrity and yield were also evaluated using the RNA 6000 Pico Chip kit with the Agilent 2100 bioanalyzer (Agilent Technologies) by calculating the RNA Integrity Number (RIN). 2.3. Reverse transcription Total RNA was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas). Briefly, 600 ng of total RNA in a maximum volume of 22 L was added to each 40-L reaction and incubated according to the manufacturer’s protocol.
2. Materials and methods
2.4. KIF5B/RET real-time PCR
2.1. Patients and samples
cDNA was subjected to real-time PCR amplification using the Taq PCR Master Mix (Qiagen). Specific primer sets for each of the analyzed KIF5B-RET variants, covering almost 90% of the fusion variants (COSF1233, COSF1231, COSF1254, COSF1256, COSF123740) [22], were designed using Primer3 software (Table 1), and 4 independent PCR reactions were performed. In addition, the gene encoding beta actin (-actin) was amplified to estimate the efficiency of cDNA synthesis. PCRs were performed in triplicate, with each 25-L reaction containing 4 L cDNA, 12.5 L of Taq PCR Master Mix (Qiagen) and 0.5 L of each primer (20 M) and water to
In total, 49 patients (15 female and 34 male) with a mean age of 66.2 years (ranging from 37 to 80) were selected from our database of patients who had undergone the molecular diagnostic routine in the last two years. The inclusion criteria required adenocarcinoma histology and a “triple-marker” negative genotype (i.e., EGFR and KRAS wild-type and EML4/ALK-negative) and the availability both of surgical formalin-fixed, paraffin-embedded tissues (FFPETs) and of cytological specimens (CSs). All of the patients were diagnosed Table 1 Varianta
Exonb
Genec
Primer sequence (5 –3 )d
Product lengthe (bp)
1
15 12
KIF5B RET
AGG AAA TGA CCA ACC ACC AG GTA CCC TGC TCT GCC TTT CA
224
2
16 12
KIF5B RET
TGC AAG CAG TTA GAA AGC ACA TCC AAA TCC GCC TTC TCC TA
165
3
22 12
KIF5B RET
GAC AGT GGC AAA AGA ACT TCA G TCC AAA TTC GCC TTC TCC TA
151
4
15 11
KIF5B RET
AGG AAA TGA CCA ACC ACC AG CGG AAG AGG AGT AGC TGA CC
189
KIF5B/RET. a Rearrangement variants. b Involved exons. c Involved gene symbols. d RT-PCR primer sequences. e Amplicons products length in base pairs (bp).
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Fig. 1. Schematic representation of the KIF5B/RET solid poorly differentiated adenocarcinomas rearranged case. A, Hematoxylin and eosin-stained (10×) section. B, Bronchial brushing, Papanicolau stain. A1, B1 real time RT-PCR amplification and amplicon melt analysis; A2, B2 amplicon electrophoresis; A3, B3 direct sequencing of the produced amplicons showing the fusion region (bolded box). A1, A2, A3 and B1, B2, B3 represent analysis on the RNA from FFPE and cytological specimen respectively.
the final volume. Then, a Rotor-Gene Q (Qiagen) thermal cycler was used under the following conditions: pre-denaturation at 95 ◦ C for 15 min, followed by denaturation at 95 ◦ C for 20 s and 57 ◦ C for 30 s. Amplicon size and specificity were assessed and visualized using the Agilent DNA 1000 Kit with an Agilent 2100 bioanalyzer (Agilent Technologies).
2.5. Sanger sequencing Sequencing analysis was performed in all amplified samples to confirm that the amplicon corresponded to the fusion transcript. PCR products were sequenced in both directions using the same primers as in real-time PCR and the BigDye
Terminator kit with an ABI 3100 DNA Sequencer (Applied Biosystems).
2.6. RET FISH testing Unstained 3–5 m sections of formalin-fixed paraffin embedded tumor tissue were subjected to dual-color FISH analysis using the KBI-60007 Tissue Digestion Kit I in combination with POSEIDONTM Repeat-FreeTM fluorescently labeled DNA probes (Kreatech Diagnostic). One hundred non-overlapping cells with hybridization signals were examined for each case with a fluorescence microscope (BX61, Olympus) at a 63× magnification (oil immersion objective). A split or break was defined as a green/red or
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yellow fusion signal splitting into separate red and green signals. Only signals that are more than one signal diameter apart from one another are counted as a break. Two co-localized green/red or yellow fusion signals identify the normal chromosome. To compile the frequency of the positive cells for RET rearrangements, 15% of the cells carrying split red and green or single red (RET 5 region) signals was considered to be a positive result [12,18]. 3. Results 3.1. Histological patterns Fourteen cases (28%) demonstrated a solid pattern; 14 (28%) had an acinar pattern; 9 (19%) had a lepidic pattern; 3 (6%) were mucinous adenocarcinomas; and 9 (19%) were unclassified adenocarcinomas. 3.2. RNA purification and cDNA synthesis All purified total RNA samples showed A260/280 ratios ranging from 1.8 to 2. The quantity of RNA purified from aspirative, exfoliative and FFPET samples eluted in 25 l of RNase free water was sufficient to reverse transcribe 600 ng of total RNA in all cases, with an average concentration of 114.8 ng/l (range 27.3–295.7 ng/l). In addition, beta actin gene amplification was successful in all of the analyzed samples, confirming the efficiency of cDNA synthesis (data not shown). 3.3. KIF5B/RET rearrangements One solid, poorly differentiated adenocarcinoma sample resulted in a positive PCR reaction when using the variant 1 primer set, i.e., the set designed for the fusion between KIF5B exon 15 and RET exon 12 (Fig. 1). The fusion amplicon was found in the cytological aspirative specimen as well as in its corresponding FFPE tissue. The reaction specificity was verified by Agilent electrophoresis, confirming the correct amplicon length (Fig. 1). PCR performed using the primer sets designed to detect variants 1, 2, 3 and 4 of the KIF5B/RET rearrangements were negative for all other samples. 3.4. Amplicon characterization The gene fusion between the KIF5B exon 15 and RET exon 12 was further confirmed by the direct sequencing of the produced amplicons. Sequences from the FFPET and cytological specimens showed the same fusion point between 2 two genes involved (Fig. 1). 3.5. Patterns of KIF5/RET in FISH Normal signals (fused 3 –5 RET) are frequently observed in lung cancer cells, and the typical KIF5/RET fusion is visualized as distinct red and green signals separated within the same cell. To compile the frequency of positive cells for RET rearrangements, 15% of the cells carrying split red and green or single red signals was considered to be a positive result. Positive cases at sequencing were confirmed by FISH testing on a FFPET sample (Fig. 2), as the cytological samples were used for the molecular analysis. 4. Discussion In the era of personalized medicine, tailored treatment based on gene alterations and molecular marker expression has become standard practice. To appropriately assign such treatments, the strategic use of small samples derived from non-surgical procedures is required to obtain adequate amounts of well-preserved
Fig. 2. FISH analysis showing cells with RET translocation with one normal (yellow) signal, and two pairs of separated signals (red and green).
material for histological diagnosis, subtyping and molecular profiling. In lung adenocarcinomas, the use of specific EGFR tyrosinekinase inhibitors (TKIs) depends on the EGFR mutational status, and KRAS mutations are well known to predict a lack of response to treatment [7,23–27]. A minority of lung tumors have been investigated for a small inversion within chromosome 2p, which gives rise to the transforming fusion gene EML4-ALK and is present in approximately 5% of lung adenocarcinomas [9,28]. Tumors with EML4-ALK translocations seem to be mutually exclusive of EGFR and KRAS mutations and have a low frequency of p53 mutations. Another ALK translocation involving KIF5B-ALK fusion has been identified in lung adenocarcinomas; however, currently, insufficient data exist to define its specific histological nature. Patients carrying this fusion oncoprotein show a decreased sensitivity to EGFR TKIs [29], an increased sensitivity to ALK TKIs [30] and an increased sensitivity to HSP90 inhibitors [29]. Recently, the KIF5B/RET fusion oncogene was discovered, although its clinical significance remains unclear. Even after Tackeuchi et al. demonstrated that vandetanib inhibited the proliferation of Ba/F3 cells expressing KIF5B/RET [12], data regarding the effect of kinase inhibitors with anti-RET activity on KIF5B/RETpositive patients are not yet available [31–35]. Consequently, several phase I or II clinical trials are currently investigating the effects of specific drugs in patients whose tumors possess this rearrangement (NCT01390818, NCT01529593, NCT01049776, NCT01639508). Accordingly, we looked for the presence of the KIF5B/RET fusion oncogene in our selected series of “triple-marker” negative adenocarcinomas. The tests conducted on resected FFPE tissues revealed the presence of 1 positive patient for variant 1 of KIF5B/RET among the 49 analyzed, thus supporting the overall low frequency of this rearrangement [10–13]. The presence of this fusion transcript was also found in an aspirative cytological specimen of the same patient and was confirmed by FISH analysis. The RET fusion tumor histology presented as a predominantly solid, poorly differentiated subtype, in agreement with a previous report [36]. These data confirm that the performance of cytology-based molecular testing is at least as effective as histology-based analysis, both with regard to the success rates for nucleic acid isolation and the ability to detect gene alterations. Although virtually all prior studies in this field have led to similar conclusions [37], the suitability of cytological material for routine molecular testing remains under discussion. Furthermore, the data collected support the appropriateness of our KIF5B/RET analysis as a source of nucleic
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