ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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Original Articles

Chromosomal rearrangements involving the NTRK1 gene in colorectal carcinoma Laurent Créancier a, Isabelle Vandenberghe a, Bruno Gomes a, Caroline Dejean a, Jean-Christophe Blanchet a, Julie Meilleroux b, Rosine Guimbaud b,c, Janick Selves b,c, Q1 Anna Kruczynski a,* a

Centre de Recherche en Oncologie Expérimentale, Institut de Recherche Pierre Fabre, 3 Avenue Hubert Curien BP13562, 31035 Toulouse Cedex 1, France Centre Hospitalier Universitaire de Toulouse, F-31300 France c Centre de Recherche en Cancérologie de Toulouse, Unité Mixte de Recherche 1037 INSERM – Université Toulouse III, France b

A R T I C L E

I N F O

Article history: Received 9 March 2015 Received in revised form 7 May 2015 Accepted 12 May 2015 Keywords: NTRK1 TRKA Gene rearrangement Colon cancer

A B S T R A C T

Chromosomal rearrangements of the NTRK1 gene, which encodes the high affinity nerve growth factor receptor (tropomyosin related kinase, TRKA), have been observed in several epithelial cancers, such as colon cancer, papillary thyroid carcinoma or non small cell lung cancer. The various NTRK1 fusions described so far lead to constitutive activation of TRKA kinase activity and are oncogenic. We further investigated here the existence and the frequency of NTRK1 gene rearrangements in colorectal cancer. Using immunohistochemistry and quantitative reverse transcriptase PCR, we analyzed a series of human colorectal cancers. We identified two TRKA positive cases over 408, with NTRK1 chromosomal rearrangements. One of these rearrangements is a TPM3–NTRK1 fusion already observed in colon cancer, while the second one is a TPR–NTRK1 fusion never described in this type of cancer. These findings further confirm that translocations in the NTRK1 gene are recurring events in colorectal cancer, although occurring at a low frequency (around 0.5%). © 2015 Published by Elsevier Ireland Ltd.

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Introduction The NTRK1 gene encodes the high affinity receptor (TRKA) for nerve growth factor (NGF). During embryogenesis, it is critical for the development and maturation of the central and peripheral nervous systems [1], whereas in adult tissues, its expression is mainly restricted to basal forebrain, sympathetic and sensory neurons, where it is implicated in pain and temperature sensing [2]. The deregulated kinase activity of trk family members has been associated with cancer [3]. More specifically, genetic alterations of the NTRK1 gene have been observed in different tumor types and associated with oncogenic activity [4]. A NTRK1 gene fusion (TPM3–NTRK1) was initially identified in a colon cancer sample [5] and later in papillary thyroid cancer, PTC (TPM3–, TPR– and TFG–NTRK1) [6]. The frequency of NTRK1 rearrangements in PTC is around 12%, with the TPM3–NTRK1 fusion being the most frequent event [7]. More recently rearrangement in NTRK1 was reported in non small cell lung cancer, NSCLC, involving fusion partners with CD74 and MPRIP [8]. These rearrangements occurred in approximately 3% NSCLC and were characterized as driver mutations [8,9]. Other NTRK1 rearrangements

have been subsequently identified in other tumor types, such as Spitz tumors, intrahepatic cholangiocarcinoma, glioblastoma and sarcoma [10–13]. In the present study, in order to further characterize the frequency and the nature of NTRK1 rearrangements in colorectal carcinoma, we investigated a large series of colon tumor specimen for the expression by immunohistochemistry of the intracellular domain of TRKA and performed the subsequent quantitative reverse transcription–polymerase chain reaction (RT-qPCR) analysis and DNA sequencing of the TRKA positive samples. Materials and methods

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Tumor models

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The colon cancer cell lines KM-12 and DLD-1 were purchased at NCI (National Cancer Institute) (Frederick, USA) and ATCC (American Type Cell Collection) (Molsheim, France), respectively and maintained in the media recommended by the suppliers in a humidified 37 °C incubator with 5% CO2 before implantation onto nude mice to establish xenograft models.

Colon cancer patient samples

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Patient samples were collected after informed consent according to institutional guidelines, and the studies were approved by the scientific committee of the Centre de Ressources Biologiques of CHU Purpan (Centre Hospitalier Universitaire de Toulouse, France).

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* Corresponding author. Tel.: +(33)534506085; fax: +(33)534503054. E-mail address: [email protected] (A. Kruczynski). http://dx.doi.org/10.1016/j.canlet.2015.05.013 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Laurent Créancier, et al., Chromosomal rearrangements involving the NTRK1 gene in colorectal carcinoma, Cancer Letters (2015), doi: 10.1016/ j.canlet.2015.05.013

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Tumor tissue microarrays

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Commercial tissue microarrays (TMA) (1191) were purchased from US Biomax (Rockville, USA), Pantomics (Richmond, USA), Biochain (Newark, USA) and SuperBiochip (Seoul, Korea). Twenty-five tissue microarrays were built from 408 cases of clinical colorectal cancer samples. Cases were screened from the Toulouse CHU collection of colorectal cancers (DC-2008-463). Grade, stage and histological type were recorded according to UICC/TNM staging, 7th edition and the WHO classification of colorectal cancer. Five hundred sixty four (564) cases of patients with a surgical resection for colorectal cancer between January 2008 and June 2013 with frozen tissue conservation were analyzed. Four hundred eight cases (408) were included after morphological selection. Formol-fixed and paraffin-embedded (FFPE) blocks were used to build TMAs. Two cores (2 mm-punch size) were punched (one on superficial area and another in front invasion of the tumor) and included in duplicate. TMAs are stored at room temperature and freshly cut for immunohistochemistry assays.

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Immunohistochemistry

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TRKA protein expression was assessed in paraffin-embedded tumor sections of KM-12 and DLD-1 xenografts (used respectively as positive and negative controls for TRKA expression) and in colorectal tumor TMAs, using a commercial anti-TRKA rabbit monoclonal antibody (Abcam ab-76291/Epitomics EP1058Y) that recognizes the carboxy-terminus domain of the protein. Immunohistochemistry detection was performed on automated BenchMark Ultra platform (Ventana Medical Systems, Tucson, USA) with heating unmasking with EDTA buffer, pH 8. Primary antibody was incubated 32’ at 1/200 dilution and after amplification with mouse anti-rabbit antibody, the detection was performed with Multimer Technology Optiview TM DAB (Ventana Medical Systems, Tucson, USA). ALK and ROS1 expression was also evaluated on Toulouse hospital’s TMA using anti-ALK mouse monoclonal antibody (clone 5A4, Labvision) and anti-ROS1 rabbit monoclonal antibody (clone D4D6, Cell signaling) at 1/50 dilution using the routine procedure of the pathological laboratory.

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RNA and DNA extraction

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Total RNA was isolated from the cells or tumor samples using Trizol (Invitrogen, Carlsbad, USA) and checked for purity, integrity and quantity. The quality of total RNA was assessed with the Agilent 2100® bio-analyzer using the RNA Nano Lab® chip and RNA 6000 Nano Assay® kit (Agilent Technologies). Genomic DNA was prepared using QIAmp DNA mini kit (Qiagen, Courtaboeuf, France). Each DNA sample was quantified by nanospectrophotometry (NanoView, GE Healthcare, Orsay, France) and qualified by 0.8% agarose electrophoresis.

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PCR analysis and sequencing

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Total RNA (1 μg) was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Marne la Coquette, France) and first-strand cDNA was used as template in the standard real-time quantitative PCR analysis (iQ Supermix, CFX96 TouchTM, Bio-Rad, Marne la Coquette, France). PCR fragments were cloned using Clone Jet PCR cloning kit (Thermo Scientific, Waltham, USA) and sequenced with Big Dye Terminator v3.1 (Applied Biosystem, Foster City, USA). For the RACE amplification, a 5′ RACE system for rapid amplification of cDNA ends (Invitrogen, France) was used to generate cDNA according to the manufacturer’s instructions. The primer sequences used were as follows. For quantitative PCR: 5′NTRK1 (forward 5′-CTGGATAGCCTCCACCACCT-3′ and reverse 5′-AGGAGAGAGACTCCAGAGCG3′); 3′NTRK1 (forward 5′-CCTACGGCAAGCAGCCCTGG-3′, and reverse 5′-ATGGCGT AGACCTCTGGTGG-3′); TPM3/NTRK1 fusion (forward 5′-CAAGGAGGCAGAGACCCGTGCT3′ and reverse 5′-CCACAGCCACCGAGACCCCAA-3′); GUSB (forward 5′-TTCAGAGCG AGTATGGAGCA-3′ and reverse 5′-TTGATCCAGACCCAGATGGT-3′); TBP (forward 5′TTGACCTAAAGACCATTGCACTTCGT-3′ and reverse 5′-TTACCGCAGCAAACCGCTTG3′); GAPDH (forward 5′-TGCTTTTAACTCTGGTAAAG-3′, and reverse 5′-TGCCATGGAA TTTGCCATGGG-3′). GUSB, TBP and GAPDH were used as internal controls. For PCR amplification: TPM3 exon 7 (5′-ACCCGTGCTGAGTTTGCTGAGA-3′); NTRK1 exon 11 (5′-TCTCCGTCCACATTTGTTGAG-3′), TPR intron 15 (5′-CAGCATCAAATGCAGCTTGT3′). The relative expression levels of the mRNAs were calculated according to the 2−(ΔCt sample – ΔCt median control) method.

Results Detection of TRKA expression in TMA of colon cancers We set up an immunohistochemical method to detect the intracellular domain of TRKA, as a readout for NTRK1 gene rearrangement in colon cancer. We selected the best commercially available antibody on the basis of sensitivity criteria. Furthermore, this

antibody corresponds to the one used by Ardini et al., who demonstrated its selectivity for TRKA detection [2]. Our method was validated using sections of colon tumor xenografts expressing TRKA, such as KM-12 (Fig. 1A) or not expressing TRKA, such as DLD1 (Fig. 1B). Using this immunohistochemical method, we assessed the expression of TRKA in 1191 commercially available TMAs of colon cancer. These samples were mainly adenocarcinomas of the colon (83%), 1.8% papillary carcinomas and 6.8% mucinous carcinomas (Table S1). Six tumor samples over 1191 samples displayed strong to moderate anti-TRKA immunoreactivity (Fig. 1C–H). These positive cases were moderately or poorly differentiated adenocarcinoma of the colon, with 4 samples/6 of stage II, 1 sample/6 of stage I and 1 sample/6 of stage III. Localization of TRKA immunostaining was cytoplasmic in all tumor samples, with two cases showing both a cytoplasmic and plasma membrane staining (Fig. 1G, G2, H, H2). TRKA immunoreactivity of these six TMA was confirmed on the corresponding whole tumor sections. However since it is technically difficult to extract RNA from TMA, we were not able to determine whether the NTRK1 gene was rearranged in these TRKA positive samples. This is why we set up a collaboration with a pathologist to further extend our investigation of TRKA expression in colorectal cancer. We selected 408 colon tumor specimen for an immunohistochemistry study and subsequent RT-qPCR analysis of TRKA positive samples. Tumors of any grade (well, moderately and poorly differentiated adenocarcinoma) and any stage (stages I, II, III, IV) were included. These samples were mainly adenocarcinomas of the colon (95.8%), with 3.8% mucinous carcinomas and 0.5% signet-ring cell carcinomas (Table S2). For each case, two cores were punched on superficial area and in front invasion of the tumor, respectively. Two samples over 408 (P10.6823 and P11.7553) displayed homogenous and strong cytoplasmic TRKA staining (Fig. 1I–L). This TRKA immunoreactivity was observed both in superficial and in front invasion cores. Overall 8 (6 + 2) colorectal samples displayed a strong TRKA immunoreactivity over a total of 1559 (1191 + 408), corresponding to a frequency of 0.5%. We also assessed NTRK1 expression by RT-qPCR in RNA extracted from colon tumor xenografts derived from patient samples (PDX). A total of 156 RNA samples were analyzed and provided by three different laboratories. None of these RNA samples was found to express detectable levels of mRNA encoding the intracellular domain of NTRK1 (data not shown). Furthermore, we also investigated the expression of other activated and rearranged RTKs in the 408 TMAs of colon cancers (Toulouse hospital collection) using immunohistochemistry (detection of the RTK intracellular domain). Only one sample of this series (P08.13390 case) displayed a strong anti-ALK immunoreactivity corresponding to an ALK-EML4 rearrangement, as revealed by FISH (data not showed). Clinical characteristics of this case are reported in Table S2 of the supplementary data. We failed to detect any positive anti-ROS1 immunoreactivity with immunohistochemistry screening in the 408 specimens. Detection of NTRK1 rearrangements in samples displaying anti-TRKA immunoreactivity A RT-qPCR method was used to quantify the relative expression level of mRNA encoding the intracellular (3′ kinase-) or extracellular (5′ regulatory-) domains of NTRK1 in RNA extracted from the two colorectal carcinoma cases showing positive TRKA immunostaining. RNA samples from the KM12 model was included as a positive control since KM12 tumors express the TPM3– NTRK1 rearrangement [2] as well as a negative TRKA immunostaining colorectal carcinoma case was included as a negative control (P08.03138, Table 1). The two samples P10.6823 and P11.7553 displaying positive TRKA immunostaining express significant detectable levels of mRNA encoding the intracellular domain of NTRK1 but not

Please cite this article in press as: Laurent Créancier, et al., Chromosomal rearrangements involving the NTRK1 gene in colorectal carcinoma, Cancer Letters (2015), doi: 10.1016/ j.canlet.2015.05.013

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Fig. 1. Immunohistochemical detection of intracellular domain of TRKA. Immunohistochemistry was performed using a commercial antibody (Abcam ab-76291). A) TRKApositive control KM12 (colon); B) TRKA-negative control DLD1 (colon); C–F) Commercial tissue microarray samples showing cytoplasmic immunohistochemical reactivity to anti-TRKA; G, H, G2, H2) Commercial tissue microarray samples showing cytoplasmic and plasma membrane immunohistochemical reactivity to anti-TRKA; I–L) Surgical colon tumor specimen (University Hospital Center of Toulouse) showing cytoplasmic immunohistochemical reactivity to anti-TRKA (I, J, P10.6823 case and K, L, P11.7553 case); Objective magnification : ×20 (A and B), ×10 (C–I and K), ×40 (J and L), ×60 (G2, H2).

6 the extracellular domain (Table 1). These results suggest a chromosomal rearrangement keeping the intracellular domain of NTRK1. Since the TPM3–NTRK1 rearrangement has already been described in colorectal and papillary carcinomas [2,14], we looked for this fusion in the P10.6823 and P11.7553 tumor samples. We were able to selectively amplify by RT-qPCR a region spanning the TPM3– NTRK1 rearrangement junction in the P11.7553 sample but not in the P10.6823 one (Table 1). This suggests that the P11.7553 tumor expresses the same TPM3–NTRK1 fusion transcript as found in KM12 and in the clinical sample of colorectal cancer described by Ardini et al. [2], whereas the P10.6823 expresses a different NTRK1 gene rearrangement.

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Table 1 Confirmation by RT-qPCR of 3′ NTRK1 over-expression and TPM3–NTRK1 fusion in the RNA of two clinical colon tumor samples.

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Q7

Clinical tumor sample/cell line

ΔCt 5′-NTRK1

ΔCt 3′-NTRK1

ΔCt TPM3/NTRK1 fusion

KM12 P08.03138

−8.6 −8 −6.6 −10.3 −6.7 −5.3 −11.7

2 −3.8 −5.1 −5.2 1.9 −3.9 2.8

2.1 −1.4 −4.1 −3.7 −7.2 −6.4 2.3

P10.6823 P11.7553

Tumor model Normal tissue Tumor Normal tissue Tumor Normal tissue Tumor

Results are presented by ΔCt between the specific target gene amplification and the mean value of the three reference genes amplification (GusB, TBP, GAPDH). KM12 cDNA was used as positive control for 3′ NTRK1 over-expression and TPM3/NTRK1 fusion expression.

Genetic characterization of NTRK1 rearrangements in samples displaying anti-TRKA immunoreactivity To confirm and characterize the fusion transcript found in the P11.7553 sample, DNA sequencing of the PCR product obtained using the primer spanning the TPM3–NTRK1 rearrangement junction was performed. The results revealed that P11.7553 sample contained two TPM3–NTRK1 fusion transcripts (Fig. 2). One of the two transcripts corresponds to the TPM3–NTRK1 rearrangement already described in colorectal carcinoma [2] (Fig. 2A). In the other TPM3– NTRK1 fusion transcript, an additional 43 base pair fragment of the TPM3 gene, coming from the 3′ portion, follows the TPM3 exon 7 and a fragment of the NTRK1 intron 9 precedes the NTRK1 exon 10 (Fig. 2B). In other words additional fragments of TPM3 and NTRK1 genes are located around the breakpoint identified in the TPM3– NTRK1 rearrangement previously described. This novel TPM3– NTRK1 fusion should also encode a functional and oncogenic chimeric protein containing the transmembrane and kinase domains of TRKA. Then we use the RACE technology to amplify the fusion transcript identified in the P10.6823 RNA sample. After clonage and DNA sequencing, we revealed that this P10.6823 RNA sample contained a TPR–NTRK1 fusion transcript, involving the 5′ portion of the TPR gene and the 3′ portion of the NTRK1 gene. Sequence analysis of this transcript allows the identification of the TPR breakpoint in exon 16, while the NTRK1 breakpoint was located in intron 9 (Fig. 3A). This result was confirmed by a genomic analysis of DNA extracted from the P10.6823 sample (Fig. 3B). This novel fusion transcript found in colorectal carcinoma corresponds to the oncogenic rearrangement of NTRK1 (TRK-T2) identified in papillary thyroid carcinoma [14].

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Fig. 2. Partial nucleotide sequence of the TPM3–NTRK1 rearrangements in the RNA P11.7553 clinical samples. A) TPM3 (exon 7)–NTRK1 (exon 10) rearrangement; B) TPM3 (exon 7-fragment 3′ TPM3 gene)-NTRK1 (partial intron 9) rearrangement. These two gene rearrangements correspond to functional fusion proteins.

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Fig. 3. Identification of TPR–NTRK1 fusions in the P10.6823 clinical sample. A) RNA sequence of TPR (exon 16)–NTRK1 (intron 9) fusion. B) Genomic DNA sequence of TPR– NTRK1 fusion. a) TPR exon 15; b) TPR intron 15; c) TPR exon 16; d) NTRK1 intron 9–exon 10. This rearrangement corresponds to the TRK-T2 described in papillary thyroid carcinoma.

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Clinical characteristics of tumors with a NTRK1 rearrangement P10.6823 case was a 53 year old female with a poorly differentiated adenocarcinoma (grade 3) of the right colon with stage II (pT3N0M0). The tumor was wild type for KRAS, NRAS, and BRAF but showed microsatellite instability with loss of MLH1/PMS2 staining, and no MLH1 promotor methylation, strongly suggesting a hereditary non polyposis colon cancer (HNPCC). However, this patient declined genetic testing for Lynch syndrome. She had no associated tumor, particularly no thyroid tumor, nor other tumor of Cowden’s syndrome but she had primitive hemochromatosis. P11.7563 case was a 66 year old male with a moderately differentiated adenocarcinoma of the left colon (grade 2) with stage II (pT3N0M0). The tumor was wild type for KRAS, NRAS, and BRAF but

MSI-positive. Moreover, this patient had two tubulous adenomas (one synchronous and another one, one year later), that did not show antiTRKA immunoreactivity, suggesting that these two adenomas did not present NTRK1 rearrangements. He had no associated tumor, particularly no thyroid tumor nor other tumor of Cowden’s syndrome. Furthermore these samples did not display anti-ALK (kinase domain), nor anti-ROS1 (kinase domain) immunoreactivity. These two patients, P10/6823 and P11/7553, did not have adjuvant therapy and were alive without relapse five and four years after surgery, respectively. Discussion Metastatic colorectal cancer is still one of the tumor types with the highest incidence and mortality. At the time of diagnosis, about

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one quarter of patients already have metastases, and the 5-year overall survival of these patients is very low [15]. Some molecularly targeted agents have demonstrated superior efficacy compared to chemotherapy but resistance mechanisms are often observed. Therefore, the identification of novel oncogenic targets to allow the design of novel and more effective targeted therapies remain a high level of priority in colorectal cancer. The success of crizotinib, an ALK/ROS1/MET targeted tyrosine kinase inhibitor, has highlighted the existence of molecular subsets of epithelial malignancies driven by rearrangement in receptor tyrosine kinase (RTK). Rearrangements of the NTRK1 gene, first biologically characterized in a colorectal patient and subsequently identified in other solid tumors, are driver mutations. Ardini et al. [2] further characterized NTRK1 rearrangement in colorectal carcinoma and out of 66 clinical samples of colorectal carcinoma has identified one sample expressing the same TPM3–NTRK1 fusion, initially discovered in colon carcinoma. These authors also showed that NTRK1 rearrangement predicted response of colorectal cancer to TRKA kinase inhibitors [2]. Our study extends this work and reveals the existence of other NTRK1 fusions in colorectal cancer. We detected different NTRK1 rearrangements in two cases of colon carcinoma out of 408 colon tumor specimen. The P11.7553 sample contained two different TPM3–NTRK1 fusion transcripts, highlighting the molecular heterogeneity of this type of tumor. The rearrangement of the TPM3 gene with different fusion partners is a frequent event since it has been detected in various tumors, such as for example in NSCLC, PTC, colorectal cancer or ALCL [1,2,16,17]. The P10.6823 sample contained a TPR–NTRK1 fusion transcript that had never been identified in colorectal cancer but that corresponds to the TPR–NTRK1 rearrangement identified in PTC [14]. As it has been described in other tumors, it is now clear that each subtype of RTK-rearranged tumor is itself a heterogenous disease made up of different fusion partners translocated to the same RTK [8,9,14,17–19]. It is likely that other NTRK1 rearrangements involving other fusion partners could be discovered in colorectal cancers or other solid tumors. These findings highlight the importance of identifying and developing new TRKA inhibitors. Overall, in this study, we detected recurrent NTRK1 fusions in 0.5% of the colon cancer samples (2/408), which also correspond to the low-frequency of fusions involving members of the NTRK family identified by Stransky et al. [13] among 20 different types of cancers. Absence of NTRK1 rearrangement in adenomas, as presumed by no surexpression of the protein, suggests that rearrangement of NRTK is a late event in the adenoma–carcinoma sequence of colorectal oncogenesis. Since TRKA expression pattern in adult tissues is very restricted, the detection by IHC of TRKA in tumors should reflect a genetic alteration or an overexpression of this protein. The P11.7553 and P10.6823 samples containing the NTRK1 rearrangements are the two positive samples displaying anti-TRKA immunoreactivity. Furthermore the anti-TRKA immunoreactivity observed was confined to the cytoplasm of the colon tumor cells, rather than being located at the cell surface, as expected for the full length receptor, when it is expressed. Therefore a cytoplasmic anti-TRKA immunoreactivity might predict a NTRK1 rearrangement. It has to be noticed that the anti-TRKA immunoreactivity detected in six commercial TMA was mainly cytoplasmic, suggesting that these six cases might also contain NTRK1 rearrangements. IHC is a technique easily performed by all pathologists, feasible to screen a high number of tumor patient samples to identify NTRK1 rearrangements and then propose a treatment with TRKA kinase inhibitors. Of course, the use of IHC to detect RTK rearrangements in tumors is appropriate only when the RTK expression pattern in adult normal tissues is very restricted and if the sensitivity of the anti-RTK antibody used is high, which is the case here.

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In conclusion, studies performed in our laboratory further support the existence of low frequency NTRK1 rearrangements in colorectal carcinoma, and identified a novel NTRK1 fusion in this solid tumor type. Patients with colorectal cancer harboring NTRK1 rearrangements may benefit from TRKA inhibitor therapy and this subset of tumors might be identifiable on the basis of immunohistochemical features. Acknowledgements The authors thank Jérôme Verdier for his skilled technical assistance and Samira Icher for her assistance for clinical annotations.

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Conflict of interest None.

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82 83 84 Supplementary data to this article can be found online at 85 Q5 86 doi:10.1016/j.canlet.2015.05.013.

Appendix: Supplementary material

87 References

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Please cite this article in press as: Laurent Créancier, et al., Chromosomal rearrangements involving the NTRK1 gene in colorectal carcinoma, Cancer Letters (2015), doi: 10.1016/ j.canlet.2015.05.013