WO2013163428A1 - Detection of ret fusions in cancer - Google Patents

Abstract

Disclosed are methods and assay systems for the identification of chromosomal rearrangements involving the RET kinase gene in cellular samples. Further described are methods for identifying cancer patients who are predicted to respond, or not respond to the therapeutic administration of a chemotherapeutic regimen including one or more kinase inhibitor(s), to treat cancer in the patient.

Description

Detection of RET fusions in Cancer

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 1 19(e) from U.S. Provisional Application Serial No. 61/638,452, filed April 25, 2012, the contents of which are incorporated herein in their entirety by this reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named "2848-140-PCT Sequence Listing ST25", has a size in bytes of - —KB, and was recorded on April 25, 2013. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to markers, methods and assay kits for the identification of non-small cell lung cancer patients predicted to respond to specific cancer therapies.

BACKGROUND OF THE INVENTION

Lung cancer remains a leading cause of mortality in cancer worldwide (Ferlay et al, 2010). About 85% to 90% of all diagnosed lung cancers are Non-Small Cell Lung Cancer (NSCLC). NSCLC arises from the epithelial cells of the lung of the central bronchi to terminal alveoli. The histological type of NSCLC correlates with site of origin, reflecting the variation in respiratory tract epithelium of the bronchi to alveoli. The most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma, but there are several other types that occur less frequently, and all types can occur in unusual histologic variants. Squamous cell carcinoma usually starts near a central bronchus. Adenocarcinoma and bronchioloalveolar carcinoma usually originate in peripheral lung tissue. Although NSCLCs are associated with cigarette smoke, adenocarcinomas may be found in patients who have never smoked. As a class, NSCLCs are relatively insensitive to chemotherapy and radiation therapy compared with Small Cell Lung Cancer (SCLC).

NSCLC is increasingly being recognized as a heterogeneous set of diseases based both upon histology as well as molecular characteristics. The identification of these subsets is relevant and critical because targeted therapy in the presence of a known oncogene driver results in remarkable clinical benefit for patients.

The first important oncogene activation via gene fusion in lung cancer was discovered in 2007 by Soda and colleagues (2007). The Anaplastic Lymphoma Kinase gene (ALK) was activated by fusion with the echinoderm microtubule-associated protein- like 4 (EML4). This fusion gene resulted in constitutive activation of the ALK tyrosine kinase domain with activation of downstream signaling pathways and transformed cell growth. Since this discovery, TFG and KIF5B have also been identified as fusion partners for ALK in NSCLC (Rikova et al., 2007; Takeuchi et al, 2009).

ROS 1 is another receptor tyrosine kinase (RTK) recently reported to be activated by gene fusions in NSCLC. The 1 st cancer-related genomic rearrangement involving ROS 1 , an intra- chromosomal deletion on chromosome 6q21 fusing the 5 ' region of GOPC to the 3 ' region of ROS 1 (Charest et al., 2003), was reported in glioblastoma. Since then, several different fusions activating ROS 1 have been identified, as summarized in Table 1. Importantly, the ROS 1 kinase domain is retained in all of these fusion events and the expressed fusion genes have been reported to be oncogenic (Rikova et al., 2007; Li et al., 201 1 ; Bergethon et al., 2012; Takeuchi et al., 2012; Gu et al. 201 1 , Birsch et al., 201 1 ; Davies et al., Clin Can Res, 2012.

Several studies (Ju et al., 2012; Kohno et al., 2012; Takeuchi et al., 2012; Lipson et al., 2012) have identified another gene activated by fusion in lung cancer, Rearranged during Transfection (RET). RET is a well known RTK, with oncogenic role in papillary thyroid carcinoma through activation by gene fusions (RET-PTC) and mutations. In the last years, a wide variety of multitargeted kinase inhibitors have entered clinical trials for patients with advanced or progressing metastatic thyroid cancers yielding higher response rates than cytotoxic chemotherapy, although responses have been confined to a subset of few patients (Schlumberger, 2010; Sherman, 2010). A review by Antonelli et al (2012) summarizes results of trials of different RET TKIs in thyroid cancer. Most of these agents also inhibit VEGFR kinases because of the structural similarity between RET and VEGF. For example, sorafenib inhibits RET, RAF, and VEGFR; imatinib inhibits RET and VEGFR-2; and vandetanib (AZ6474) inhibits RET, VEGFR-2, and EGFR. The reports on RET fusions in lung adenocarcinoma are summarized in Table 2, in which total of 33 patients were reported harboring KIF5B-RET fusions involving 7 different breakpoints.

Table 2

*one pt had both K15R12 and K15Rl l .

Cancer therapies targeting a specific gene result in improved clinical benefit for patients. ALK+ lung cancers responded well to crizotinib (Kwak et al., 2010) and this drug was approved in Aug 26, 201 1 by the US FDA for the treatment of patients with advanced NSCLC proven to be ALK positive (ALK+). Crizotinib is superior to standard chemotherapy in ALK+ lung cancer patients. Additionally, it was reported that lung adenocarcinomas carrying ROS1 also respond well to crizotinib (Bergethon et al., 2012, Davies et al, Clin Can Res, 2012).

However, application of such cancer therapies is dependent upon development of reliable diagnostic methods for identification of underlying gene fusions and there continues to be a need in the art for reliable and sensitive methods for detection of oncogenic gene fusion marker, such as RET gene fusions.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method of identifying a NSCLC tumor as likely to be responsive to tyrosine kinase inhibitors comprising (a) contacting a NSCLC tissue test sample with at least one KIF5B probe and at least two RET probes; (b) detecting chromosomal rearrangement of the RET gene in the DNA from the test sample based on the hybridization pattern between the probes and the DNA from the test sample; and (c) identifying the NSCLC tumor as likely to respond to tyrosine kinase inhibitors when chromosomal rearrangement is detected.

In some embodiments, at least two of the KIF5B and RET probes are labeled with a fluorescent label. In some embodiments, the detecting step comprises fluorescence in situ hybridization (FISH) using break-apart (BA) probes specific for regions 5' and 3' of the common breakpoints in a rearranged RET gene. In some embodiments, the KIF5B probes map at 1 Op 11.2 and the RET probes map at 10ql l .2. In some embodiments, the probes comprise: a) at least one 5 ' KIF5B probe that hybridizes with a sequence selected from the group consisting of nucleotide position 32,304,999 to position 32,493,021 of chromosome 10 (BAC clone RP11-367F12), nucleotide position 32,494,678 to position 32,649,872 of chromosome 10 (BAC clone RP11-166N17), and, a nucleotide sequence that is at least 70% identical to either of these sequences; b) at least one 5' RET probe that hybridizes with a sequence selected from the group consisting of nucleotide position 43,205,232 to position 43,412,434 of chromosome 10 (BAC clone RP13-397N12), nucleotide position

43,404,932 to position 43,588,263 of chromosome 10 (BAC clone RP13-368N15), nucleotide position 43,550,471 to position 43,605,967 of chromosome 10 (BAC clone

CTD-3181E16), and, a nucleotide sequence that is at least 70% identical to any of these sequences; and, c) at least one 3' RET probe that hybridizes with a sequence selected from the group consisting of nucleotide position 43,591,858 to position 43,780,564 of chromosome 10 (BAC clone RP11-322A19), nucleotide position 43,782,565 to position 43,958,528 of chromosome 10 (BAC clone RP11-347G11), and a nucleotide sequence that is at least 70% identical to any of these sequences.

In some embodiments, the probes a, b and c are labeled with fluorescent labels that are fluorescent in different regions of the spectrum. In some embodiments, each of the probes a, b and c are labeled with one fluorescent label selected from SpectrumYellow, SpectrumGreen and SpectrumRed. In some embodiments, probe a is labeled with SpectrumYellow, probe b is labeled with SpectrumGreen and probe c is labeled with SpectrumRed label.

In some embodiments, a hybridization pattern comprising fused signals corresponding to probes a and c with separated signal corresponding to probe b indicates chromosomal rearrangement. In some embodiments, a hybridization pattern comprising fused signals corresponding to probes b and c with a separated signal corresponding to probe a indicates non-rearrangement of chromosomal genes. In some embodiments, a hybridization pattern comprising the separation of signals corresponding to probes b and c indicates a break in RET. In some embodiments, RET is fused with KIF5B. In some embodiments, RET is fused with another gene that is not KIF5B. In some embodiments, the detecting step identifies any RET chromosomal rearrangement. In some embodiments, the detecting step identifies a RET chromosomal rearrangement that activates the RET gene. In some embodiments, the chromosomal rearrangement comprises fusion of the KIF5B and RET genes.

In some embodiments, the tyrosine kinase inhibitors are selected from the group consisting of sorafenib, imatinib, cabozantinib, ponatinib, and vandetanib. In some embodiments, the sample comprises a surgical or biopsy specimen, a fine needle aspiration, fresh, frozen or paraffin embedded tissue, a tissue imprint, sputum, , or a bronchial lavage.

In some embodiments, the method further comprises confirming chromosomal rearrangement by reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, the RT-PCR is performed using mRNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections or frozen tissue sections from the NSCLC tissue test sample. In some embodiments, a RT-PCR polynucleotide product indicates the presence of a fusion transcript that spans a fusion point that contains sequence from both a

5' partner gene and the RET gene. In some embodiments, the RT-PCR polynucleotide product is analyzed by evaluating the size of the product. In some embodiments, the RT- PCR polynucleotide product is analyzed by direct sequencing of the product.

In some embodiments, where the hybridization pattern of the probes to the DNA from the test sample is indicative of a chromosomal rearrangement and a RT-PCR polynucleotide product indicates the absence of a fusion transcript that spans a fusion point that contains sequence from both a 5' partner gene and the RET gene, the method further comprises conducting inverse PCR to amplify cDNA from known nucleotide sequence in the RET gene to amplify and detect unknown, adjacent sequence from the 5' fusion gene partner.

In some embodiments, the present invention includes an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 70%, at least 75%, 80%, at least 85%o, 90%), at least 95%, or at least 100%> identical to a sequence selected from the group consisting of: SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24. In some embodiments, the present invention includes an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic drawing of the tri color RET probe design.

Figure 2 shows representative images for single BAC clones FISH assays in AG09391 cell line showing the locations of each BAC clone. Clones RP1 1 -367F12B and RP1 1 - 166N17, yellow) were mapped at 10p l l .22 (2A and 2B). Clones RP 13-397N12, RP13-368N15 and CTD-3181E16, green) were mapped at l Oql 1.21 (2A, 2B and 2C). Clones F and G (RP1 1 -322A19, RP 1 1-347G1 1, red) were mapped at l Oql 1.21 (2A and 2B).

Figure 3 shows representative images for typical signals from single BAC clone FISH assays in the AG09391 cell line showing typical compact round, sharp signals in yellow for BAC clones RP 1 1 -367F 12 (A), RP1 1 - 166N17 (B); green for RP13-397N12 (C), RP13-368N15 (D) and CTD3181E16 (E); and red for RP1 1 -322A19 (F) and RP1 1 -347G1 1 (G).

Figure 4 shows representative images for atypical signals from single BAC clone probes hybridized with the AG09391 cell line, showing atypical signals: splits (for R and Y); patchy ( for Y and G) and stringy (for Y and G). Figure 5 shows representative images showing typical and atypical signals using Color- coded probe combo FISH assay in AG09391 cell line

Figure 6 shows 3 -Color KIF5B-RET probe hybridized with the AG09391 cell line, showing typical negative signal pattern (fused R and G with separated Y; distance between fused R/G and Y >1 signal diameters; distance between R/G and Y < 1 diameter) and inconclusive signal pattern including Fused RGY; and Separated R-G-Y.

Figure 7 shows Tri-Color KIF5B-RET probe hybridized with FFPE lung adenocarcinomas, showing Positive (A) and Negative (B) specimens. White arrows indicate Fused RY-G triplets (positive pattern); green arrows indicate RG-Y (negative pattern); orange arrows indicate inconclusive triplets (excluded from analyses).

Figure 8 shows representative images of the Tri-Color KIF5B-RET probe hybridization patterns indicative of typical and atypical patterns of chromosomal rearrangement and non- rearrangement in clinical samples.

Figure 9 shows representative patterns of the Tri-Color KIF5B-RET probe hybridization patterns indicative of CCDC6.RET fusion and RET fusion with an unknown partner in clinical samples. [Figures 10 A and B show the results of administration of RET inhibitor vandetanib in NSCLC patients in which RET fusion was diagnosed.

Figure 1 1 shows the results of administration of RET inhibitor cabozantinib in NSCLC patients in which RET fusion was diagnosed.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are novel methods and systems for detection of chromosomal rearrangements involving the RET gene. Since NSCLC exhibits gene fusions involving the RET gene and a partner gene, further described herein are methods for identifying subtypes of NSCLC tumors that exhibit RET gene rearrangements and for identifying NSCLC tumors that are likely to be responsive to tyrosine kinase inhibitors.

In one embodiment, the present invention comprises a FISH system for detection of chromosomal rearrangements involving the RET gene. Chromosomal rearrangements include fusions of RET with other genes. The RET fusion may be with genes present on the same chromosome such as KIF5B or CCDC6, or genes present on other chromosomes. Additionally, the term chromosomal rearrangements may include fusions of RET with non-coding sequences, which may be present on the same chromosome as RET or may be present on other chromosomes. In some instances, the chromosomal rearrangements involving the RET gene lead to activation of the RET gene.

About 1~2% of lung adenocarcinomas (ADCs) reveal somatic fusions of the KIF5B introns with RET introns resulting in in-frame fusion transcripts of KIF5B and RET with constitutive activation of RET. The KIF5B gene is located at 1 Op 1 1.22 (starting at 32,345,959 and ending at 32,297,338) and consists of 25 exons; the RET gene is located at 1 Oql 1.21 (starting at 43,571,875 and ending at 43,626,399) and consists of 20 exons. The chromosomal rearrangement originating the KIF5B-RET fusion is a small pericentric inversion. As described in Examples land 2, a tri-color KIF5B-RET FISH probe system was designed and optimized. A detailed analysis regarding signal configuration and pattern analyses was performed and scoring criteria were identified and validated. The system was demonstrated to be efficient for analyses in cell suspensions and formalin-fixed, paraffin-embedded (FFPE). Significantly, the tri-color KIF5B- RET FISH probe set was also able to identify the KIF5B-RET using a dual-color format. This indicated that the probe set was able to detect any chromosomal rearrangement involving RET, including RET fusions with unknown genes or non-coding sequences.

It is noted that the probes disclosed herein are meant to be exemplary. Based on the disclosure, one skilled in the art will be able to design and validate additional probes for detection of chromosomal rearrangement involving the RET gene. All such probes are encompassed in the present invention.

In one embodiment, the probes comprise (a) a KIF5B probe, (b) a 5 ' RET probe and (c) a 3 ' RET probe. The design of the probes is explained in Example 1 and Figure 1. The KIF5B probes map at 10pl l .2 and the 5' and 3' RET probes map at 10ql l .2. The RET probes are specific for regions 5 ' and 3 ' of the common breakpoints in a rearranged RET gene.

In some embodiments, the KIF5B probe specifically hybridizes to the sequence corresponding to nucleotide position 32,304,999 to position 32,493,021 of chromosome 10 (BAC clone RP1 1-367F12) or to the sequence corresponding to nucleotide position 32,494,678 to position 32,649,872 of chromosome 10 (BAC clone RPl 1-166N17). In some embodiments, the 5' RET probe specifically hybridizes to the sequence corresponding to nucleotide position 43,205,232 to position 43,412,434 of chromosome 10 (BAC clone RP13-397N12), or to nucleotide position 43,404,932 to position 43,588,263 of chromosome 10 (BAC clone RP13-368N15), or to nucleotide position 43,550,471 to position 43,605,967 of chromosome 10 (BAC clone CTD- 3181 E 16) . In some embodiments, the 3' RET probe specifically hybridizes to the sequence corresponding to nucleotide position 43,591,858 to position 43,780,564 of chromosome 10 (BAC clone RPl 1-322A19), or to nucleotide position 43,782,565 to position 43,958,528 of chromosome 10 (BAC clone RPl 1-347G1 1).

The probes are labeled to detect hybridization. The label group can be a fluorophore, radioisotope, an enzyme, or an enzyme co-factor. In some embodiments, the probes are labeled with fluorescent labels and hybridization of the probes is detected using FISH. A number of fluorescent labels are commercially available, and methods for fluorescent labeling of probes and FISH protocols are well known in the art. In some embodiments, at least two of the probes are labeled with fluorescent labels that fluoresce in different regions of the spectrum and the spatial pattern of the hybridization signals indicates the chromosomal rearrangement of the RET gene. In one embodiment the three probes a, b and c may be labeled with three different fluorescent labels that fluoresce in three different regions of the spectrum. A number of such fluorescent labels are known and commercially available, including without limitation, the Vysis'™ series including SpectrumRed™, SpectrumGold™, SpectrumOrange™, SpectrumGreen™, SpectrumAqua™, and SpectrumBlue™ and the Alexa™ series. Other label tags such as quantum dots are also applicable. Example 1 explains one such combination of probes. In Example 1, the KIF5B probe was labeled with SpectrumYellow, 5^' 'RET probe was labeled with SpectrumGreen and 3 'RET probe was labeled with SpectrumRed label. FISH assays were performed using the labeled probes in cell suspensions or in tissue samples. A hybridization signal pattern comprising fused red (3'R£7) and green (5' RET) signals with a separated yellow (KIF5B) signal indicates non-rearrangement of the RET gene. A hybridization pattern comprising separation of red (3'R£T) and green (5' RET) signals indicates a break in RET. A hybridization pattern comprising fused red (3 'RET) and yellow (KIF5B) signals with a separated green (5 'RET) signal indicates chromosomal rearrangement of the RET gene involving a fusion between the RET and KIF5B genes.

Additionally, the spatial relationship among red, green and yellow signals is indicative of chromosomal rearrangement of the RET gene involving RET gene fusions with other partner genes, that are not KIF5B. For example, since CCDC6 and NCOA4 are located close to RET on chromosome arm lOq, the spatial relationship among red, green and yellow signals will suggest whether CCDC6 or NCOA4 is fused with RET. It is also possible that RET is fused with another gene mapped in other chromosome, in which case red and green will be seen randomly distributed in the nucleus. A number of such spatial signal patterns and their interpretations are shown in Figure 8. Based on the guidance provided here one in the art will be able to design probes using different labels to detect chromosomal rearrangement of the RET gene.

The chromosomal rearrangement of the RET gene can be further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) using mRNA obtained from the sample. The RT-PCR utilizes a reverse primer that hybridizes to a single location within the 3' end of RET and a fusion partner-specific 5' forward primer. All PCR products are evaluated by evaluating the size of the product and/or direct sequencing to confirm the presence of a fusion transcript that spans the fusion point and that contains sequence from both the 5' partner gene and the 3' RET kinase gene.

In cases where hybridization pattern of the probes is indicative of chromosomal rearrrangement but the RT PCR product indicates the absence of a fusion transcript that spans a fusion point that contains a sequence from both a 5 ' partner gene and the RET gene, an inverse PCR is performed to amplify cDNA from the known sequence in the RET gene to amplify and detect the unknown, adjacent sequence from the 5' fusion gene partner.

In one embodiment, the present invention comprises a method of identifying a NSCLC tumor as likely to be responsive to tyrosine kinase inhibitors. The method comprises identifying chromosomal rearrangement involving the RET gene using probes described above. In one embodiment, the method comprises contacting a NSCLC tissue test sample with at least one KIF5B probe and at least two RET probes. The sample may be a surgical or biopsy specimen, a fine needle aspiration, fresh, frozen or paraffin embedded tissue, a tissue imprint, sputum or a bronchial lavage from a subject. The method further comprises detecting chromosomal rearrangement involving the RET gene in the DNA from the test sample based on the hybridization pattern between the probes and the DNA from the test sample and identifying the NSCLC tumor as likely to respond to a RET inhibitor when a chromosomal rearrangement is detected. RET inhibitors include a tyrosine kinase inhibitor or an inhibitor that targets tyrosine kinase downstream signalling cascade, or combinations thereof. A number of tyrosine kinase inhibitors that inhibit RET are known in the art and are included in the present invention. Examples of such inhibitors include, without limitation, vandetanib, cabozantinib, sunitinib, crizotinib, sorafenib, imatinib, ponatinib, and other similar agents. Additionally, RET inhibitors include molecules that inhibit RET, including without limitation, antibodies or fragments thereof, or aptamers that recognize and bind to molecular epitopes of RET.

In some embodiments, the method further comprises treating a subject with NSCLC. The method comprises determining the subtype of the NSCLC in the subject by identifying whether the NSCLC comprises a chromosomal rearrangement involving the RET gene as described above and administering a chemotherapeutic regimen with a suitable RET kinase inhibitor to the subject. Example 5 shows that NSCLC patients harboring a RET gene fusion, show clinical response to administration of chemotherapy with RET inhibitors.

The terminology used herein is for describing particular embodiments and is not intended to be limiting. As used herein, the singular forms "a," "and" and "the" include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to "a marker" includes a combination of two or more such markers. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain.

Reference to a nucleic acid sequence includes a sequence that can consist or consist essentially of the said sequence. Also included are nucleic acid sequences that possess at least about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 percent or more identity to the said sequence or SEQ ID NO. Further included are nucleic acid molecules that hybridize to, or are the complements of the said sequence.

The practice of the invention employs, unless otherwise indicated, conventional methods of analytical biochemistry, microbiology, molecular biology and recombinant DNA generally known techniques within the skill of the art. Such techniques are explained fully in the literature. (See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual. 3rd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000; DNA Cloning: A Practical Approach, Vol. I & II (Glover, ed.); Oligonucleotide Synthesis (Gait, ed., Current Edition); Nucleic Acid Hybridization (Hames & Higgins, eds., Current Edition); Transcription and Translation (Hames & Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, Vol. I & II (Tijessen, ed.); Fundamental Virology, 2nd Edition, Vol. I & II (Fields and Knipe, eds.)).

Hybridization experiments are well known in the art. High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). One of skill in the art can calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between about 20°C and about 35°C (lower stringency), more preferably, between about 28°C and about 40°C (more stringent), and even more preferably, between about 35°C and about 45°C (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between about 30°C and about 45°C, more preferably, between about 38°C and about 50°C, and even more preferably, between about 45°C and about 55°C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%. Alternatively, Tm can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25°C below the calculated Tm of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated Tm of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37°C in about 0.1X-

0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC). Other hybridization conditions, and for example, those most useful with nucleic acid arrays, will be known to those of skill in the art.

Using the methods of the present invention, administration a chemotherapeutic drug or drug combination can be evaluated or re-evaluated in light of the assay results of the present invention. For example, the tyrosine kinase inhibitor drug(s) can be administered differently to different subject populations, depending on the presence of the chromosomal rearrangement involving RET gene in tumor samples from the subjects tested. Results from the different drug regimens can also be compared with each other directly. Alternatively, the assay results may indicate the desirability of one drug regimen over another, or indicate that a specific drug regimen should or should not be administered to a cancer patient. In one embodiment, the finding of the presence of the RET gene fusion is indicative of a predictor for response to treatment with chemotherapeutic agents comprising specific tyrosine kinase inhibitors ("tyrosine kinase inhibitor chemotherapeutic agents"). In another preferred embodiment, the absence of the RET gene fusion in a cancer patient is indicative of a predictor of poor response to treatment with tyrosine kinase inhibitor chemotherapeutic agents, and may further recommend not administering tyrosine kinase inhibitor chemotherapeutic agent drug regimens.

In another aspect, the invention provides a kit for identifying cancer patients predicted to respond or not respond to tyrosine kinase inhibitor drugs, based on the presence or absence of chromosomal rearrangement involving RET gene.

The kits of the invention may comprise one or more of the following: a polynucleotide probe that can hybridize to a polynucleotide marker, pairs of primers that under appropriate reaction conditions can prime amplification of at least a portion of a gene fusion polynucleotide marker (e.g., by PCR), instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic or therapeutic use.

The Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.

EXAMPLES

Example 1 illustrates the design of FISH probes to detect chromosomal rearrangements involving the KIF5B and RET genes in NSCLC.

Probe Development

Clones selection and PCR verification

We proposed to design a KIF5B-RET FISH probe to identify this inversion in formalin- fixed, paraffin-embedded (FFPE) tissue specimens. For this probe set, we selected seven BAC clones: two for the KIF5B 5' probe (labeled with SpectrumGold, sY), recognizing sequences at or near the 5' of KIF5B gene [RP1 1-367F12 (clone A) and RP1 1-166N17 (clone B)], three for the RET 5 ' probe (labeled with SpectrumGreen, sG), recognizing sequences at and near the 5 ' of the RET gene [RP13-397N12 (clone C), RP13-368N15 (clone D) and CTD-3181E16 (clone E)] and two for the RET 3' probe (labeled with SpectrumRed, sR), recognizing sequences at or near the 3 ' of RET gene [RP 1 1 -322A19 (clone F) and RP1 1 -347G1 1 (clone G)]. Detailed information for all 7 clones is listed in Table 3. See also Figure 1.

Table 3

All BAC clones were purchased from CHORI or Invitrogen. To verify that the BAC clones encompassed the regions of interest, specific primers were designed and synthesized by Integrated DNA Technologies (Table 3). The glycerol stabs were plated on agar plates containing selected antibiotic and 10 single-cell colonies from each BAC clone were selected for PCR verification. Two PCR-validated single colonies were frozen in glycerol stocks at -86°C and one or 2 of them were subject to cloning.

Genomic DNA extraction, amplification, and labeling

Mini-cultures of validated single-cell colonies from each BAC clone were performed in antibiotic-containing LB medium, and genomic DNA was extracted and purified using QIAamp DNA Mini Kit from Qiagen (Cat# 51306). The purified genomic DNAs from each BAC clone were subject to whole genomic amplification using the REPLI-g Midi Kit from Qiagen (Cat# 150045).

Amplified DNA from each BAC clone was labeled in 1 μg aliquots with SpectrumGold (all KIF5B 5' probe clones), SpectrumGreen conjugated dUTPs (all RET 5' probe clones), and SpectrumRed conjugated dUTPs (all RET 3' probe clones), using the Vysis Nick translation kit (Cat# 32-801300), according to the manufacturer's instructions. Each reaction was then treated according to the planned use in validation assays for single clones or combos. Labeled DNAs were co-precipitated with herring sperm DNA as carrier (1 :50) and human Cot-1 DNA (1 : 10) for blocking repetitive sequences.

Definition of Scoring System.

A. Expected patterns based on the KIF5B/RET probe design

The 5' KIF5B probe covered 343.2 Kb of the 5 'genomic region from the break points of KIF5B gene; the 5' RET probe covered 446.0 Kb of the 5 'genomic region from the break points of RET gene and the 3' RET probe covered 364.7 Kb of the 3 'genomic region from the break points of RET gene. In normal diploid cells, the 5' RET probe (G) and the 3' RET probe (R) are 14.1 Kb overlapped, while 5: KIF5B probe (Y) is 10.55 Mb away from the end of 5' RET probe. The normal diploid (2N) cells should have two triplets of Red (R), Green (G) and Yellow (Y) signals, the typical triplets pattern should be fused R and G signals with a separated Y signal in non-rearrangement cells (recorded as R/G-Y) (Figure 1).

If the rearrangement occurs between the KIF5B and RET genes, the 5' KIF5B probe (Y) could be broken into a small (0-12.3 Kb) and a large fragment (330.9-343.2 Kb); the 5' RET probe (G) would be completed; the 3' RET probe (R) could be broken into a small (15.3~20.2 Kb) and a large (344.5~349.4 Kb) fragment. The signal pattern of rearrangement would be paired fused large R and large Y with separated G, a diagram representing the design of the KIF5B-RET probe is shown in Figure 1. However, due to technical artifacts and level of chromatin condensation, atypical patterns may also be seen. Atypical triplet patterns may include all fused R, G and Y signals (recorded as R/G/Y); separated R, G and Y signals (R-G-Y); fused G and Y with separated R (R-G/Y); and fused R and Y with separated G (R/Y-G). B. Analyses of Signal Configuration on Normal Specimens

Before classifying the triplet signal pattern, we need to understand the individual signal configuration and the paired signal pattern. The typical signal configuration for R, G or Y probes should be a round and sharp compact "Dot". However, atypical patterns may be seen in nuclei due to technical variations, probe quality and chromatin stretching. Because the KIF5B-RET probe is designed for differentiating between normal and rearranged chromosomes in tumor cells, any atypical configuration of the signals or the relationship (signal pattern) between KIF5B and RET from the probe should be evaluated in details in normal specimens.

In interphase nuclei, signals were classified in four different categories according to the displayed configuration:

Dot Split Patchy Stringy

The configuration "Dot" is a round and compact signal. The other categories of split, patchy, and stringy are uncompact signals: split is a divided signal, usually in 2 or 3 fragments; patchy is a diffuse signal with irregular presentation and multiple tiny spots; stringy is an elongated fibrous-like signal. All these four signal configurations were presented in assays with single BAC clones probe and color-coded probes. For signal scoring, one dot was counted as 1 signal; a signal with split, patchy or stringy configuration was also counted as 1 even if it consists of 2 or more small spots.

C. Analyses of the Relationship between Pairs of Signals (Signal Pattern) in Normal Specimens

Classification of two related signals as fused or separated was based on how far apart of each other they laid. The distance between KIF5B (Y) and RET (R/G) is only 10.66 Mb, thus the physical co-localization of these genes was investigated in normal specimen to build a specific analysis criteria for the tri-color KIF5B-RET probe in lung cancer. For this analyses, signal pairs (R and G, R and Y, and G and Y) were initially evaluated according to the distance between them and classified in 5 categories: (1) overlapped (O), (2) touching (T), (3) Adjacent 1 (Al) when the 2 signals were apart by a gap <1 diameter of the larger signal, (4) Adjacent 2 (A2) when they were apart by a gap >1 to <2 diameters of the larger signal and (5) Adjacent 3 (A3) when they were apart by a gap >2 diameters of the larger signal. The distance between 2 round and sharp typical signals is easy to assess by using the criteria described above. However, classifying the distance between signals in a pair with 1 or 2 atypical signals is more challenging and requires detailed guidelines. For better identifying the distance between the signals in a pair with atypical signals, we considered the following guidelines: a) The signal diameter was always taken at the longest axis. For instance, the diameter of a stringy signal was its long length.

b) If multiple spots were present, and one or more small spots from the paired signals were overlapped or touching, the pair was considered overlapped or touching.

c) If multiple spots were present, the gap distance was measured between the closest small spots from different colors.

D. Analyses of the tri-color KIF5B-RET Configuration in Normal Specimens - Cell Suspensions and FFPE Tissue Sections

For the analysis in cell line, only normal diploid cells (2N) with 2 R, 2 G and 2 Y signals were scored. Nuclei missing signals or with extra signals were excluded.

For analysis in FFPE specimens, the following guidelines were developed:

1. Cell Selection a) Nuclei showing acceptable morphological features for scoring were identified in a field. b) The number of red, green and yellow signals in each nucleus was assessed. Only nuclei with same number of R, G and Y signals, thus potential carriers of tri-color triplets were selected for analyses. Nuclei missing signals or with only dual-color doublets were excluded.

2. Signal Interpretation: Each tri-color triplet in each nucleus was evaluated according to the following steps: a. R signals were identified under single red interference filter and counted to verify the number of triplets under analyses in that nucleus.

b. G signals were identified in single filter, counted and their physical localization compared with the R signals using the dual filter red-green. The green signal could be fused to the red (RG) or split apart from the red (R-G)

c. Yellow signals were investigated for their association with the classified red and green pair, either fused with only red, fused with only green, fused with both or separated from both.

d. The final classification of each tri-color triplet followed the char below: e.

RG-Y (Negative, N)

RGY (Inconclusive, I)

Count R Compare R and G

An Analysis Worksheet was generated to identify, both in cell suspensions and in FFPE specimens, how each tri-color triplet in each nucleus was classified. Nuclei with only Inconclusive triplets were excluded for final scoring. At least 80 scorable nuclei were considered per FFPE specimen.

Example 2 illustrates that the FISH fusion probes for detection of chromosomal rearrangements involving the KIF5B and RET genes works efficiently in both cell suspensions and FFPE specimens Validation of single clones, color-coded combo and the KIF5B-RET probe set

For validation of the single BAC clones, labeled DNAs were co-precipitated with herring sperm DNA as carrier (1 :50) and human Cot-1 DNA (1 : 10) for blocking repetitive sequences and each pellet was diluted in 10 μΐ of tDenHyb™-2 hybridization buffer from Insitus Biotechnologies (Cat#D002) for a final concentration of 100 ng/μΐ. Triple-color FISH assays were performed using combinations of one KIF5B 5' probe clone (Yellow), one RET 5' probe clone (Green) and one RET 3' probe clone (Red) in the cell line AG09391 (normal karyotype). The ACF 3-color probe was a mixture of 100 ng of A (Y), 100 ng of C (G), lOOng of F (R), and 1.5 μΐ of cDenHyb™-2 hybridization buffer for a final volume of 4.5 μΐ for a hybridization area of 1 13mm²; BDG set was made using similar strategy; Clone E probe (Green) was validated by single-color FISH using lOOng of the probe in 4.5 μΐ for a hybridization area of 1 13mm².

For evaluation of the color-coded combo set, the labeling reactions of all clones with the same color were co-precipitated, the two Gold (Y) labeled KIF5B 5 'probe clones (A+B), the three Green (G) labeled RET 5 'probe clones (C+D+E), and the two Red (R) labeled RET 3 'probe clones (F+G). Each color combo was precipitated with human Cot- 1 DNA (1 : 10) and herring sperm DNA in a variable amount to adjust to a final amount of 6 lug DNA). Each color combo DNA was then diluted in 10 μΐ of tDenHyb -2 hybridization buffer for a final concentration of lOOng/μΙ for each BAC clone.

For validation the color-coded combo probes (5' KIF5B Y, 5' RET G and 3' RET R), single- color FISH assays were performed in AG09391 cell line using combinations of 100 ng of each BAC clone. The 3-color KIF5B-RET probe set was prepared as a mixture of 1 μΐ of KIF5B 5' probe (Y), 1 μΐ of RET 5' probe (G), 1 μΐ of RET 3' probe (R) and 1.5 μΐ of cDenHybTM-2 hybridization buffer to a final volume of 4.5 μΐ for a hybridization area of 1 13mm².

FISH assays

All FISH assays in cell suspensions were performed according to standard protocol. Briefly, the slides were treated in a solution of 70% acetic acid for 30~60 sec, incubated in 0.008% pepsin/0.01 M HCL at 37°C for 7~9 min, fixed in 1% formaldehyde solution for 10 min and dehydrated in graded ethanol series. Probe mix was applied to the selected 12 mm² diameter hybridization areas, which were covered with glass cover slips and sealed with rubber cement. DNA co-denaturation was performed for 10 minutes in an 85°C dry oven and hybridization was allowed to occur in a moist chamber at 37°C for 40 hours. Post-hybridization washes were performed with 2><SSC/0.3% NP-40 at 72°C for 2 min, and 2><SSC for 2 min at room temperature, and dehydrated in graded ethanol series. Chromatin was counterstained with DAPI (0^g/ml in Vectashield Mounting Medium, Vector Laboratories).

FFPE specimens were also tested according to standard lab protocols. Specimens were incubated at 56°C for 2 hours, dewaxed in CitriSolv, dehydrated and air-dried, then slides were soaked in 2xSSC at 75°C for 10-13 min and digested in 0.6 mg/ml proteinase K at 45°C for 20-22 min. After dehydration, the KIF5B/RET probe which contained 100 ng/BAC for R-color-coded probe, 150 ng/BAC for G-color-coded and Y-color-coded probe in 4.5 μΐ of hybridization buffer was applied onto selected 1 13 mm² hybridization areas and hybridization for 48 hours, post-hybridization washes performed as described above for the cell line.

Evaluation of Single BAC Clone probes

Chromosome Mapping

The quality of the preparations and the intensity of the fluorescence signal were excellent in all the slides. Chromosomal mapping was investigated in 50 karyotypically normal metaphase spreads and all of the individual BAC clones mapped correctly: the BAC clones A, B for 5' KIF5B mapped at lOpl 1.2, the BAC clones C, D, E, F and G mapped at lOql 1.2. The AG09391 cell line had about 65% of the cells with diploid (2N) and 35% of the cells with tetraploid (4N) chromosome content. These cells had, respectively, 2 and 4 copies of each of the clones tested. Representative images of these results are shown in Figures 2A, 2B and 2C, respectively for the ACF, BCG and E probe sets. Figure 2 shows Single BAC clones FISH assays in AG09391 cell line showing the locations of each BAC clone. Clones RP1 1-367F12B and RP1 1- 166N17, yellow) were mapped at 10pl l.22 (2A and 2B). Clones RP13-397N12, RP13-368N15 and CTD-3181E16, green) were mapped at lOql 1.21 (2A, 2B and 2C). Clones F and G (RP1 1-322A19, RP11-347G1 1, red) were mapped at lOql 1.21 (2A and 2B).

Evaluation of Probe Configuration for Single Clone and Color-Coded Combo Probes

The signal quality and pattern classification were investigated in 100 diploid interphase nuclei (2N) and only nuclei with 2R, 2G and 2Y were selected for analysis. As shown in Table 4 A and B, the prevalent form for the individual clones was typical as a dot (with 93.5~99% of the signals included in this category). About 0.5%~4.0% of the signals presented as split, 0.5-1.5% as patchy, and 0%~1.5% as stringy, making the frequency of signals with irregular configuration between 1.0% and 6.5% and the cells carrying signals with atypical configuration between 8.0%~13.0%.

Table 4. Single BAC probe signal analysis in 100 (2N) interphase nuclei from the AG09391 cell line.

A. Classification er si nal; B. Classification er cell.

Representative images for typical signals from all single BAC clone probes are shown in Figure 3 and representative images for atypical signals are shown in Figure 4. Figure 3 shows single BAC clones FISH assays in the AG09391 cell line showing typical compact round, sharp signals in yellow for BAC clones RP1 1-367F12 (A), RP1 1-166N17 (B); green for RP13-397N12 (C), RP13- 368N15 (D) and CTD3181E16 (E); and red for RP1 1-322A19 (F) and RP1 1-347G1 1 (G). Figure 4 shows single BAC clone probes hybridized with the AG09391 cell line, showing atypical signals: splits (for R and Y); patchy ( for Y and G) and stringy (for Y and G).

When all same color probes were combined, dot was still the prevalent signal but frequencies dropped down to 87.5%, 83.0% and 85.5%, respectively, for the R, G and Y color-coded probe set. Frequencies of split, patchy and stringy signals were, respectively, 7.5%, 3.5% and 1.5% for R signals; 7.5%, 8.0%, 1.5% for G signals, and 10.5%, 3.0%, 1.0% for Y signals. The frequency of cells with irregular R, G and Y signals, respectively, hit 20%, 29% and 25%. Therefore, atypical signals were much more frequent with the combo probe than with individual clones. The detailed information is shown in Table 5 A and B. Representative images for typical and atypical signals from all color- coded probes are shown in Figure 5.

Table 5. Color coded combo probe analysis in 100 (2N) interphase nuclei in AG09391 cell line. A. Classification per signal; B. Classification per cell.

scored.

Evaluation of the tri-color KIF5B-RET Combo Probe in normal cell line

Next, analyses of the spatial relationship between KIF5B (Y), RET 5' (G) and RET 3' (R) were performed for the tri-color probe set in 100 normal diploid cells of AG0939. Nuclei with diploid (2N) typically should display 2 triplets of R/G-Y. Initially, each pair of signals was considered individually and classified as Fused when overlapped or touching, and separated when adjacent Al, A2 or A3. Among the 200 triplets, about 94.0% showed R and G signals overlapped and touching (Fused), in only 6.0% of R and G pairs they were classified as separated (Al to A3), mimicking a false positive pattern (split 3 'RET-5 'RET). The pairs R and Y and G and Y were designed as fused in, respectively, 19.5% (mimicking a false positive pattern (KIF5B-3 'RET) and 16.5% of the triplets. Detailed information is shown in Table 6.

Table 6. KIF5B-RET Combo probe signal pattern analysis in 100 (2N) cells in AG09391 cell line

( analysis for distance between signal R, G and Go)

When analyses was performed by triplet, 74.0% of triplets had the typical negative pattern fused R and G with separated Y (R/G-Y) and the most common atypical pattern was all Fused (R/G/Y). The typical positive pattern for the KIF5B-RET fusion (R/Y-G) was only detected in 0.5% of the triplets in the normal specimen. Considering analyses per cell, 56.0% of the cells presented only typical signal pattern; while 37.0% of the cells presented at least one triplet with all fused R/G/Y (all 3 colors fused). The typical positive pattern for the KIF5B-RET fusion (R/Y-G) was only detected in 1 % of the cells in the normal specimen. Detailed results for this analysis were showed in Table 7 A and B. Representative images are shown in Figure 6.

Table 7. KIF5B-RET Combo probe signal pattern analysis in 100 (2N) cells in AG09391 cell line.

Evaluation of the tri-color KIF5B 3-Color Probe in FFPE specimens.

The results on cell line were encouraging and supported to move the analyses to formalin-fixed, paraffin-embedded tissue sections. To test the KIF5B/RET probe in formalin-fixed, paraffin- embedded (FFPE) specimens, five FFPE specimens from NSCLC previously tested negative for ALK rearrangement were randomly selected for validation. The intensity of the signals in those specimens was good to excellent.

One specimen was identified as carrying the KIF5B/RET rearrangement (Sp #1), with 64% of cells displaying tri-color triplets with Positive pattern (R/Y-G) (Figure 7A). All other specimens were classified as negative for the rearrangement, with less than 5% nuclei displaying the positive pattern. A representative image of the negative specimens is shown in Figure 7B.

Thus, the probe set developed for detection of the KIF5B-RET fusion was demonstrated efficient both in cell suspensions and FFPE specimens. Additionally, it also proved to be efficient to detect breaks occurring within the RET exons 8 to 12 as evidenced by the dual-color analyses in FFPE sections. This finding supports the use of this tri-color probe also for detection of any RET rearrangements that are potentially mechanism of activation of this gene, independently of knowing the partner gene.

Example 3 illustrates the detection of gene fusions using mRNA-based assays with RT-PCR and Inverse PCR.

The presence of RET gene fusions was confirmed in multiple NSCLC patient cases using mRNA extracted from FFPE or frozen tissue. The assays utilize a reverse primer that hybridizes to a single location within the 3' end of RET and multiple different fusion partner-specific 5' forward primers. This assay generates a PCR product that is detected and sized using the bioanalyzer from Agilent. All PCR products are evaluated by direct sequencing to confirm the presence of a fusion transcript that spans the fusion point and that contains sequence from both the 5 ' partner gene and the 3' gene containing the kinase (ALK, ROS1, or RET). In cases where FISH detects evidence of a gene fusion event but no PCR product is detected using our assay, an inverse PCR is performed to amplify cDNA from known sequence in the 3' RET to amplify and detect unknown, adjacent sequence from the 5' fusion gene partner (Davies et al., submitted to Clin Can Res).

RNA extraction from FFPE tissue: RNA from either FFPE or Frozen tissue was processed using the RecoverAll™ Total Nucleic Acid Isolation Kit [Ambion (Austin, TX)] as previously described.⁵¹ For FFPE tissue, sections were initially deparaffinized in xylene and washed with 100% ethanol prior to the Protease K digest.

RET Gene Fusion Multiplex RT-PCR Assay: To confirm the presence of a RET gene fusion and to identify the fusion partner of RET from RNA samples, RT-PCR was carried out using the Superscript® III First-Strand Synthesis System (Invitrogen) with a previously published RET primer located in exon 13 of Ret (Ret-2381R)⁵². For first strand synthesis, RNA, dNTPs and RET-2381R primer were initially denatured at 65°C for 5mins and then placed on ice for 2 mins. Superscript® III reserve transcriptase, RNasin, DTT and reaction buffer was then added to the denatured samples and first strand synthesis was carried out in a PCR machine under the following conditions: 55°C, lOmins; 50°C 120mins; 85°C, lOmins; 4°C hold. Following first strand synthesis, the duplexed RNA was remove by an RNase H digest at 37°C for 20 min. Multiplex RT-PCR was then performed to amplify either KIF5B-RET variants or the CCDC6-Ret fusion using the primers (KIF5B-867F, KIF5B-1241F, KIF5B 2265F, KIF5B-272F CCDC6-197F, and RET-2381R).² PCR conditions for detecting the Ret fusion partners included an initial denaturation at 95°C for 5 min. followed by 10 cycles of touchdown PCR (annealing temperature ranging from 60°C to 55°C with a 0.5 decrease per cycle and a lmin extension at 72°C) and 30 cycles of PCR (annealing temperature at 55°C and lmin extension at 72°C). PCR products were resolved on a 1.5% agarose gel. PCR products were excised from agarose gel, gel purified (Wizard SV Gel and PCR Clean Up Kit; Promega) and sequenced at the University of Colorado DNA Sequencing Core.

RET Multiplex RT-PCR primers:

RET-2381 R: 5 '-CAGGCCCCATACAATTTGAT-3 ' (SEQ ID NO : 1 )

KIF5B 2265F: 5 '-AGCCACAGATCAGGAAAAGA-3 ' (SEQ ID NO:2)

KIF5B- 1241F: 5'-CTGATGCTGAAAGAAGAAAGTGT-3' (SEQ ID NO:3)

KIF5B-867F: 5 '- ATTAGGTGGCAACTGTAGAACC-3 ' (SEQ ID NO:4)

KIF5B-272F: 5 '-AGACACACACAATGGAGGGTA-3 ' (SEQ ID NO:5)

CCDC6-197F: 5 '-TGCAGCAAGAGAACAAGGTG-3 ' (SEQ ID NO:6)

Inverse PCR Assay

To determine the RET partner gene in RET split FISH-positive patients, we used an inverse

RT-PCR method. Double-stranded cDNA was synthesized from 2 μg of total RNA with 1 pM of the primer RET-2746R (SEQ ID NO:7, 5 '-TAACTGGAATCCGACCCTGG-3 ') and a cDNA Synthesis System (Roche), and was self-ligated by incubation overnight with T4 DNA ligase (TaKaRa Bio). We subjected the resulting circular cDNA to PCR (35 cycles at 94°C for 15 S, 62°C for 30 S, and 72°C for 1 min) with primers RET-2381R (SEQ ID NO: l) and RET-2271F (SEQ ID NO:8, 5 '-GAAGATGCTGAAAGAGAACGC-3 ') in a final volume of 20 μί. We subjected 1 μΐ, of the 1 : 100 diluted reaction products to a second PCR step (the same settings as above) with primers RET-2208R (SEQ ID NO:9, 5'-TCCAAATTCGCCTTCTCCTA-3') and RET-2605F (SEQ ID NO: 10, 5 '-AAGCTCGTTCATCGGGACT-3 ') in a final volume of 20 μί. The resulting products were purified by gel extraction and directly sequenced in both directions with primers RET-2605F and RET-2208R.

Example 4 illustrates the application of FISH probes to clinical specimens to detect all chromosomal rearrangements involving RET genes in NSCLC independently of the fusion partner.

A 3 'RET signal separated from the 5 'RET signal, indicates that the tyrosine kinase domain

(at 3 'RET) is free to fuse with any other gene that potentially could keep it active and therefore oncogenic. The FISH probes were used to detect the RET gene fusions in clinical samples obtained from NSCLC patients. The patient cohort was pan-negative, i.e. tested negative for EGFR and KRAS mutations and ALK and ROS1 fusions. As summarized in the table 5 below, RET rearrangement was detected in 8 out of 50 adeno- carcinoma specimens. Thus, RET rearrangement was detected in approximately 15% of samples. Frequency of RET rearrangements in this enriched lung adenocarcinoma cohort was considerably higher than reported in unselected, unenriched cohort of Wang ei al, JCO, 2012).

See figures 8 A and B for representative fluorescence images showing fusion of the green signal (5' RET) and red signal (3 'RET) indicating non-rearrangement of RET, and figures 8C, 9 A and B, for representative images showing a separation of the green signal (5' RET) and red signal (3 'RET) indicating rearrangement of RET. KIF5B:RET was detected in 5 samples (approximately 10% of samples) (Figure 8C). Figure 9A shows detection of CCDC6:RET fusion, whereas figure 9B shows a pattern indicative of RET fusion with an unknown partner.

Example 5 illustrates that NSCLC patients harboring a RET gene fusion, show clinical response to RET inhibitors.

To investigate the efficacy of RET inhibitors in the treatment of RET fusion-positive tumors, RET positive patients (from Example 4) were selected for administration of RET inhibitors. Four patients were given FDA approved drugs of vandetanib (2 patients) and sunitinib (2 patients), and the remaining four received no RET therapy. Figures 10A and 10B show case summaries of the two patients who received vandetanib. Patient LB (slide 42) was positive for KIF5B:RET fusion and was given therapy comprising administration of vandetanib. The disease was observed to be stable after 6 months. In patient MF, the disease was found to remain stable after two months. Out of the four patients who received no RET therapy, two died, one was unavailable and one is undergoing evaluation.

In another study, three patients were treated with the RET inhibitor cabozantinib on a prospective phase 2 trial for patients with RET fusion-positive NSCLCs. (Drillon et al., Cancer

Discovery Online, March 2013.) Confirmed partial responses were observed in two patients, including one harboring a novel TRIM33 :RET fusion. A third patient with a KIF5B:RET fusion has had prolonged stable disease approaching 8 months (31 weeks). All three patients remained progression- free on treatment. Details are shown in figure 1 1.

Those skilled in the art will appreciate, or be able to ascertain using no more than routine experimentation, further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references are herein expressly incorporated by reference in their entirety.

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Claims

What is claimed is

1. A method of identifying a NSCLC tumor as likely to be responsive to tyrosine kinase

inhibitors comprising:

(a) contacting a NSCLC tissue test sample with at least one KIF5B probe and at least two RET probes;

(b) detecting chromosomal rearrangement of the RET gene in the DNA from the test sample based on the hybridization pattern between the probes and the DNA from the test sample; and

(c) identifying the NSCLC tumor as likely to respond to specific tyrosine kinase inhibitors when chromosomal rearrangement is detected.

2. The method of claim 1 wherein at least two of the KIF5B and RET probes are labeled with a fluorescent label.

3. The method of claim 1 wherein the detecting step comprises fluorescence in situ hybridization (FISH) using break-apart (BA) probes specific for regions 5 ' and 3 ' of the common breakpoints in a rearranged RET gene.

4. The method of claim 1 wherein the KIF5B probes map at 1 Op 1 1.2 and the RET probes map at

10ql l .2.

5. The method of claim 1 wherein the probes comprise:

a) at least one 5 ' KIF5B probe that hybridizes with a sequence selected from the group consisting of:

nucleotide position 32,304,999 to position 32,493,021 of chromosome 10 (BAC clone

RP1 1-367F12),

nucleotide position 32,494,678 to position 32,649,872 of chromosome 10 (BAC clone RP1 1-166N17), and,

a nucleotide sequence that is at least 70% identical to either of these sequences;

b) at least one 5' RET probe that hybridizes with a sequence selected from the group consisting of:

nucleotide position 43,205,232 to position 43,412,434 of chromosome 10 (BAC clone RP13-397N12),

nucleotide position 43,404,932 to position 43,588,263 of chromosome 10 (BAC clone RP13-368N15),

nucleotide position 43,550,471 to position 43,605,967 of chromosome 10 (BAC clone CTD-3181E16), and,

a nucleotide sequence that is at least 70% identical to any of these sequences; and, c) at least one 3 ' RET probe that hybridizes with a sequence selected from the group consisting of:

nucleotide position 43,591,858 to position 43,780,564 of chromosome 10 (BAC clone RP1 1-322A19), nucleotide position 43,782,565 to position 43,958,528 of chromosome 10 (BAC clone RP1 1-347G1 1), and,

a nucleotide sequence that is at least 70% identical to any of these sequences.

6. The method of claim 5 wherein the probes a, b and c are labeled with fluorescent labels that are fluorescent in different regions of the spectrum.

7. The method of claim 5 wherein each of the probes a, b and c are labeled with one fluorescent label selected from SpectrumYellow, SpectrumGreen and SpectrumRed.

8. The method of claims 6 or 7 wherein probe a is labeled with SpectrumYellow, probe b is labeled with SpectrumGreen and probe c is labeled with SpectrumRed label.

9. The method of claim 5, wherein a hybridization pattern comprising fused signals

corresponding to probes a and c with separated signal corresponding to probe b indicates chromosomal rearrangement.

10. The method of claim 5, wherein a hybridization pattern comprising fused signals

corresponding to probes b and c with a separated signal corresponding to probe a indicates non-rearrangement of chromosomal genes.

1 1. The method of claim 5, wherein a hybridization pattern comprising the separation of signals corresponding to probes b and c indicates a break in RET.

12. The method of claim 11 , wherein RET is fused with another gene that is not KIF5B.

13. The method of Claim 1, wherein the detecting step identifies any RET chromosomal

rearrangement.

14. The method of Claim 1, wherein the detecting step identifies a RET chromosomal

rearrangement that activates the RET gene.

15. The method of claim 1, wherein the chromosomal rearrangement comprises fusion of the KIF5B and RET genes.

16. The method of claim 1, wherein the tyrosine kinase inhibitors are selected from the group consisting of sorafenib, imatinib, cabozantinib, ponatinib, and vandetanib.

17. The method of claim 1, wherein the sample comprises a surgical or biopsy specimen, a fine needle aspiration, fresh, frozen or paraffin embedded tissue, a tissue imprint, sputum, , or a bronchial lavage.

18. The method of claim 1, further comprising:

(d) confirming chromosomal rearrangement by reverse transcription polymerase chain reaction (RT-PCR).

19. The method of claim 15, wherein the RT-PCR is performed using mRNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections or frozen tissue sections from the NSCLC tissue test sample.

20. The method of claim 15, wherein a RT-PCR polynucleotide product indicates the presence of a fusion transcript that spans a fusion point that contains sequence from both a 5 ' partner gene and the RET gene.

21. The method of claim 17, wherein the RT-PCR polynucleotide product is analyzed by

evaluating the size of the product.

22. The method of claim 17, wherein the RT-PCR polynucleotide product is analyzed by direct sequencing of the product.

23. The method of claim 15, wherein the hybridization pattern of the probes to the DNA from the test sample is indicative of a chromosomal rearrangement and a RT-PCR polynucleotide product indicates the absence of a fusion transcript that spans a fusion point that contains sequence from both a 5' partner gene and the RET gene, further comprising:

(e) conducting inverse PCR to amplify cDNA from known nucleotide sequence in the RET gene to amplify and detect unknown, adjacent sequence from the 5' fusion gene partner.

24. An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 70%

identical to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.