WO2015038710A1 - Methods for diagnosing and treating intrahepatic cholangiocarcinoma - Google Patents

Abstract

Disclosed herein are methods for detecting in a biological sample from a patient the presence or absence of a fusion gene having a 5' portion from a fibroblast growth factor receptor 2 (FGFR2) gene or fragment thereof and a 3' portion from a Periphilin-1 (PPHLN1) gene or fragment thereof. The methods can further include diagnosing and treating the patient for intrahepatic cholangiocarcinoma.

Description

Methods for Diagnosing and Treating Intrahepatic

Cholangiocarcinoma

RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Patent

Application Serial No. 61/876,451, filed September 11, 2013, which is herein incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to the field of molecular biology, and more particularly to a novel fusion gene identified in intrahepatic cholangiocarcinoma (ICC).

STATEMENT RE SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 11, 2014, is named 27527-0130WOl_SL.txt and is 51,405 bytes in size.

BACKGROUND

Fusion gene events are known to be potent driver oncogenes involved in the pathogenesis of human cancer. The translational relevance of such fusion gene events has been limited so far, however, by the misguided notion that they are exclusively associated with hematological malignancies. The recent discovery of novel fusion gene events associated with different types of solid cancers, such as, e.g., prostate, lung, and breast cancer has resulted in a growing interest in these genetic alterations. In some cancers, the discovery of specific oncogenic loops has yielded striking benefits in terms of therapy, as recently reported in small cell lung cancer where the identification of the EML4-ALK tyrosine fusion gene has led to the successful development of a specific inhibitor, crizotinib, with clinical efficacy. Recently, certain fibroblast growth factor receptor (FGFR) fusion genes have been identified in a broad spectrum of solid cancers, including intrahepatic cholangiocarcinoma (ICC), suggesting that these fusion gene events may represent novel candidate therapeutic targets in solid cancers. ICC is a poorly understood bile duct cancer with very dismal prognosis and extremely limited therapeutic options. Without resection of the tumor at an early stage, this disease is uniformly fatal, and only palliative treatment is available. A first line conclusive treatment has not been established, and, even when curative resection is feasible, intrahepatic recurrence is highly frequent, worsening the outcome of these patients. Thus, improved methods for diagnosing ICC, as well as novel therapies for treating this disease, are greatly needed in the art.

SUMMARY

As follows from the Background section above, there is a need in the art for novel methods for diagnosing and treating ICC. It is presently discovered that a subset of ICC patients have a tyrosine kinase fusion gene containing portions of the fibroblast growth factor receptor 2 (FGFR2) (10q26) and Periphilin-1 (PPHLN1) (12ql2) genes (FGFR2-PPHLN1 fusion gene). It is also discovered that

overexpression of the FGFR2-PPHLN1 fusion gene promoted proliferation of ICC cell lines in vitro, and that treatment with specific FGFR2 inhibitors exhibited potent anti-tumoral effects. Thus, provided herein are novel methods for treating a patient with ICC, based on the presence of the FGFR2-PPHL 1 fusion gene in the subject. These and other related benefits are presently provided, and discussed in detail below.

In some aspects, provided herein is a method for treating ICC in a patient. The method can include (a) providing a biological sample from the patient; (b) detecting the presence or absence in the sample of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof or the polypeptide encoded by the fusion gene; and (c) treating the ICC if the presence of the fusion gene or polypeptide is detected.

In another aspect, provided herein is a method for diagnosing intrahepatic cholangiocarcinoma (ICC) in a patient. The method can include (a) providing a biological sample from the patient; (b) detecting the presence or absence in the sample of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof or the polypeptide encoded by the fusion gene; and (c) diagnosing the patient as suffering from ICC if the presence of the fusion gene or polypeptide is detected. In some aspects, the diagnosis is made in conjunction with detecting the presence of one or more other markers of ICC. N some aspects, the method further includes treating the patient's ICC. In some aspects, treating ICC includes administering a cancer therapy. For example, cancer therapies that may be administered can include e.g., chemotherapy (e.g.,

administering a chemotherapeutic agent), administering a biologic agent, e.g., antigen, vaccine, antibody etc., administering a cytokine, radiation therapy, immunotherapy, and/or surgery, etc.. The cancer therapy can be a therapy that inhibits at least one biological function of an FGFR2 fusion gene (e.g., FGFR2 -PPHLNl), e.g., an inhibitor of an FGFR2-PPHLN1 fusion gene or polypeptide.

In some aspects of the above methods for treating or diagnosing ICC, the FGFR2 gene or fragment thereof comprises the first 19 exons of FGFR2. In some aspects, step (b) can include detecting chromosomal rearrangements of genomic DNA having a 5' DNA portion from the FGFR2 gene or fragment thereof and a 3' DNA portion from the PPHLNl gene or fragment thereof. In some aspects, step (b) can include detecting chimeric mRNA transcripts having a 5' RNA portion transcribed from the FGFR2 gene or fragment thereof and a 3' RNA portion transcribed from the PPHLNl gene or fragment thereof. In some aspects, the biological sample is selected from the group consisting of tissue, blood, plasma, serum, urine, urine supernatant, urine cell pellet, tumor cells, and liver cells.

In some aspects of the above methods, step (b) can include determining the presence of the fusion gene using an automated sequencer. In some aspects, the detecting in step (b) can include detecting a polypeptide encoded by the fusion gene. In some aspects, the method can further include prescribing or administering therapy for ICC to the subject when it is determined that the fusion gene is present in the subject.

Also provided herein is a method for detecting the presence of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHLNl gene or fragment thereof in a human subject. In some aspects, the method can include: (a) obtaining nucleic acid from a biological sample collected from the subject; (b) analyzing the nucleic acid to determine its nucleotide sequence with an electronic computer sequencing device; (c) comparing, with software programmed to perform a base-by -base comparison, the nucleotide sequence of the nucleic acid to a control sequence comprising at least 25 consecutive nucleic acids of one of the following nucleic acid sequence: (i) if the nucleic acid comprises genomic DNA: TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

TATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAACA GTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13); (ii) if the nucleic acid comprises mRNA:

CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACUAGUU AA (SEQ ID NO: 12); and (iii) if the nucleic acid sequence comprises cDNA:

CGAATTCTCACTCTCACAACCAATGAGGATGGCTACAATAGACTAGTTAA

(SEQ ID NO: 14) or its complement:

TTAACTAGTCTATTGTAGCCATCCTCATTGGTTGTGAGAGTGAGAATTCG

(SEQ ID NO: 15); (d) displaying the comparison between the nucleotide sequence of the nucleic acid and the control sequence on an electronically operated (computer monitor) screen; and (e) determining that the fusion gene is present in the subject when the compared sequences are substantially identical; or (f) determining that the fusion gene is not present in the sample when the compared sequences are the not substantially identical. The skilled artisan will appreciate that shorter or longer sequences (relative to SEQ ID NOs: 9 and 12-15 or their respective complements) covering the fusion junction can be used to detect the fusion protein, depending on the specific assay being used and the desired level of specificity.

In the above method for detecting the presence of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHLNl gene or fragment thereof in a human subject, the biological sample can be obtained from a tumor biopsy. The method for detecting the presence of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHLNl gene or fragment thereof in a human subject can further include prescribing or

administering therapy for ICC to the subject when it is determined that the fusion gene is present in the subject.

In other aspects, provided herein is a method for determining the susceptibility of a cancer in a patient to a cancer therapy comprising administration of an inhibitor of the FGF pathway. In some aspects, the method can include (a) providing a biological sample from the patient; (b) detecting the presence or absence in the sample of a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHLNl gene or fragment thereof or the polypeptide encoded by the fusion gene; and (c) identifying the cancer as likely to be susceptible to the cancer therapy if the fusion gene or polypeptide is detected in the sample. In some aspects of the method for determining the susceptibility of a cancer in a patient to a cancer therapy, the cancer is ICC. In some aspects of the method for determining the susceptibility of a cancer in a patient to a cancer therapy, the method further includes prescribing or administering the therapy comprising administration of an FGF pathway inhibitor to the patient when the fusion gene or polypeptide is detected in the sample.

In any of the above methods the PPHL 1 gene or fragment thereof can encode a polypeptide that is capable of inducing the oligomerization of FGFR2. In any of the above methods, the FGFR2 gene or fragment thereof can encode one or more kinase domains and/or one or more tyrosine phosphorylation sites.

In any of the above methods, the FGFR2 gene can have a nucleic acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence variant thereof. In any of the above methods, the PPHLN1 gene can have a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence variant thereof. In some aspects of any of the above methods, the PPHLN1 gene can have a nucleic acid sequence comprising residues 178-1663 of SEQ ID NO: 3.

In any of the above methods, the amino terminal portion of the polypeptide encoded by the fusion gene can include a carboxy-terminally truncated portion of an FGFR2 polypeptide having the amino acid sequence identified by SEQ ID NO: 6 or a sequence variant thereof.

In any of the above methods, the carboxy terminal portion of the polypeptide encoded by the fusion gene can include an amino-terminally truncation portion of the PPHLN1 polypeptide having an amino acid sequence identified by SEQ ID NO: 7 or a sequence variant thereof.

In certain of the above methods, the therapy for treating ICC can include administering a therapy that targets the fusion gene or polypeptide encoded by the fusion gene. In certain of the above methods for identifying, diagnosing and/or treating ICC, the therapy for treating ICC can be an antisense therapy, a gene therapy, or an antibody therapy.

Also provided herein is a method for treating ICC in a subject in need thereof, wherein the method includes targeting in the subject a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof, or targeting a polypeptide encoded by the fusion gene. In some aspects, the targeting includes administering to the subject an inhibitor of the fusion gene or polypeptide encoded by the fusion gene. In some aspects, the inhibitor is an antisense molecule specific for the fusion gene. In some aspects, the inhibitor is an antibody specific for the polypeptide encoded by the fusion gene. In some aspects, the targeting can include administering gene therapy to the subject. In some aspects, the gene therapy is targeted to the FGFR2-PPHL 1 fusion gene.

Also provided herein is a method for inhibiting the development of ICC in a subject in need thereof. In some aspects, the method can include targeting in the subject a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof, or targeting a polypeptide encoded by the fusion gene. In some aspects, the targeting can include administering to the subject an inhibitor of the fusion gene or polypeptide encoded by the fusion gene. In some aspects, the inhibitor is an antisense molecule specific for the fusion gene. In some aspects, the inhibitor is an antibody specific for the polypeptide encoded by the fusion gene. In some aspects, the targeting comprises administering gene therapy to the subject. In some aspects, the gene therapy is targeted to the FGFR2-PPHLN1 fusion gene.

In any of the above methods, the fusion gene can include a fusion junction genomic nucleic acid sequence comprising:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13).

Also provided herein is a composition comprising at least one of the following: (a) an oligonucleotide probe comprising a sequence that hybridizes to a junction of a chimeric genomic DNA or chimeric mRNA in which a 5' portion of the chimeric genomic DNA or chimeric mRNA is from an FGFR2 gene or fragment thereof and a 3' portion of the chimeric genomic DNA or chimeric mRNA is from a PPHLN1 gene or fragment thereof; (b) a first oligonucleotide probe comprising a sequence that hybridizes to a 5' portion of a chimeric genomic DNA or chimeric mRNA from an FGFR2 gene or fragment thereof and a second oligonucleotide probe comprising a sequence that hybridizes to a 3' portion of the chimeric genomic DNA or chimeric mRNA from a PPHLN1 gene or fragment thereof; and (c) a first amplification oligonucleotide comprising a sequence that hybridizes to a 5' portion of a chimeric genomic DNA or chimeric mRNA from an FGFR2 gene or fragment thereof and a second amplification oligonucleotide comprising a sequence that hybridizes to a 3' portion of the chimeric genomic DNA or chimeric mRNA from a PPHLN1 gene or fragment thereof.

In some aspects of the composition, the junction of a chimeric genomic DNA comprises the sequence:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13).

In some aspects of the composition, the junction of a chimeric mRNA comprises the sequence:

CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACUAGUU AA (SEQ ID NO: 12).

In some aspects, the FGFR2 gene has a nucleic acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence variant thereof. In some aspects, the PPHLN1 gene has a nucleic acid sequence comprising SEQ ID NO: 3, or a sequence variant thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the present document, including definitions, will control.

All publications, patent applications, patents, and other references (e.g., GenBank® Accession Numbers) mentioned herein are incorporated by reference in their entireties. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. Other features, objects, and advantages of the present disclosure will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram illustrating the components of the FGFR2- PPHLN1 fusion mRNA. The FGFR2 portion is inverted relative to its normal orientation. FGFR2 is missing the last exon whereas PPHL l is missing the first 3 exons.

FIG. 2 is a photograph of a PCR gel, showing the presence or absence of the FGFR2-PPHL 1 fusion gene in 7 tumoral tissue samples (lanes 1 to 7 from left to right on right side of 100 base pair (bp) ladder) and in 7 matched normal tissue samples (lanes 1 to 7 from left to right on the left side of the ladder). The matched normal tissue sample in Lane 1 (left side of ladder) corresponds to the tumoral tissue sample in Lane 1 (right side of ladder), the matched normal tissue sample in Lane 2 (left side of ladder) corresponds to the tumoral tissue sample in Lane 2 (right side of ladder), and so on.

FIG. 3 contains a schematic diagram illustrating the FGFR2 -PPHLNl fusion gene, and the inset shows the cDNA sequence of the region around the fusion junction of the most abundant isoform of the fusion gene. The sequence in the inset corresponds to SEQ ID NO: 14:

CGAATTCTCACTCTCACAACCAATGAGGATGGCTACAATAGACTAGTTAA. The numbering in the diagram corresponds to the exons of each gene (from 5' to 3': FGFR2 exons 3b to 18 and exons 4 to l ib of PPHLNl).

FIG. 4 is photograph of a PCR gel showing the PCR products for the FGFR2- PPHLN1 fusion gene. In lane 2, the upper band corresponds to the long isoform of the fusion gene and the lower band corresponds to the shorter isoform of the fusion gene. A DNA size ladder is in Lane 1.

FIG. 5 contains schematic diagrams of the long and short isoforms of the FGFR2-PPHLN1 fusion gene. The long isoform (upper diagram) contains, from 5' to 3', exons 3b to 19 of FGFR2 (transcript variant 2, also known as FGFRIIIb, corresponding to GenBank® Accession No. NM_022970) (SEQ ID NO: 2) and exons 4 to l ib of PPHLNl (corresponding to GenBank® Accession No. NM_201439.1) (SEQ ID NO: 3). The short isoform (lower diagram) contains, from 5' to 3', exons 3b to 19 of FGFR2 (transcript variant 1 , corresponding to GenBank® Accession No. NM_001144913.1) (SEQ ID NO: 4) and exons 4 to l ib of PPHLNl (corresponding to GenBank® Accession No. NM_201439.1) (SEQ ID NO: 3).

FIG. 6A shows the predicted amino acid sequence of the long isoform of the FGFR2-PPHLN1 fusion gene, corresponding to SEQ ID NO: 5. The predicted fusion protein contains 1,111 amino acids (122 kDa), with the amino-terminal portion (residues 1-768) identical to that of the long isoform of FGFR2 (GenBank® accession number NP_075259.4) (SEQ ID NO: 6), whereas the carboxy-terminal portion (boxed portion in figure) (residues 769-1111 of the fusion protein) is identical to PPHLNl starting at residue 25 of the wild-type protein (accession number NP_958847.1) (SEQ ID NO: 7), up to the first stop codon in the boxed portion. The predicted amino acid sequences of four or more amino acid residues following each stop codon correspond to SEQ ID NO: 8, and 32-38, from left to right.

FIG. 6B contains a schematic diagram of the predicted domains of the polypeptide encoded by the FGFR2 -PPHLNl fusion gene, including three IgG domains and tyrosine phosphorylation sites (TK1 and TK2) in the FGFR2 region (amino acid residues 1 to 768 of the fusion gene). This diagram also shows that amino acid residues 769-821 of native FGFR2 and residues 1 to 24 of native PPHLNl are not present in the long isoform of the FGFR2 -PPHLNl fusion gene.

FIG. 7 is a schematic diagram illustrating the components of the FGFR2- PPHLN1 gene fusion genomic DNA and the nucleic acid sequence around the fusion junction (TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTT- CAGGTCTACTTAATTTAGTAATTCAGTTATATACATATTCCATAT) (SEQ ID NO: 9) (the dash in this sequence indicates that junction between the FGFR2 portion and the PPHLNl portion— not shown in Figure). The FGFR2 portion is inverted relative to its normal orientation. The numbers 123242678 and 42736824 in the diagram correspond to the DNA coordinates where the breakpoint occurs.

FIG. 8A-8B contain graphs of the Sanger sequencing results for six ICC tumor mRNA samples. These mRNA samples had an identical fusion cDNA sequence around the breakpoint (fusion junction) (TCACAACCAATGAG - GATGGCTACAATAG) (SEQ ID NO: 10).

FIG. 9 is an illustration of the ligand-dependent activation of FGFR2 and the constitutive activation in the case of the FGFR2 fusion polypeptides (e.g., FGFR2- PPHL 1 fusion polypeptide). FGFR2 signaling (including phosphorylation (circles labeled "P") of the tyrosine phosphorylation sites ("TK")) leads to proliferation, survival, and angiogenesis of tumor cells.

FIG. 10 is a photograph of a Western blot result in an immunoprecipitation experiment. Lysates of 293T cells that were transiently transfected with a control (empty vector) or the FGFR2-PPHL 1 fusion gene (long isoform) containing a V5 tag were prepared. Immunoprecipitation of the lysates was performed using an anti- V5 tag antibody, followed by immunoblotting for the V5 tag (left 2 lanes of blot) using anti-V5 antibody, or using an anti- phosphorylated tyrosine antibody (Anti- Phospho Tyr) (right two lanes of blot). The total lysates were also blotted and stained using antibodies specific for phospho- and total ERK, and for tubulin, as a loading control.

FIG. 11 is the complete predicted protein sequence of one isoform of the FGFR2-PPHLN1 fusion gene (corresponding to SEQ ID NO: 5).

FIG. 12 is a line graph quantifying the proliferation relative to day 1 on the Y- axis versus time (day) on the X-axis of 293T cells that were stably transfected with FGFR2-PPHLN1 fusion gene or empty vector ("ctr").

FIG. 13A is a schematic representation of the fusion protein FGFR2-PPHLN1. The normal FGFR2 and PPHLN1 protein are also represented. FIGs. 13B-C are bar graphs quantifying number of colonies (13B) and percent (%) viability of HUCCTl cell line transfected to overexpress FGFR2-PPHLN1 (right bar in each pair of bars) compared to its parental cell line transfected with the empty vector (left bar in each pair of bars). Results were normalized to the empty vector transfected control and presented as the mean of three independent experiments (mean +s.d.). P values are indicated in the bar graphs.

FIG. 14A and FIG 14B are bar graphs quantifying the percent (%) viability (14A) and number of migrated cells per field (14B) in HUCCTl cell line transfected to overexpress FGFR2-PPHLN1 and cultured in the presence of the indicated concentration of BGJ398 (0, 1, 2.5, and 5 μΜ) (14A) or 1 μιη (14B) (right bar in each pair of bars) compared to its parental cell line transfected with the empty vector (DMSO control) (left bar in each pair of bars). Results were normalized to the DMSO-treated control and presented as the mean of three independent experiments (mean +s.d.). P-values are indicated.

FIG. 15: contains the complete coding DNA sequence (CDS) of the fusion gene FGFR2-PPHL 1 amplified from the iCCA case where it was identified. The first underlined nucleotides "ATG" indicate the start codon. The second underlined sequence "GAT" indicates the start of the PPHL 1 sequence, following the FGFR2 sequence. The sequence shown corresponds to SEQ ID NO: 1.

FIG. 16 contains the genomic sequence around the breakpoint. The underlined nucleotides ("CAGG") indicate the start of the PPHL 1 portion around the breakpoint of the fusion, which corresponds to intron #3 of PPHL 1. This portion follows the sequence of FGFR2 around the breakpoint, which corresponds to intron #19 of FGFR2. The sequence shown corresponds to SEQ ID NO: 31.

DETAILED DESCRIPTION

Overview

As discussed above, ICC is a fatal bile duct cancer with very dismal prognosis and extremely limited therapeutic options. For patients at advanced stage, only palliative treatment is available, and a first line conclusive treatment has not been established. As described in the Examples, below, a novel tyrosine kinase fusion gene containing FGFR2 (10q26) and PPHLN1 (12ql2) genes in a subset of patients with ICC has been discovered. Further, overexpression of the fusion gene promoted proliferation of ICC cell lines in vitro. Treatment with a specific FGFR2 inhibitor also exhibited potent anti-tumoral effects. Thus, FGFR2-PPHLN1 fusion gene is presently identified as a novel therapeutic target for treating ICC and related cancers as well as a novel marker for identifying/diagnosing/treating a subset of ICC patients whose cancers are susceptible to treatment with an FGF pathway inhibitor.

Definitions:

As used herein, the term "biological sample" means a material obtained from the tissue (e.g., organ, connective tissue, bone, skin, tumor, etc.) and/or bodily fluid

(e.g., urine, blood, plasma, serum, urine supernatant, urine cell pellet, lymph, etc.) of a subject (e.g., mammal, e.g., human, e.g., patient). Non-limiting examples of such biological samples can be, e.g., a cell lysate obtained from a tissue or bodily fluid, nucleic acid, amino acid, protein, or other product obtained from and/or prepared from cells (e.g. tumor cells, e.g., ICC tumor cells, or, e.g., normal (non-cancerous) cells) obtained from the subject, or the cells themselves.

As used herein, the term "fusion gene" refers to a chimeric genomic DNA, a chimeric messenger RNA, a truncated protein or a chimeric protein resulting from the fusion of at least a portion of a first gene to at least a portion of a second gene. The fusion gene need not include entire genes or exons of genes.

As used herein, a cancer that is "susceptible" to a cancer therapy disclosed herein (e.g., treatment for cancer with an FGF pathway inhibitor (e.g., an inhibitor of FGF or an inhibitor of FGFR2)) means that the cancer cell, as a result of exposure to the cancer therapy, will have decreased growth and/or survival.

As used herein "decreasing the growth of a cancer cell" includes inhibiting the proliferation of a cancer cell and/or the killing of a cancer by, for example, the induction of necrosis or of apoptosis of the cancer cell.

The terms "therapeutically effective" and "effective amount," used interchangeably, refer to that quantity of a composition, compound or pharmaceutical formulation that is sufficient to reduce or eliminate at least one symptom of a disease or condition specified herein, e.g., cancer. When a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The dosage of the therapeutic formulation will vary, depending upon the nature of the disease or condition, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered, e.g., weekly, biweekly, daily, semi-weekly, etc., to maintain an effective dosage level. Therapeutically effective dosages can be determined stepwise by combinations of approaches such as (i) characterization of effective doses of the composition or compound in in vitro cell culture assays using tumor cell growth and/or survival as a readout followed by (ii) characterization in animal studies using tumor growth inhibition and/or animal survival as a readout, followed by (iii) characterization in human trials using enhanced tumor growth inhibition and/or enhanced cancer survival rates as a readout. As used herein, the "presence" of a fusion gene (e.g., FGFR2-PPHLN1 fusion gene) in a sample (e.g. a sample obtained from a subject (e.g., cancer patient)), means that DNA, mRNA, and/or cDNA encoding the fusion gene and/or the protein product of the fusion gene can be detected in a sample by any suitable method known in the art. For example but not limited to Northern blot, polymerase chain reaction (PCR), e.g., quantitative real-time, "QPCR", Western blot, immunoassay (e.g., ELISA), immunohistochemistry, cell immunostaining and fluorescence activated cell sorting (FACS), etc.

As used herein, the term "subject" means any mammal, and, in particular, a human, and can also be referred to, e.g., as an individual or patient. A non-human mammal can be, for example, and without limitation, a non-human primate (such as a monkey, baboon, gorilla, or orangutan), a bovine animal, a horse, a whale, a dolphin, a sheep, a goat, a pig, a dog, a feline animal (such as a cat), a rabbit, a guinea pig, a hamster, a gerbil, a rat, or a mouse.

As used herein, the term "wild-type", e.g., in the context of wild-type human FGFR2 and wild-type human PPHL 1 nucleic acid sequences, means that the sequence is the naturally occurring, non-mutated, non-translocated "normal" sequence.

As used herein, "treating" or "treatment" of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical or sub-clinical symptoms of the state, disorder or condition developing in a mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and/or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of

maintenance treatment) or at least one clinical or sub-clinical symptom thereof; and/or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms; and/or (4) causing a decrease in the severity of one or more symptoms of the disease. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

As used herein, the term "treating cancer" means causing a partial or complete decrease in the rate of growth of a tumor, and/or in the size of the tumor and/or in the rate of local or distant tumor metastasis, and/or the overall tumor burden in a subject, and/or any decrease in tumor survival, in the presence of an inhibitor (e.g., an FGF pathway inhibitor, e.g., an FGFR2 inhibitor) or other cancer therapy described herein. As used herein, "inhibiting the development of a cancer (e.g., ICC)" means arresting the development of the cancer prior to metastasis or a relapse thereof (in case of maintenance treatment).

The therapeutic methods disclosed herein can be administered with an additional therapy as part of a "combination therapy." For example, additional therapies can include an additional treatment for cancer (e.g., chemotherapy (e.g., administering a chemotherapeutic agent), administering a biologic agent, e.g., antigen, vaccine, antibody etc., administering a cytokine, radiation therapy, immunotherapy, and/or surgery, etc.) including e.g., a therapy that inhibits at least one biological function of an FGFR2 fusion gene (e.g. FGFR2-PPHLN1). Such combination therapy can be sequential therapy wherein the patient is treated first with one therapy and then the other, and so on, or all therapies can be administered simultaneously. In either case, these therapies are said to be coadministered. It is to be understood that "coadministered" does not necessarily mean that the drugs and/or therapies are administered in a combined form (i.e., they may be administered separately or together to the same or different sites at the same or different times).

As used herein, the term "inhibits at least one biological activity of a fusion gene" refers to any agent that decreases any activity of a fusion gene disclosed herein (e.g., FGFR2-PPHLN1) (e.g., including, but not limited to, the activities described herein), via directly contacting fusion gene protein, contacting fusion gene mRNA or genomic DNA, causing conformational changes of fusion gene polypeptides, decreasing fusion gene protein levels, or interfering with fusion gene interactions with signaling partners, and affecting the expression of fusion gene target genes. Inhibitors also include molecules that indirectly regulate fusion gene biological activity by intercepting upstream or downstream signaling molecules. In a specific embodiment, an inhibitor is an inhibitor of an FGF signaling pathway (e.g., an FGFR2 or FGF inhibitor). Such inhibitors are known in the art (see, e.g., Daniele et al. Curr Oncol Rep. 2012 Apr; 14(2): l l l-9).

As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. As used herein, the term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, solvate or prodrug, e.g., ester, of a compound of the present disclosure, which upon administration to the recipient is capable of providing (directly or indirectly) a compound described herein, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1 : Principles and Practice. Pharmaceutically acceptable derivatives include salts, solvates, esters, carbamates, and/or phosphate esters.

The terms, "polypeptide" and "protein" are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.

As used herein, the term "nucleic acid" or "oligonucleotide" refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term also encompasses nucleic-acid-like structures with synthetic backbones, examples of which are described below. The term nucleic acid is used

interchangeably with cDNA, cRNA, mRNA, oligonucleotide, probe and amplification product.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity and/or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length polypeptide or fragment thereof are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

As used herein, the term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

The term "nucleic acid hybridization" refers to the pairing of complementary strands of nucleic acids. The mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of nucleic acids. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). See Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length. Examples of high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, followed by washing in 0. lx SSC/0.1% SDS (where lx SSC is 0.15 M NaCl, 0.15 M Na citrate) at 68°C or for oligonucleotide (oligo) inhibitors washing in 6xSSC/0.5% sodium pyrophosphate at about 37°C (for 14 nucleotide-long oligos), at about 48°C (for about 17 nucleotide- long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60°C (for 23 nucleotide-long oligos).

Conditions of intermediate or moderate stringency (such as, for example, an aqueous solution of 2xSSC at 65°C; alternatively, for example, hybridization to filter- bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C followed by washing in 0.2 x SSC/0.1% SDS at 42°C) and low stringency (such as, for example, an aqueous solution of 2xSSC at 55°C), require correspondingly less overall

complementarity for hybridization to occur between two sequences. Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA or RNA molecule and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol. 1975;98:503; Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 2, ch. 9.50, CSH

Laboratory Press, 1989; Ausubel et al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes part I, chapt 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, N.Y ("Tijssen").

As used herein, the term "standard hybridization conditions" refers to hybridization conditions that allow hybridization of two nucleotide molecules having at least 50% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 75% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

As used herein, the term "substantially identical" in the context of two or more nucleic acid sequences, means having at least 90% sequence identity. As used herein, a sequence variant of a given sequence, e.g. an FGFR2 or PPHLNl nucleic acid or amino acid sequence, is a sequence that has at least 90% sequence identity, and can be shorter or longer than the given sequence. As used herein, sequence variants of a gene are also referred to as "isoforms." Various isoforms of FGFR2 and PPHLNl have been determined to be present in the human population. For example, certain isoforms of FGFR2 (identified herein by SEQ ID NO: 3) and PPHLNl (e.g., SEQ ID NO: 7) are prevalent in the human population; however, other isoforms including isoforms that can be longer or shorter than those prevalent isoforms, and that can have one or more nucleic acid and/or amino acid substitutions, are also encompassed by the present disclosure. In particular, the methods and composition disclosed herein encompass FGFR2-PPHLN1 gene fusions that comprise any known or to-be- discovered sequences of FGFR2 and/or PPHLNl genes, so long as the sequence at the breakpoint (fusion junction) is conserved, as discussed below.

As used herein, the phrase "under hybridization conditions" means under conditions that facilitate specific hybridization of a subset of capture oligonucleotides to complementary sequences present in the cDNA or cRNA. The terms "hybridizing specifically to" and "specific hybridization" and "selectively hybridize to," as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under at least moderately stringent conditions, and preferably, highly stringent conditions, as discussed above.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base- pairing rules. For example, the sequence "5'-A-G-T-3'," is complementary to the sequence "3'-T-C-A-5'." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.

The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid is "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target that is substantially non- complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

A gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other (i.e. substantially identical to each other as defined above). When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "amplification oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid, or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligonucleotide is a "primer" that hybridizes to a template nucleic acid and contains a 3' OH end that is extended by a polymerase in an amplification process. Another example of an amplification oligonucleotide is an oligonucleotide that is not extended by a polymerase (e.g., because it has a 3' blocked end) but participates in or facilitates amplification. Amplification oligonucleotides may optionally include modified nucleotides or analogs, or additional nucleotides that participate in an amplification reaction but are not complementary to or contained in the target nucleic acid.

Amplification oligonucleotides may contain a sequence that is not complementary to the target or template sequence. For example, the 5' region of a primer may include a promoter sequence that is non-complementary to the target nucleic acid (referred to as a "promoter-primer"). Those skilled in the art will understand that an amplification oligonucleotide that functions as a primer may be modified to include a 5' promoter sequence, and thus function as a promoter-primer. Similarly, a promoter-primer may be modified by removal of, or synthesis without, a promoter sequence and still function as a primer. A 3' blocked amplification oligonucleotide may provide a promoter sequence and serve as a template for polymerization (referred to as a "promoter-provider").

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing to at least a portion of another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present disclosure will be labeled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present disclosure be limited to any particular detection system or label.

The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature, and includes, e.g., cDNA. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single- stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

As used herein, the term "purified" or "to purify" refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target- reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

The following examples are meant to illustrate, not limit, the disclosure.

I. Fusion Genes

The present disclosure identifies recurrent FGFR2-PPHL 1 fusion genes indicative of the presence of ICC. While not intending to be limited to any one particular theory or mechanism of action, the FGFR2 -PPHL l fusion genes are presently discovered to be the result of a chromosomal rearrangement of a fibroblast growth factor receptor 2 (FGFR2) gene and Periphilin 1 (PPHLNl) gene. Despite their recurrence, the length of the FGFR2-PPHLN1 fusion gene and the encoded fusion polypeptide vary, as a result of the occurrence of multiple transcript variants of FGFR2 present in the human population. The fusion genes disclosed herein typically comprise a 5' portion from FGFR2 gene and a 3' portion from PPHLNl gene. The recurrent fusion genes have use as diagnostic markers and clinical targets for ICC.

As discussed above, various isoforms of FGFR2 and PPHLNl have been determined to be present in the human population. Certain isoforms of FGFR2 (identified herein by SEQ ID NO: 3) and PPHLNl (e.g., SEQ ID NO: 7) are prevalent in the human population; however, other isoforms including isoforms that are longer or shorter than the prevalent isoform are also present, as well as sequence variants of any of these isoforms that can have one or more nucleic acid and/or amino acid substitutions. Thus, the methods and composition disclosed herein encompass FGFR2-PPHL 1 gene fusions that comprise any known or to-be-discovered sequences of the FGFR2 and/or PPHLN1 genes. It is discovered that the FGFR2- PPHL 1 genes comprise a conserved fusion junction sequence at the site where FGFR2 is fused to PPHL 1 in the genomic DNA. This feature is shared by the FGFR2-PPHL 1 fusion genes encompassed by the present disclosure.

As discussed above, the fusion genes and encoded fusion polypeptides encompassed by the present disclosure are not limited to any one particular nucleic acid or amino acid sequence for the FGFR2 and PPHL 1 portions present therein. However, by way of non-limiting example, the following sequences for those genes and encoded polypeptides are provided. The skilled artisan will appreciate how to determine other possible sequences (e.g., sequence variants, e.g., isoforms) for those genes and encoded polypeptides by referring to publicly available sequence databases (e.g., NBCI) and the literature.

By way of example, a human FGFR2 gene can have, comprise, or consist of the nucleic acid sequence set forth in GenBank® Accession No. NM_022970 (SEQ ID NO: 2), which has 4,657 base pairs (bp). In another example, the human FGFR2 gene has a nucleic acid sequence set forth in GenBank® Accession No.

NM_001 144913.1 (SEQ ID NO: 4). The corresponding polypeptides encoded by these human FGFR2 gene sequences are set forth in GenBank® Accession No.

NP_075259.4 (SEQ ID NO: 6) and GenBank® Accession No. NP_001 138385.1 (SEQ ID NO: 1 1), respectively.

In some embodiments, a fusion gene according to the present disclosure comprises a fragment of an FGFR2 gene. Fragments of an FGFR2 gene can comprise any portion of the FGFR2 gene that has a shorter nucleic acid sequence than a full length FGFR2 gene sequence (e.g. as set forth in SEQ ID NOs: 2 and 4). For example a fragment of an FGFR2 gene, can comprise 4,656 bp of SEQ ID NO: 2, or fewer. In some embodiments, a fusion gene of the present disclosure comprises 10-4656, 10- 4500, 50-4500, 100-4000, 200-3500, 300-3000, 400-2500, 500-2000, or 1000-1500 consecutive bp of an FGFR2 gene (e.g., SEQ ID NO: 2 and SEQ ID NO: 4). In an exemplary, non-limiting embodiment, a fusion gene comprises an FGFR2 portion that encodes a polypeptide that lacks residues 769-821 of the native FGFR2 polypeptide sequence set forth in SEQ ID NO: 6. In some embodiments, the FGFR2 gene or fragment thereof contains one or more (e.g., 1 or 2) tyrosine kinase (TK) domains of the native gene. In another embodiment, the FGFR2 gene or fragment thereof comprises all two of the TK domains of the native gene. In some embodiments, the FGFR2 gene or fragment thereof comprises one or more (e.g., 1, 2, or 3) immunoglobulin domains (e.g., IgG domains). Typically, although not necessarily, a fragment of an FGFR2 gene encompassed herein encodes a polypeptide that retains the ability to initiate a signaling cascade following oligomerization (e.g., dimerization). Typically, although not necessarily, a fragment of an FGFR2 gene encompassed herein encodes a polypeptide is constitutively activated (e.g., constitutive FGFR2 signaling in the absence of the receptor's ligand).

The FGFR2 gene is highly conserved. Thus, also encompassed herein are fusion genes which comprise a homolog of the human FGFR2 gene. Such homologs include, e.g., FGFR2 genes from chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, fruit fly, and mosquito.

By way of non-limiting example, a fusion gene of the present disclosure can comprise all or a portion (e.g., fragment) of a PPHLNl gene. In some embodiments, the PPHLNl gene comprises exons 4 to 1 lb of human PPHLNl (corresponding to GenBank® Accession No. NM_201439.1) (SEQ ID NO: 3) or a fragment thereof. However, as discussed above, also encompassed herein are sequence variants of PPHLNl and isoforms thereof. By way of non-limiting example, the fusion genes encompassed herein can comprise a fragment that encodes a polypeptide comprising residues 25 - 458 of the sequence set forth in GenBank® Accession Number NP_958847.1) (SEQ ID NO: 7); of course, the skilled artisan will appreciate that fragments of other lengths are also encompassed by the FGFR2 -PPHLNl fusion genes and fusion polypeptides encoded by the FGFR2-PPHLN1 fusion genes disclosed herein.

Fragments of PPHLNl gene can comprise any portion of the PPHLNl gene that has a shorter nucleic acid sequence than a full length PPHLNl gene sequence (e.g. as set forth in SEQ ID NO: 3). For example a fragment of a human PPHLNl gene can comprise 1,676 bp of SEQ ID NO: 3, or fewer. In some embodiments, a fusion gene of the present disclosure comprises 10-1,676, 10-1600, 50-1500, 100- 1500, 200-1500, 300-1500, 400-1500, 500-1500, or 1000-1500 consecutive bp of an PPHLNl gene (e.g., as set forth in SEQ ID NO: 3). Thus, in some embodiments, a fragment of a human PPHLNl gene encodes a fragment of the polypeptide having an amino acid sequence set forth in GenBank® Accession Number NP_958847.1 (SEQ ID NO: 7).

Preferably, although not necessarily, a fragment of a PPHLNl gene encompassed by the fusion genes disclosed herein encodes a polypeptide that retains that ability to cause constitutive activation of the FGFR2 polypeptides in the fusion genes disclosed herein. In some embodiments, the PPHLNl gene or fragment thereof encodes a polypeptide that is capable of inducing the oligomerization of FGFR2.

The PPHLNl gene is highly conserved. Thus, also encompassed herein are fusion genes which comprise a homolog of the human PPHLNl gene. Such homologs include, e.g., PPHLNl genes from chimpanzee, Rhesus monkey, dog, mouse, rat, chicken, and zebrafish.

In one embodiment, the FGFR2-PPHLN1 fusion gene comprises a fusion junction that comprises a DNA sequence that encodes an mRNA sequence corresponding to the following cDNA sequence:

TCACAACCAATGAGGATGGCTACAATAG (SEQ ID NO: 10) (as shown in Fig. 8), or its complement: CTATTGTAGCCATCCTCATTGGTTGTGA (SEQ ID NO: 32).

In another embodiment, the FGFR2 -PPHLNl fusion gene comprises a fusion junction which comprises the following mRNA sequence:

CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACU AGUUAA (SEQ ID NO: 12).

In one embodiment, an FGFR2 -PPHLNl fusion gene of the present disclosure has a fusion junction which comprises the following genomic nucleic acid sequence: TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9) (as shown in Fig. 7), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO : 13).

In one embodiment, a fusion gene of the present disclosure comprises a nucleic acid sequence encoding an mRNA sequence at the fusion junction which comprises a sequence corresponding to the following cDNA sequences:

CGAATTCTCACTCTCACAACCAATGAGGATGGCTACAATAGACTAGTTAA

(SEQ ID NO: 14) (as shown in Fig. 3), or its complement:

TTAACTAGTCTATTGTAGCCATCCTCATTGGTTGTGAGAGTGAGAATTCG

(SEQ ID NO: 15).

In certain embodiments, the fusion junction of FGFR2-PPHLN1 fusion gene comprises at least 25 consecutive nucleic acids of any of the above sequences for the fusion junction.

In another embodiment, a fusion polypeptide encoded by an FGFR2-PPHLN1 fusion encompassed by the present disclosure comprises the following amino acid sequence at the fusion junction:

LYMMMRDCWHAVPSQRPTFKQLVEDLDRILTLTTNEDGYNRLVNIVPKKPPL LDRPGEGSYNRYYSHVDY (SEQ ID NO: 18).

By way of example, an FGFR2-PPHLN1 fusion gene product (fusion protein) encompassed herein can comprise the amino acid sequence set forth in SEQ ID NO: 5 (shown in Fig. 6A and Fig. 11). In this embodiment, the fusion protein comprises 1,111 amino acids (122 kDa), with the amino-terminal portion (residues 1-768) identical to that of the long isoform of FGFR2 (accession number NP_075259.4) (SEQ ID NO: 6), and the carboxy-terminal portion (residues 769-1111 of the fusion protein) is identical to PPHLN1 at residue 25 - 458 of the wild-type protein (accession number NP_958847.1) (SEQ ID NO: 7). However, as discussed above, it is to be appreciated that the FGFR2-PPHLN1 fusion genes and encoded fusion polypeptides can comprise FGFR2 and PPHLN1 amino acid sequences that differ from the above exemplary amino acid sequences, since many isoforms of the FGFR2 and PPHLN1 gene are present in the human population.

II. Inhibitors

i) Small Molecules

Chemical agents, referred to in the art as "small molecule" compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000 Da, preferably less than 5,000 Da, more preferably less than 1,000 Da, and most preferably less than 500 Da. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified utilizing the screening methods known in the art. Methods for generating and obtaining small molecules are well known in the art (see, e.g., Schreiber, Science 2000; 151 : 1964- 1969; Radmann et al, Science 2000; 151 : 1947-1948).

In certain embodiments, encompassed herein are small molecule inhibitors of FGFR2, e.g., FGFR2 in the context of an FGFR2-PPHLN1 fusion polypeptide. Non- limiting examples of small molecule inhibitors encompassed by the methods disclosed herein include, e.g., AZD4547 (N-(5-(3,5-dimethoxyphenethyl)-lH-pyrazol-3-yl)-4- ((3S,5R)-3,5-dimethylpiperazin-l-yl)benzamide) (AstraZeneca), Ponatinib (3-(2- Imidazo[l,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methylpiperazin-l- yl)methyl]-3-(trifluoromethyl)phenyl]benzamide) (Ariad), Dovitinib (4-Amino-5- fluoro-3 - [6-(4-methyl- 1 -piperazinyl)- 1 H-benzimidazol-2-yl] -2(1 H)-quinolinone 2- hydroxypropanoate) (Novartis), BGJ398 (3-(2,6-dichloro-3,5-dimethoxyphenyl)-l-(6- ((4-(4-ethylpiperazin- 1 -yl)phenyl)amino)pyrimidin-4-yl)- 1 -methylurea) (Novartis), E- 3810 (6-((7-(( 1 -aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yl)oxy)-N-methyl- 1-naphthamide ) (EOS Pharmaceuticals), JNJ-42756493 (Astex/Janssen), ARQ 807 (ArQule), ENMD2076 ((E)-N-(5-methyl- 1 H-pyrazol-3 -yl)-6-(4-methylpiperazin- 1 - yl)-2-styrylpyrimidin-4-amine)(CASI Pharmaceuticals), LY2874455 ((R)-(E)-2-(4-(2- (5-( 1 -(3 ,5 -Dichloropyridin-4-yl)ethoxy)- 1 H-indazol-3yl)vinyl)- 1 H-pyrazol- 1 - yl)ethanol) (Lilly), Brivanib ((S)-(R)-l-((4-((4-fluoro-2-methyl-lH-indol-5-yl)oxy)-5- methylpyrrolo[2,l-f][l,2,4]triazin-6-yl)oxy)propan-2-yl 2-aminopropanoate) (Bristol- Myers Squibb), Nintedanib (BIBF 1120) (methyl (3Z)-3-{[(4-{methyl[(4- methylpiperazin-l-yl)acetyl]amino}phenyl)amino](phenyl)methylidene}-2-oxo-2,3- dihydro-lH-indole-6-carboxylate) (Boehringer Ingelheim), and Stivarga (BAY 734506) (4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3- fluorophenoxy]-N-methylpyridine-2-carboxamide hydrate) (Bayer).

Other suitable small molecules and methods for screening for suitable small molecule inhibitors are known in the art. By way of example, in vitro screening assays can be used to identify small molecules that inhibit FGFR2 or PPHLN1 expression and/or activity in a cancer cell. For an exemplary assay, see Example 5, below. Other suitable assays known in the art may also be used. ii) Antibodies

The fusion proteins of the present disclosure, including fragments, derivatives and analogs thereof, may be used as immunogens to produce antibodies having use in the diagnostic, research, and therapeutic methods described below. The antibodies may be, by way of example, and without limitation, polyclonal or monoclonal, chimeric, humanized, or single chain or Fab fragments. Various procedures known to those of ordinary skill in the art may be used for the production and/or labeling of such antibodies and fragments. [See, e.g., Burns, ed., Immunochemical Protocols, 3^rd ed., Humana Press (2005); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Kozbor et al, Immunology Today 4: 72 (1983); Kohler and Milstein, Nature 256: 495 (1975).]

In some embodiments, an antibody encompassed herein is an inhibitory antibody that binds to a portion of an FGFR2-PPHLN1 fusion protein and inhibits (is an antagonist of) at least one function of the fusion protein. The antibody may bind to a portion of FGFR2, or a portion of PPHLN1, or a portion that includes the junction region between the two polypeptides and thus includes a portion of FGFR2 and a portion of PPHL 1 in the binding region of the antibody. In a specific embodiment, an antagonistic anti-FGFR2-PPHL l antibody inhibits a pro-oncogenic function of the fusion protein and/or has anti-tumoral effects. In one embodiment,the antagonistic antibody inhibits tumor cell proliferation.

In another embodiment, a therapeutic antibody encompassed herein is an antibody-drug conjugate. For example, an antibody targeted to FGFR2 or PPHL 1 can be conjugated to an agent that inhibits one or more oncogenic properties of a cancer cell and/or that causes cell death, and/or mediates another desirable function for treating IHCC. A non-limiting example of such an antibody is FGFR2-ADC, available from Bayer (Pittsburgh, PA). Other FGFR2-specific antibodies are well known in the art, e.g., BAY 1163877 (Bayer), and BAY1 179470 (Bayer). iii) DNA and RNA Detection

The fusion genes of the present disclosure may be detected as chromosomal rearrangements of genomic DNA or chimeric mRNA using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and, nucleic acid amplification. A. Sequencing

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing [see, e.g., Sanger F, Coulson AR (May 1975). "A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase". J. Mol. Biol. 94 (3): 441-8; and Sanger F, et al (December 1977). "DNA sequencing with chain-terminating inhibitors". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5463-7]. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack, experimentally, RNA is usually reverse transcribed to DNA before sequencing.

Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive, or other labeled, oligonucleotide primer complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, standard four deoxynucleotide bases, and a low concentration of one chain terminating nucleotide, most commonly a di-deoxynucleotide. This reaction is repeated in four separate tubes with each of the bases taking turns as the di-deoxynucleotide. Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular di- deoxynucleotide is used. For each reaction tube, the fragments are size-separated by electrophoresis in a slab polyacrylamide gel or a capillary tube filled with a viscous polymer. The sequence is determined by reading which lane produces a visualized mark from the labeled primer as you scan from the top of the gel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di- deoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength.

B. Hybridization

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot. In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. The probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.

C. FISH

In some embodiments, fusion sequences are detected using fluorescence in situ hybridization (FISH). For example, FISH assays for the present disclosure can utilize bacterial artificial chromosomes (BACs). These have been used extensively in the human genome sequencing project (see Nature 409: 953-958 (2001)) and clones containing specific BACs are available through distributors that can be located through many sources, e.g., NCBI. Each BAC clone from the human genome has been given a reference name that unambiguously identifies it. These names can be used to find a corresponding GenBank® sequence and to order copies of the clone from a distributor.

Guidance regarding methodology may be obtained from many references including, e.g.: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In situ Hybridization: In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England

(1992) ); Kuo, et al, Am. J. Hum. Genet. 49: 112-1 19 (1991); Klinger, et al, Am. J. Hum. Genet. 51 :55-65 (1992); and Ward, et al, Am. J. Hum. Genet. 52:854-865

(1993) ). There are also kits that are commercially available and that provide protocols for performing FISH assays (available from e.g., Oncor, Inc., Gaithersburg, Md.). Patents providing guidance on methodology include U.S. Pat. Nos. 5,225,326;

5,545,524; 6, 121,489 and 6,573,043.

D. Microarrays

In some embodiments, fusion genes can be detected using microarray (e.g., DNA microarrays (e.g., cDNA microarrays and oligonucleotide microarrays). A DNA microarray, commonly known as gene chip, DNA chip, or biochip, is a collection of microscopic DNA spots attached to a solid surface (e.g., glass, plastic or silicon chip) forming an array for the purpose of expression profiling or monitoring expression levels for thousands of genes simultaneously. The affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray. Microarrays can be used to identify disease genes by comparing gene expression in disease and normal cells. Microarrays can be fabricated using a variety of technologies, including but not limiting: printing with fine-pointed pins onto glass slides; photolithography using pre-made masks; photolithography using dynamic micromirror devices; ink-jet printing; or, electrochemistry on microelectrode arrays.

E. Southern and Northern Blotting

Southern and Northern blotting is used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from a sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter. The filter bound DNA or RNA is subject to hybridization with a labeled probe complementary to the sequence of interest. Hybridized probe bound to the filter is detected. A variant of the procedure is the reverse Northern blot, in which the substrate nucleic acid that is affixed to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from a tissue and labeled.

F. Amplification

Chromosomal rearrangements of genomic DNA and chimeric mRNA may be amplified prior to or simultaneous with detection. Illustrative, non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), RNA sequencing (see, e.g., Example 1), and nucleic acid sequence based amplification (NASBA). Those of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require that RNA be reversed transcribed to DNA prior to amplification (e.g., RT-PCR), whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800, 159 and 4,965, 188), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. For other various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683, 195, 4,683,202 and 4,800,159; Mullis et al, Meth. Enzymol. 155: 335 (1987); and, Murakawa et al, DNA 7: 287 (1988)).

Transcription mediated amplification (see, e.g., U.S. Pat. Nos. 5,480,784 and 5,399,491), commonly referred to as TMA, synthesizes multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH in which multiple RNA copies of the target sequence autocatalytically generate additional copies. See, e.g., U.S. Pat. Nos.

5,399,491 and 5,824,518. In a variation described in U.S. Pub. No. 2006/0046265, TMA optionally incorporates the use of blocking moieties, terminating moieties, and other modifying moieties to improve TMA process sensitivity and accuracy.

The ligase chain reaction (see, e.g., Weiss, R., Science 254: 1292 (1991)), commonly referred to as LCR, uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid. The DNA

oligonucleotides are covalently linked by a DNA ligase in repeated cycles of thermal denaturation, hybridization and ligation to produce a detectable double-stranded ligated oligonucleotide product.

Strand displacement amplification (see, e.g., Walker, G. et al., Proc. Natl. Acad. Sci. USA 89: 392-396 (1992); and U.S. Pat. Nos. 5,270, 184 and 5,455, 166), commonly referred to as SDA, uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTPaS to produce a duplex hemiphosphorothioated primer extension product, endonuclease- mediated nicking of a hemimodified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3' end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product.

Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequence based amplification (see, e.g., U.S. Pat. No. 5,130,238), commonly referred to as NASBA; one that uses an RNA replicase to amplify the probe molecule itself (see, e.g., Lizardi et al, BioTechnol. 6: 1197 (1988)), commonly referred to as QP replicase; a transcription based amplification method (see, e.g., Kwoh et al, Proc. Natl. Acad. Sci. USA 86: 1 173 (1989)); and, self-sustained sequence replication (see, e.g., Guatelli et al, Proc. Natl. Acad. Sci. USA 87: 1874 (1990)). For further discussion of known amplification methods, see, e.g., Persing, David FL, "In vitro Nucleic Acid Amplification Techniques" in Diagnostic Medical Microbiology:

Principles and Applications (Persing et al, Eds.), pp. 51-87 (American Society for Microbiology, Washington, D.C. (1993)).

In addition to the detection methods described above, non-amplified or amplified fusion gene nucleic acids can be detected by any conventional means. For example, the fusion genes can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Illustrative non-limiting examples of detection methods are described below.

One illustrative detection method, the Hybridization Protection Assay (HP A) involves hybridizing a chemiluminescent oligonucleotide probe (e.g., an acridinium ester-labeled (AE) probe) to the target sequence, selectively hydrolyzing the chemiluminescent label present on unhybridized probe, and measuring the chemiluminescence produced from the remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283, 174 and Norman C. Nelson et al, Nonisotopic Probing, Blotting, and Sequencing, ch. 17 (Larry J. Kricka ed., 2d ed. 1995).

Another illustrative detection method provides for quantitative evaluation of the amplification process in real-time. Evaluation of an amplification process in "real-time" involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction, and using the determined values to calculate the amount of target sequence initially present in the sample. A variety of methods for determining the amount of initial target sequence present in a sample based on real-time amplification are well known in the art. These include methods are disclosed, e.g., in U.S. Pat. Nos. 6,303,305 and 6,541,205.

Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification, is disclosed, e.g., in U.S. Pat. No. 5,710,029.

Amplification products may be detected in real-time through the use of various self-hybridizing probes, most of which have a stem-loop structure. Such self- hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence. By way of non-limiting example, "molecular torches" are a type of self-hybridizing probe that includes distinct regions of self-complementarity (referred to as "the target binding domain" and "the target closing domain") which are connected by a joining region (e.g., non-nucleotide linker) and which hybridize to each other under predetermined hybridization assay conditions. In one embodiment, molecular torches contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification reaction under strand displacement conditions. Under strand displacement conditions, hybridization of the two complementary regions, which may be fully or partially complementary, of the molecular torch is favored, except in the presence of the target sequence, which will bind to the single-stranded region present in the target binding domain and displace all or a portion of the target closing domain. The target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e.g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized molecular torches. Molecular torches and a variety of types of interacting label pairs are disclosed, e.g., in U.S. Pat. No. 6,534,274.

Another example of a detection probe having self-complementarity is a "molecular beacon." Molecular beacons include nucleic acid molecules having a target complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are disclosed, e.g., in U.S. Pat. Nos. 5,925,517 and 6, 150,097.

Other self-hybridizing probes are well known to those of ordinary skill in the art. By way of non-limiting example, probe binding pairs having interacting labels, such as those disclosed, e.g., in U.S. Pat. No. 5,928,862, might be adapted for use in the present disclosure. Probe systems used to detect single nucleotide polymorphisms (SNPs) may also be utilized in the present disclosure. Additional detection systems include "molecular switches," as disclosed, e.g., in U.S. Pub. No. 2005/0042638. Other probes, such as those comprising intercalating dyes and/or fluorochromes, are also useful for detection of amplification products in the present disclosure. See, e.g., U.S. Pat. No. 5,814.

III. Protein Detection

The fusion genes of the present disclosure may be detected as truncated FGFR2 and/or truncated PPHLNl polypeptides or as chimeric proteins using a variety of protein techniques known to those of ordinary skill in the art, including but not limited to: protein sequencing; and, immunoassays.

A. Sequencing

Illustrative, non-limiting examples of protein sequencing techniques include, but are not limited to, mass spectrometry and Edman degradation.

Mass spectrometry can, in principle, sequence any size protein but becomes computationally more difficult as size increases. A protein is digested by an endoprotease, and the resulting solution is passed through a high pressure liquid chromatography column. At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high positive potential into the mass spectrometer. The charge on the droplets causes them to fragment until only single ions remain. The peptides are then fragmented and the mass-charge ratios of the fragments measured. The mass spectrum is analyzed by computer and often compared against a database of previously sequenced proteins in order to determine the sequences of the fragments. The process is then repeated with a different digestion enzyme, and the overlaps in sequences are used to construct a sequence for the protein.

In the Edman degradation reaction, the peptide to be sequenced is adsorbed onto a solid surface (e.g., a glass fiber coated with polybrene). The Edman reagent, phenylisothiocyanate (PTC), is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine, and reacts with the amine group of the N-terminal amino acid. The terminal amino acid derivative can then be selectively detached by the addition of anhydrous acid. The derivative isomerizes to give a substituted phenylthiohydantoin, which can be washed off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.

B. Immunoassays

Illustrative, non-limiting examples of immunoassays include, but are not limited to: immunoprecipitation; Western blot; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and, immuno-PCR. Polyclonal or monoclonal antibodies detectably labeled using various techniques known to those of ordinary skill in the art (e.g., calorimetric, fluorescent, chemiluminescent or radioactive) are suitable for use in the immunoassays.

Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody specific for that antigen. The process can be used to identify protein complexes present in cell extracts by targeting a protein believed to be in the complex. The complexes are brought out of solution by insoluble antibody- binding proteins isolated initially from bacteria, such as Protein A and Protein G. The antibodies can also be coupled to sepharose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using mass spectrometry, Western blotting, or any number of other methods for identifying constituents in the complex.

A Western blot, or immunoblot, is a method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane, typically polyvinyldifluoride or nitrocellulose, where they are probed using antibodies specific to the protein of interest. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups.

An ELISA, short for Enzyme-Linked Immunosorbent Assay, is a biochemical technique to detect the presence of an antibody or an antigen in a sample. It utilizes a minimum of two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme. The second antibody will cause a chromogenic or fluorogenic substrate to produce a signal. Variations of ELISA include sandwich ELISA, competitive ELISA, and ELISPOT. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations and also for detecting the presence of antigen.

Immunohistochemistry and immunocytochemistry refer to the process of localizing proteins in a tissue section or cell, respectively, via the principle of antigens in tissue or cells binding to their respective antibodies. Visualization is enabled by tagging the antibody with color producing or fluorescent tags. Typical examples of color tags include, but are not limited to, horseradish peroxidase and alkaline phosphatase. Typical examples of fluorophore tags include, but are not limited to, fluorescein isothiocyanate (FITC) or phycoerythrin (PE).

Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus. A beam of light (e.g., a laser) of a single frequency or color is directed onto a hydrodynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source. The combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector, one for each fluorescent emission peak, it is possible to deduce various facts about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC correlates with the density or inner complexity of the particle (e.g., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

Immuno-polymerase chain reaction (IPCR) utilizes nucleic acid amplification techniques to increase signal generation in antibody-based immunoassays. Because no protein equivalence of PCR exists, that is, proteins cannot be replicated in the same manner that nucleic acid is replicated during PCR, the only way to increase detection sensitivity is by signal amplification. The target proteins are bound to antibodies which are directly or indirectly conjugated to oligonucleotides. Unbound antibodies are washed away and the remaining bound antibodies have their oligonucleotides amplified. Protein detection occurs via detection of amplified oligonucleotides using standard nucleic acid detection methods, including real-time methods.

IV. In Vivo Imaging

The fusion genes of the present disclosure may also be detected using in vivo imaging techniques, including but not limited to: radionuclide imaging; positron emission tomography (PET); computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection. In some embodiments, in vivo imaging techniques are used to visualize the presence of or expression of a fusion gene or fusion protein in an animal (e.g., a human or non- human mammal). For example, in some embodiments, a fusion protein is labeled using a labeled antibody specific for the fusion protein. A specifically bound and labeled antibody can be detected in an individual using an in vivo imaging method, including, but not limited to, radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection.

V. Diagnostic Applications

Presently disclosed are methods for identifying and diagnosing ICC in a patient. The methods can include detecting the presence or absence in a biological sample from a patient of a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a Periphilin-1 (PPHL 1) gene or fragment thereof (e.g., any of the FGFR2-PPHLN1 fusion genes described above). The methods further include diagnosing the patient as suffering from ICC if the presence of the fusion gene is detected. Alternatively, or in addition, the method includes treating ICC in the patient.

The methods disclosed herein may also be useful for identifying and diagnosing other forms of liver cancer, such as, e.g., extrahepatic and mixed hepatocellular cholangiocarcinoma.

The fusion of an FGFR2 gene and a PPHL 1 gene is detectable as DNA, RNA or protein. Initially, the fusion gene is detectable as a chromosomal rearrangement of genomic DNA having a 5' portion from an FGFR2 gene (e.g., fragment of an FGFR2 gene) and a 3' portion from the PPHLN1 gene (e.g., a fragment of the PPHL 1 gene). Once transcribed, the fusion gene is detectable as a chimeric mRNA having a 5' portion from an FGFR2 gene (e.g., fragment of an FGFR2 gene) and a 3' portion from the PPHLN1 gene (e.g., a fragment of the PPHLN1 gene). Once translated, the fusion gene is detectable as a carboxy- terminally truncated FGFR2 protein fused to an amino-terminally truncated PPHLN1 polypeptide; a chimeric polypeptide having an amino-terminal portion from FGFR2 and a carboxy-terminal portion from PPHL 1. The truncated FGFR2 and PPHL 1 polypeptides may differ from their respective native proteins in amino acid sequence, post-translational processing and/or secondary, tertiary or quaternary structure. Such differences, if present, can be used to identify the presence of the fusion gene. In a specific embodiment, the fusion gene is a human fusion gene. Specific methods of detection are described in more detail below.

In some embodiments, the fusion gene can be detected in a sample by detecting the fusion junction of the FGFR2 and PPHLN1 genes and/or polypeptides. To detect the fusion junction at the nucleic acid level, probes can be designed to target the unique sequence around the fusion junction (e.g. if the nucleic acid comprises mRNA:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13); if the nucleic acid comprises mRNA:

CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACUAGUU AA (SEQ ID NO: 12); and if the nucleic acid sequence comprises cDNA:

CGAATTCTCACTCTCACAACCAATGAGGATGGCTACAATAGACTAGTTAA

(SEQ ID NO: 14) or its complement:

TTAACTAGTCTATTGTAGCCATCCTCATTGGTTGTGAGAGTGAGAATTCG

(SEQ ID NO: 15).

To detect the fusion junction at the protein level, antibodies specific for the junction region can be used in an immunoassay described herein. Other suitable detection methods known in the art for detecting protein sequences may also be used.

The present disclosure provides DNA, RNA and protein based diagnostic methods that either directly or indirectly detect the fusion genes. The present disclosure also provides compositions and kits for diagnostic purposes.

The diagnostic methods of the present disclosure may be qualitative or quantitative. Where applicable, qualitative or quantitative diagnostic methods may also include amplification of target, signal or intermediary (e.g., a universal primer).

An initial assay may confirm the presence of a fusion gene but not identify the specific fusion. A secondary assay is then performed to determine the identity of the particular fusion, if desired. The second assay may use a different detection technology than the initial assay.

The fusion genes of the present disclosure may be detected along with other markers in a multiplex or panel format. Markers are selected for their predictive value alone or in combination with the fusion genes. Exemplary potential ICC cancer markers include, but are not limited to mucins 4 and metalloproteinases 7 and 9. Other markers that may be used in combination with detection of the FGFR2-PPHLN fusion gene include, e.g., CK7 and CK19. Any suitable ICC marker known or to be discovered in the art can be used. Markers for other cancers, diseases, infections, and metabolic conditions are also contemplated for inclusion in a multiplex panel format.

The diagnostic methods of the present disclosure may also be modified with reference to data correlating particular fusion genes with the stage, aggressiveness or progression of the disease or the presence or risk of metastasis. Ultimately, the information provided by the methods of the present disclosure will assist a physician in choosing the best course of treatment for a particular patient. Thus, in some aspects, the diagnostic methods further include prescribing or administering therapy for ICC to the subject when it is determined that the fusion gene is present in the subject.

Any patient sample suspected of containing the fusion genes may be tested according to the methods of the present disclosure. By way of non-limiting examples, the sample may be tissue (e.g., tumor tissue, e.g., a liver biopsy, or blood, urine, plasma, serum, urine supernatant, urine cell pellet or liver cells).

The patient sample typically requires preliminary processing designed to isolate or enrich the sample for the fusion genes or cells that contain the fusion genes. A variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited to: centrifugation; immunocapture; cell lysis; and, nucleic acid target capture (See, e.g., EP Pat. No. 1 409 727).

The in vivo imaging methods of the present disclosure are useful in the diagnosis of cancers that express the FGFR2-PPHL 1 fusion genes or proteins of the present disclosure (e.g., ICC). Such techniques allow for diagnosis without the use of a biopsy. The in vivo imaging methods of the present disclosure are also useful for providing prognoses to cancer patients. For example, the presence of a marker indicative of a cancer (e.g., ICC) and/or the presence of a FGFR2-PPHLN1 fusion gene or protein is indicative that a cancer is likely to be susceptible to a particular cancer therapy (e.g., FGF therapy (e.g., inhibitor of FGF and/or FGFR2 and/or an inhibitor of PPHLNl).

In some embodiments, reagents (e.g., antibodies) specific for the fusion genes of the present disclosure are fluorescently labeled. The labeled antibodies are introduced into a subject (e.g., orally or parenterally). Fluorescently labeled antibodies are detected using any suitable method (e.g., using the apparatus described in U.S. Pat. No. 6, 198,107).

In other embodiments, antibodies are radioactively labeled. The use of antibodies for in vivo diagnosis is well known in the art. Sumerdon et al, (Nucl. Med. Biol 17:247-254 [1990] have described an optimized antibody-chelator for the radioimmunoscintographic imaging of tumors using Indium- 1 11 as the label. Griffin et al, (J Clin One 9:631-640 [1991]) have described the use of this agent in detecting tumors in patients suspected of having recurrent colorectal cancer. The use of similar agents with paramagnetic ions as labels for magnetic resonance imaging is known in the art (Lauffer, Magnetic Resonance in Medicine 22:339-342 [1991]). The label used will depend on the imaging modality chosen. Radioactive labels such as Indium- 1 11, Technetium-99m, or Iodine- 131 can be used for planar scans or single photon emission computed tomography (SPECT). Positron emitting labels such as Fluorine- 19 can also be used for positron emission tomography (PET). For MRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used.

Radioactive metals with half-lives ranging from 1 hour to 3.5 days are available for conjugation to antibodies, such as scandium-47 (3.5 days) gallium-67 (2.8 days), gallium-68 (68 minutes), technetiium-99m (6 hours), and indium- 1 1 1 (3.2 days), of which gallium-67, technetium-99m, and indium- 1 11 are preferable for gamma camera imaging, gallium-68 is preferable for positron emission tomography.

A useful method of labeling antibodies with such radiometals is by means of a bifunctional chelating agent, such as diethylenetriaminepentaacetic acid (DTP A), as described, for example, by Khaw et al. (Science 209:295 [1980]) for In- 111 and Tc- 99m, and by Scheinberg et al. (Science 215: 1511 [1982]). Other chelating agents may also be used, but the l-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPA are advantageous because their use permits conjugation without affecting the antibody's immunoreactivity substantially.

Another method for coupling DPTA to proteins is by use of the cyclic anhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl. Radiat. Isot. 33:327 [1982]) for labeling of albumin with In-11 1, but which can be adapted for labeling of antibodies. A suitable method of labeling antibodies with Tc-99m which does not use chelation with DPTA is the pretinning method of Crockford et al. (see, e.g., U.S. Pat. No. 4,323,546).

A preferred method of labeling immunoglobulins with Tc-99m is that described by Wong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978]) for plasma protein, and applied successfully by Wong et al. (J. Nucl. Med., 23:229 [1981]) for labeling antibodies.

In the case of the radiometals conjugated to the specific antibody, it is likewise desirable to introduce as high a proportion of the radiolabel as possible into the antibody molecule without destroying its immunospecificity. A further improvement may be achieved by effecting radiolabeling in the presence of the specific FGFR2- PPHL 1 fusion gene or protein of the present disclosure, to ensure that the antigen binding site on the antibody will be protected. The antigen is separated after labeling. In still further embodiments, in vivo biophotonic imaging (Xenogen, Almeda, Calif.) is utilized for in vivo imaging. This real-time in vivo imaging utilizes luciferase. The luciferase gene is incorporated into cells, microorganisms, and animals (e.g., as a fusion protein with a FGFR2-PPHLN1 fusion gene or protein of the present disclosure). When active, it leads to a reaction that emits light. A CCD camera and software is used to capture the image and analyze it.

As disclosed herein, diagnostic methods can further include administering a treatment (e.g., cancer therapy) to the subject for treating ICC.

VI. Data Analysis

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given fusion gene or other markers) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present disclosure provides the further benefit that the clinician need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject. Thus, in some embodiments, provided herein are methods for detecting the presence of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHLNl gene or fragment thereof in a human subject, wherein the method includes: (a) obtaining nucleic acid from a biological sample collected from the subject; (b) analyzing the nucleic acid to determine its nucleotide sequence with an electronic computer sequencing device; (c) comparing, with software programmed to perform a base-by- base comparison, the nucleotide sequence of the nucleic acid to a control sequence comprising at least 25 consecutive nucleic acids of one of the following nucleic acid sequence: (i) if the nucleic acid comprises genomic DNA:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13); (ii) if the nucleic acid comprises mRNA: CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACUAGUU AA (SEQ ID NO: 12); and (iii) if the nucleic acid sequence comprises cDNA:

CGAATTCTCACTCTCACAACCAATGAGGATGGCTACAATAGACTAGTTAA

(SEQ ID NO: 14) or its complement:

TTAACTAGTCTATTGTAGCCATCCTCATTGGTTGTGAGAGTGAGAATTCG

(SEQ ID NO: 15); (d) displaying the comparison between the nucleotide sequence of the nucleic acid and the control sequence on an electronically operated (computer monitor) screen; and (e) determining that the fusion gene is present in the subject when the compared sequences are substantially identical; or (f) determining that the fusion gene is not present in the sample when the compared sequences are the not substantially identical. In some embodiments, the method further comprises prescribing or administering therapy for ICC to the subject when it is determined that the fusion gene is present in the subject.

Electronic computer sequencing devices are known in the art and described above. Non-limiting examples include, e.g., HiSeq 2000 (Illumina, San Diego, CA), and the Applied Biosystems 3700 DNA sequencer (ABI PRISM® 3730XL; Applied Biosystems, Grand Island, NY). Further, methods for comparing the nucleic acid sequences are known in the art, and exemplary methods are described above (e.g., sequencing, hybridization, microarray). Such methods are also described, e.g., in U.S. Patent No. 6,045,996; and Olson, Nat Methods 2007, 4:891-892. Methods for obtaining biological samples from the subject are well known in the art, and examples of suitable samples are described above.

The present disclosure contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects. For example, in some embodiments of the present disclosure, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., expression data), specific for the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of cancer being present and/or susceptibility of the cancer to a particular cancer therapy (e.g., to treatment with an inhibitor of an FGF signaling pathway) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some

embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease. VII. Prognostic Applications

In some embodiments, experiments conducted during the course of development of the present disclosure demonstrated a close correlation between the presence of the fusion genes of the present disclosure and the increased susceptibility of liver cancer cells (e.g., ICC) to killing with FGFR inhibitors (see e.g., Example 4, below). Thus, in some embodiments, provided herein are methods for determining the susceptibility of a liver cancer (e.g., ICC, or extrahepatic or mixed hepatocellular cholangiocarcinoma) in a patient to a cancer therapy comprising an FGFR2 inhibitor. In some embodiments, these methods can include: (a) providing a biological sample from the patient; and (b) detecting the presence or absence in the sample of a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof; (c) identifying the cancer as likely to be susceptible to the therapy if the fusion gene is detected in the sample; and (d) identifying the cancer as unlikely to be susceptible to the therapy if the fusion gene is not detected in the sample.

Any suitable assay may be used to determine whether cells are present having an FGFR2-PPHLN1 fusion gene of the present disclosure (e.g., those described above).

Although the present disclosure will most preferably be used in connection with obtaining a prognosis (e.g., determining likelihood of responding to a cancer therapy, such as, e.g., treatment with an FGFR2 inhibitor and/or a PPHL 1 inhibitor and/or a specific inhibitor of the FGFR2-PPHLN1 fusion protein) for ICC patients and other liver cancer patients (e.g., extrahepatic and mixed hepatocellular cholangiocarcinoma), other solid tumors may also be examined and the assays and probes described herein may be used in determining whether the cells are likely to respond to treatment with an inhibitor of the FGF signaling pathway. Examples of tumors that may be characterized using this procedure include tumors of the breast, lung, colon, ovary, uterus, esophagus, stomach, prostate, kidney, brain, skin and muscle. The assays will also be of value to researchers studying these cancers in cell lines and animal models. VIII. Drug Screening Applications

In some embodiments, the present disclosure provides drug screening assays (e.g., to screen for anticancer drugs). The screening methods of the present disclosure utilize FGFR2-PPHL 1 fusion genes or proteins identified using the methods of the present disclosure. For example, in some embodiments, the present disclosure provides methods of screening for compounds that alter (e.g., decrease) the expression of fusion genes. The compounds or agents may interfere with transcription, by interacting, for example, with the promoter region. The compounds or agents may interfere with mRNA produced from the fusion (e.g., by RNA interference, antisense technologies, etc.). The compounds or agents may interfere with pathways that are upstream or downstream of the biological activity of the fusion. In some

embodiments, candidate compounds are antisense or interfering RNA agents (e.g., oligonucleotides) directed against FGFR2-PPHLN1 fusion genes, e.g., as disclose herein. In other embodiments, candidate compounds are antibodies or small molecules that specifically bind to a FGFR2-PPHL 1 fusion gene or protein and inhibit its biological function.

In one screening method, candidate compounds are evaluated for their ability to alter FGFR2-PPHL 1 fusion gene or protein expression by contacting a compound with a cell expressing an FGFR2-PPHL 1 fusion gene or protein and then assaying for the effect of the candidate compounds on expression. In some embodiments, the effect of candidate compounds on expression of a fusion protein gene is assayed for by detecting the level of fusion protein mRNA expressed by the cell. mRNA expression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression of fusion protein genes is assayed by measuring the level of polypeptide encoded by the fusion gene. The level of polypeptide expressed can be measured using any suitable method, including but not limited to, those disclosed herein.

Specifically, the present disclosure provides screening methods for identifying modulators, i.e., candidate or test compounds or agents (e.g., antisense

oligonucleotides, proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to the FGFR2-PPHLN1 fusion gene/polypeptide), have an inhibitory (or stimulatory) effect on, for example, fusion gene expression and/or activity, or have a stimulatory or inhibitory effect on, for example, the fusion gene/polypeptide. Compounds thus identified can be used to modulate the activity of target gene products (e.g., FGFR2-PPHLN1 fusion gene/polypeptide) either directly or indirectly in a therapeutic protocol, to inhibit the biological function of the target FGFR2-PPHL 1 fusion polypeptide, or to identify compounds that disrupt normal target gene interactions. Compounds that inhibit the activity or expression of FGFR2- PPHL 1 fusion gene/polypeptides as disclosed herein are useful in the treatment of proliferative disorders, e.g., cancer, particularly liver cancer, such as ICC.

In one embodiment, the present disclosure provides assays for screening candidate or test compounds that inhibit expression of FGFR2-PPHLN1 fusion gene or expression or activity of the encoded polypeptides. In another embodiment, the disclosure provides assays for screening candidate or test compounds that bind to or modulate the activity of the FGFR2-PPHLN1 fusion gene/polypeptide.

The test compounds of the present disclosure can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al, J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ^"one- bead one-compound^" library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al, Proc. Nad. Acad. Sci. USA 91 : 11422 [1994]; Zuckermann et al, J. Med. Chem. 37:2678 [1994]; Cho et al, Science 261 : 1303 [1993]; Carrell et al, Angew. Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061 [1994]; and Gallop et al, J. Med. Chem. 37: 1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13 :412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores (see, e.g., U.S. Pat. No. 5,223,409), plasmids (see, e.g., Cull et al, Proc. Nad. Acad. Sci. USA 89: 18651869 [1992]) or on phage (see, e.g., Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et al, Proc. Natl. Acad. Sci. 87:6378-6382

[1990]; and Felici, J. Mol. Biol. 222:301 [1991]).

In one embodiment, an assay is a cell-based assay in which a cell that expresses a FGFR2-PPHLN1 fusion gene mRNA or protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to the modulate FGFR2-PPHLN1 fusion polypeptide activity is determined. Determining the activity can be accomplished by monitoring, for example, changes in functional activity, changes in expression levels, or the like.

The ability of the test compound to modulate binding of a fusion protein to a binding partner, e.g., a substrate or modulator, can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to the fusion protein can be determined by detecting the labeled compound, e.g., substrate, in a complex.

Alternatively, the FGFR2-PPHLN1 fusion gene polypeptideis coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate binding of the fusion protein to a cancer marker substrate in a complex. For example, compounds (e.g., substrates) can be labeled with ¹²⁵1, ³⁵S, ¹⁴C or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

The ability of a compound to interact with a FGFR2-PPHLN1 fusion proteinwith or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with the fusion protein without the labeling of either the compound or the fusion protein (McConnell et al. Science 257: 1906-1912). A "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and the fusion protein.

In yet another embodiment, a cell-free assay is provided in which a fusion protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the fusion protein, mRNA, or biologically active portion thereof is evaluated. Preferred biologically active portions of the fusion proteins or mRNA to be used in assays of the present disclosure include fragments that participate in interactions with substrates or other proteins.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FRET) (see, for example, Lakowicz et al, U.S. Pat. No. 5,631, 169; Stavrianopoulos et al, U.S. Pat. No. 4,968, 103). A fluorophore label is selected such that a first donor molecule's emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy.

Alternately, the ^" donor^" protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ^" acceptor^" molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ^"acceptor^" molecule label should be maximal. A FRET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the ability of the fusion protein or fusion gene (e.g. mRNA) to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem. 63 :2338-2345 [1991] and Szabo et al. Curr. Opin. Struct. Biol. 5:699-705 [1995]). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize an FGFR2-PPHL 1 fusion gene protein, an anti-FGFR2-PPHL l fusion protein antibody or its target molecule to facilitate separation of complexed from non-complexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a FGFR2-PPHLN1 fusion protein, or interaction of a FGFR2-PPHLN1 fusion protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase- can be fused to the fusion proteins disclosed herein or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or fusion protein, and the mixture incubated under conditions conducive for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.

Alternatively, the complexes can be dissociated from the matrix, and the level of FGFR2-PPHL 1 fusion protein binding or activity determined using standard techniques. Other techniques for immobilizing either FGFR2-PPHL 1 fusion protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated FGFR2-PPHL 1 fusion protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, EL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-IgG antibody).

This assay is performed utilizing antibodies reactive with FGFR2-PPHLN1 fusion protein or target molecules but which do not interfere with binding of the FGFR2-PPHLN1 fusion protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or FGFR2-PPHLN1 fusion protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the fusion protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the fusion protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including, but not limited to: differential

centrifugation (see, for example, Rivas and Minton, Trends Biochem Sci 18:284-7 [1993]); chromatography (gel filtration chromatography, ion-exchange

chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York); and immunoprecipitation (see, for example, Ausubel et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (See e.g., Heegaard J. Mol. Recognit 1 1 : 141-8 [1998]; Hage and Tweed J. Chromatogr. Biomed. Sci. Appl 699:499-525 [1997]). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

The assay can include contacting the FGFR2-PPHL 1 fusion gene or protein or biologically active portion thereof with a known compound that binds the FGFR2- PPHL 1 fusion protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FGFR2-PPHL 1 fusion gene or protein, wherein determining the ability of the test compound to interact with a FGFR2-PPHL 1 fusion gene or proteinincludes determining the ability of the test compound to preferentially bind FGFR2-PPHLN1 fusion gene or protein or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

To the extent that FGFR2-PPHL 1 fusion genes or proteins can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins, inhibitors of such an interactions are useful.

For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared such that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4, 109,496, which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

Alternatively, FGFR2-PPHL 1 fusion proteincan be used as a "bait protein" in a two- hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al, Cell 72:223-232 [1993]; Madura et al, J. Biol. Chem. 268.12046-12054 [1993]; Bartel et al, Biotechniques 14:920-924 [1993]; Iwabuchi et al, Oncogene 8: 1693- 1696 [1993]; and Brent WO 94/10300), to identify other proteins, that bind to or interact with FGFR2-PPHLN1 fusion protein and are involved in FGFR2-PPHLN1 fusion protein activity. Such binding proteins can be activators or inhibitors of signals by the FGFR2-PPHLN1 fusion protein or targets as, for example, downstream elements of a FGFR2-PPHLN1 fusion protein-mediated signaling pathway. Modulators of FGFR2-PPHL 1 fusion gene or protein expression can also be identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of FGFR2-PPHL 1 fusion gene or protein is evaluated relative to the level of expression of FGFR2-PPHLN1 fusion gene or protein in the absence of the candidate compound. When expression of FGFR2-PPHL 1 fusion gene or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of cFGFR2-PPHLNl fusion gene or protein expression. Alternatively, when expression of FGFR2- PPHLN1 fusion gene or protein is less (i.e., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of FGFR2-PPHL 1 fusion gene or protein expression. The level of FGFR2-PPHL 1 fusion gene or protein expression can be determined by methods described herein for detecting FGFR2-PPHL 1 fusion gene or protein expression.

A modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a FGFR2-PPHL 1 fusion gene or protein can be confirmed in vivo, e.g., in an animal such as an animal model for a disease (e.g., an animal with ICC; or an animal harboring a xenograft of ICC from an animal (e.g., human) or cells from a cancer resulting from metastasis of ICC (e.g., to a lymph node, bone, lung), or cells from a ICC cell line.

This disclosure further pertains to novel agents identified by the above- described screening assays (See e.g., below description of cancer therapies).

Accordingly, it is within the scope of this disclosure to further use an agent identified as described herein (e.g., a FGFR2-PPHLN1 fusion gene or protein modulating agent, an antisense FGFR2-PPHLN1 fusion gene nucleic acid molecule, a siRNA molecule, a FGFR2-PPHL 1 fusion protein-specific antibody, or a FGFR2-PPHL 1 fusion gene or protein-binding partner) in an appropriate animal model (such as those described herein) to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be, e.g., used for treatments as described herein. IX. Therapeutic Applications

In some embodiments, the present disclosure provides therapies for cancer (e.g., ICC). In some embodiments, therapies directly or indirectly target fusion genes of the present disclosure. In some embodiments, these methods (therapies) are applied for the treatment of cancer (e.g., ICC) and/or for inhibiting the development of cancer (e.g., ICC).

Thus, in some embodiments, provided herein are methods for treating ICC in a subject in need thereof. In other embodiments, methods are provided for inhibiting the development of ICC. These methods can include targeting in the subject a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof, or targeting a polypeptide encoded by the fusion gene. In some embodiments, the targeting comprises administering to the subject an inhibitor of the fusion gene or polypeptide encoded by the fusion gene. In some embodiments, the inhibitor is an antisense molecule specific for the fusion gene. In some embodiments, the inhibitor is an antibody specific for the polypeptide encoded by the fusion gene.

A. RNA Interference and Antisense Therapies

In some embodiments, the present disclosure targets the expression of fusion genes. For example, in some embodiments, the present disclosure employs compositions comprising oligomeric antisense or RNAi compounds, particularly oligonucleotides (e.g., those identified in the drug screening methods described above), for use in modulating the function of nucleic acid molecules encoding FGFR2-PPHL 1 fusion genes or proteins of the present disclosure, ultimately modulating the amount of FGFR2-PPHLN1 fusion gene or protein expressed. In some embodiments, these methods are applied for the treatment of cancer (e.g., ICC) or for inhibiting the development of cancer (e.g., ICC).

1. RNA Interference (RNAi)

In some embodiments, RNAi is utilized to inhibit fusion protein function. RNAi represents an evolutionary conserved cellular defense for controlling the expression of foreign genes in most eukaryotes, including humans. RNAi is typically triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single-stranded target RNAs homologous in response to dsRNA. The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), which are normally produced from long dsRNA by enzymatic cleavage in the cell. siRNAs are generally approximately twenty-one nucleotides in length (e.g. 21-23 nucleotides in length), and have a base-paired structure characterized by two nucleotide 3'-overhangs. Following the introduction of a small RNA, or RNAi, into the cell, it is believed the sequence is delivered to an enzyme complex called RISC(RNA-induced silencing complex). RISC recognizes the target and cleaves it with an endonuclease. It is noted that if larger RNA sequences are delivered to a cell, RNase III enzyme (Dicer) converts longer dsRNA into 21-23 nt ds siRNA fragments. In some embodiments, RNAi oligonucleotides are designed to target the junction region of fusion proteins.

Chemically synthesized siRNAs have become powerful reagents for genome- wide analysis of mammalian gene function in cultured somatic cells. Beyond their value for validation of gene function, siRNAs also hold great potential as gene- specific therapeutic agents (Tuschl and Borkhardt, Molecular Intervent. 2002; 2 (3): 158-67).

The transfection of siRNAs into animal cells results in the potent, long-lasting post-transcriptional silencing of specific genes (Caplen et al, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al, Nature. 2001; 411 :494-8; Elbashir et al, Genes Dev. 2001; 15: 188-200; and Elbashir et al, EMBO J. 2001 ; 20: 6877-88). Methods and compositions for performing RNAi with siRNAs are described, for example, in U.S. Pat. No. 6,506,559.

siRNAs are extraordinarily effective at lowering the amounts of targeted RNA, and by extension proteins, frequently to undetectable levels. The silencing effect can last several months, and is extraordinarily specific, because one nucleotide mismatch between the target RNA and the central region of the siRNA is frequently sufficient to prevent silencing (Brummelkamp et al, Science 2002; 296:550-3; and Holen et al, Nucleic Acids Res. 2002; 30: 1757-66).

An important factor in the design of siRNAs is the presence of accessible sites for siRNA binding. Bahoia et al, (J. Biol. Chem., 2003; 278: 15991-15997) describe the use of a type of DNA array called a scanning array to find accessible sites in mRNAs for designing effective siRNAs. These arrays comprise oligonucleotides ranging in size from monomers to a certain maximum, usually Corners, synthesized using a physical barrier (mask) by stepwise addition of each base in the sequence. Thus the arrays represent a full oligonucleotide complement of a region of the target gene. Hybridization of the target mRNA to these arrays provides an exhaustive accessibility profile of this region of the target mRNA. Such data are useful in the design of antisense oligonucleotides (ranging from 7mers to 25mers), where it is important to achieve a compromise between oligonucleotide length and binding affinity, to retain efficacy and target specificity (Sohail et al, Nucleic Acids Res., 2001; 29 (10): 2041-2045). Additional methods and concerns for selecting siRNAs are described for example, in WO 05054270, WO05038054A1, WO03070966A2, J Mol Biol. 2005 May 13; 348 (4):883-93, J Mol Biol. 2005 May 13; 348 (4):871-81, and Nucleic Acids Res. 2003 Aug. 1; 31 (15):4417-24. In addition, software (e.g., the MWG online siMAX siRNA design tool) is commercially or publicly available for use in the selection of siRNAs.

2. Antisense

In other embodiments, fusion protein expression is modulated using antisense compounds that specifically hybridize with one or more nucleic acids encoding FGFR2-PPHLN1 fusion proteins of the present disclosure. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as "antisense." The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of FGFR2-PPHLN1 fusion genes or proteins of the present disclosure. In the context of the present disclosure, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. For example, expression may be inhibited to potentially prevent tumor proliferation.

Specific nucleic acids are typically targeted for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of the present disclosure, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present disclosure, the target is a nucleic acid molecule encoding an FGFR2-PPHL 1 fusion gene of the present disclosure. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present disclosure, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'- CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the present disclosure, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a tumor antigen of the present disclosure, regardless of the sequence(s) of such codons.

Translation termination codon (or "stop codon") of a gene may have one of three sequences (i.e., 5'-UAA, 5'-UAG and 5'-UGA; the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. The open reading frame (ORF) or "coding region," which refers to the region between the translation initiation codon and the translation termination codon, is also a region that may be targeted effectively. Other target regions include the 5' untranslated region (5' UTR), referring to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3' UTR), referring to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of an mRNA comprises an N7- methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap. The cap region may also be a preferred target region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," that are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites (i.e., intron-exon junctions) may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets (e.g., the junction between the truncated FGFR2 gene and the truncated PPHLNl gene in the fusion genes disclosed herein). It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

In some embodiments, target sites for antisense inhibition are identified using commercially available software programs (e.g., Biognostik, Gottingen, Germany; SysArris Software, Bangalore, India; Antisense Research Group, University of Liverpool, Liverpool, England; GeneTrove, Carlsbad, Calif). In other embodiments, target sites for antisense inhibition are identified using the accessible site method described in PCT Publ. No. WO0198537A2. Once one or more target sites have been identified, oligonucleotides are chosen that are sufficiently complementary to the target (i.e., hybridize sufficiently well and with sufficient specificity) to give the desired effect. For example, in preferred embodiments of the present disclosure, antisense oligonucleotides are targeted to or near the start codon.

It is understood that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired (i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed).

The specificity and sensitivity of antisense can be applied for therapeutic uses. For example, antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides are useful therapeutic modalities that can be configured to be useful in treatment regimens for treatment of cells, tissues, and animals, especially humans.

The present disclosure also contemplates the use of other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics, such as are described below. The antisense compounds in accordance with this disclosure preferably comprise from about 8 to about 30 nucleobases (i.e., from about 8 to about 30 linked bases), although both longer and shorter sequences may find use with the present disclosure. Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 25 nucleobases.

Specific examples of preferred antisense compounds useful with the present disclosure include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Modified oligonucleotide backbones can include, for example,

phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates,

thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. See Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36: 1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211 ; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss- Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156).

Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

In other oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e., the backbone) of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. See, e.g., U.S. Pat. Nos.

5,539,082; 5,714,331; and 5,719,262 and Nielsen et al, Science 254: 1497 (1991).

Other backbone modifications are known in the art, and encompassed by the methods disclosed herein.

Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C, and can be combined with 2'-0-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the present disclosure involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the

oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, (e.g., hexyl-S-tritylthiol), a

thiocholesterol, an aliphatic chain, (e.g., dodecandiol or undecyl residues), a phospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generate oligonucleotides containing the above-described modifications. The present disclosure is not limited to the antisense oligonucleotides described above. Any suitable modification or substitution may be utilized.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present disclosure also includes antisense compounds that are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of the present disclosure, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to

phosphorothioate deoxyoligonucleotides hybridizing to the same target region.

Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric antisense compounds of the present disclosure may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above.

The present disclosure also includes pharmaceutical compositions and formulations that include the antisense compounds of the present disclosure as described below.

B. Gene Therapy

The present disclosure contemplates the use of any genetic manipulation for use in modulating the expression of fusion genes (e.g., FGFR2-PPHLN1 fusion gene) of the present disclosure. In some embodiments, these methods are applied for the treatment of cancer (e.g., ICC) or for inhibiting the development of cancer (e.g., ICC). Examples of genetic manipulation include, but are not limited to, gene knockout (e.g., removing the fusion gene from the chromosome using, for example, recombination), expression of antisense constructs with or without inducible promoters, and the like. Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method. A suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g., expression of an antisense construct). Genetic therapy may also be used to deliver siRNA or other interfering molecules that are expressed in vivo (e.g., upon stimulation by an inducible promoter (e.g., an androgen-responsive promoter)).

Introduction of molecules carrying genetic information into cells is achieved by any of various methods including, but not limited to, directed injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, and the like. Exemplary methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno- associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are the preferred gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo. Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice. Examples of adenoviral vectors and methods for gene transfer are described in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat. Appl. Nos.

6,033,908, 6,019,978, 6,001,557, 5,994, 132, 5,994, 128, 5,994, 106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544.

Vectors may be administered to subject in a variety of ways. For example, in some embodiments of the present disclosure, vectors are administered into tumors or tissue associated with tumors using direct injection. In other embodiments, administration is via the blood or lymphatic circulation (See e.g., PCT publication 99/02685). Exemplary dose levels of adenoviral vector are preferably 10⁸ to 10¹¹ vector particles added to the perfusate.

C. Antibody Therapy

In some embodiments, the present disclosure provides antibodies that target prostate tumors that express an FGFR2-PPHL 1 fusion gene of the present disclosure. In some embodiments, these methods are applied for the treatment of cancer (e.g., ICC) or for inhibiting the development of cancer (e.g., ICC). Any suitable antibody (e.g., monoclonal, polyclonal, or synthetic) may be utilized in the therapeutic methods disclosed herein. In preferred embodiments, the antibodies used for cancer therapy are humanized antibodies. Methods for humanizing antibodies are well known in the art (see e.g., U.S. Pat. Nos. 6, 180,370, 5,585,089, 6,054,297, and 5,565,332).

In some embodiments, the therapeutic antibodies comprise an antibody generated against an FGFR2-PPHLN1 fusion gene of the present disclosure, wherein the antibody is conjugated to a cytotoxic agent. In such embodiments, a tumor specific therapeutic agent is generated that does not target normal cells, thus reducing many of the detrimental side effects of traditional chemotherapy. For certain applications, it is envisioned that the therapeutic agents will be pharmacologic agents that will serve as useful agents for attachment to antibodies, particularly cytotoxic or otherwise anticellular agents having the ability to kill or suppress the growth or cell division of tumor cells. The present disclosure contemplates the use of any pharmacologic agent that can be conjugated to an antibody, and delivered in active form. Exemplary anticellular agents include chemotherapeutic agents, radioisotopes, and cytotoxins. The therapeutic antibodies of the present disclosure may include a variety of cytotoxic moieties, including but not limited to, radioactive isotopes (e.g., iodine-131, iodine-123, technicium-99m, indium-I l l, rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125 or astatine-211), hormones such as a steroid, antimetabolites such as cytosines (e.g., arabinoside, fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycin C), vinca alkaloids (e.g., demecolcine; etoposide; mithramycin), and antitumor alkylating agent such as chlorambucil or melphalan. Other embodiments may include agents such as a coagulant, a cytokine, growth factor, bacterial endotoxin or the lipid A moiety of bacterial endotoxin. For example, in some embodiments, therapeutic agents will include plant-, fungus- or bacteria-derived toxin, such as an A chain toxins, a ribosome inactivating protein, a- sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention just a few examples. In some preferred embodiments, deglycosylated ricin A chain is utilized.

The above-disclosed, exemplary agents may, if desired, be successfully conjugated to an antibody, in a manner that will allow their targeting, internalization, release or presentation to blood components at the site of the targeted tumor cells as required using known conjugation technology (see, e.g., Ghose et al, Methods Enzymol., 93:280 [1983]).

For example, in some embodiments the present disclosure provides immunotoxins targeted to a FGFR2-PPHLN1 protein of the present disclosure.

Immunotoxins are conjugates of a specific targeting agent typically a tumor-directed antibody or fragment, with a cytotoxic agent, such as a toxin moiety. The targeting agent directs the toxin to, and thereby selectively kills, cells carrying the targeted antigen. In some embodiments, therapeutic antibodies employ crosslinkers that provide high in vivo stability (Thorpe et al, Cancer Res., 48:6396 [1988]).

In other embodiments, particularly those involving treatment of solid tumors, antibodies are designed to have a cytotoxic or otherwise anticellular effect against the tumor vasculature, by suppressing the growth or cell division of the vascular endothelial cells. This attack is intended to lead to a tumor-localized vascular collapse, depriving the tumor cells (e.g., ICC tumor cells), e.g., those tumor cells distal of the vasculature, of oxygen and nutrients, ultimately leading to cell death and tumor necrosis. Further, as discussed above, the treatment methods disclosed herein can be administered with an additional therapy as part of a "combination therapy." For example, additional therapies can include an additional treatment for cancer (e.g., chemotherapy (e.g., administering a chemotherapeutic agent), administering a biologic agent, e.g., antigen, vaccine, antibody etc., administering a cytokine, radiation therapy, immunotherapy, and/or surgery, etc.) including e.g., a therapy that inhibits at least one biological function of an FGFR2 fusion gene (e.g. FGFR2- PPHLN1). Such combination therapy can be sequential therapy wherein the patient is treated first with one therapy and then the other, and so on, or all therapies can be administered simultaneously. In either case, these therapies are said to be coadministered. It is to be understood that "coadministered" does not necessarily mean that the drugs and/or therapies are administered in a combined form (i.e., they may be administered separately or together to the same or different sites at the same or different times).

Certain embodiments of the disclosure provide compositions containing (a) one or more antisense compounds (e.g., described above) and (b) one or more other additional cancer therapies.

In one embodiment, the additional therapy is a chemotherapeutic agent that functions by a non-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). Antiinflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the disclosure. Other non-antisense chemotherapeutic agents are also within the scope of this disclosure. Two or more combined compounds may be used together or sequentially.

In another embodiment, the additional therapy for coadministration is an antibody based therapeutic (e.g., an antibody described above). Such antibody-based therapeutics can be formulated as a pharmaceutical composition, as described below. In some embodiments, administration of the combination therapy results in a measurable decrease in cancer (e.g., decrease or elimination of tumor) (i.e., treats the cancer).

X. Compositions & Kits

Compositions for use in the methods of the present disclosure include, but are not limited to, probes, amplification oligonucleotides, and antibodies. Particularly preferred compositions detect a product only when an FGFR2 gene or fragment thereof fuses to PPHLN1 gene or fragment thereof. These compositions include, e.g., an oligonucleotide probe comprising a sequence that hybridizes to a fusion junction of a chimeric genomic DNA or chimeric mRNA in which a 5' portion of the chimeric genomic DNA or chimeric mRNA is from an FGFR2 gene or fragment thereof and a 3' portion of the chimeric genomic DNA or chimeric mRNA is from a PPHLN1 gene or fragment thereof; a first oligonucleotide probe comprising a sequence that hybridizes to a 5' portion of a chimeric genomic DNA or chimeric mRNA from an FGFR2 gene or fragment thereof and a second oligonucleotide probe comprising a sequence that hybridizes to a 3' portion of the chimeric genomic DNA or chimeric mRNA from a PPHLN1 gene or fragment thereof; and a first amplification oligonucleotide comprising a sequence that hybridizes to a 5' portion of a chimeric genomic DNA or chimeric mRNA from a transcriptional regulatory region of an FGFR2 gene or fragment thereof and a second amplification oligonucleotide comprising a sequence that hybridizes to a 3' portion of the chimeric genomic DNA or chimeric mRNA from a PPHLN1 gene or fragment thereof. Other useful

compositions, however, include: a pair of labeled probes wherein the first labeled probe comprises a sequence that hybridizes to a transcriptional regulatory region of an FGFR2 gene and the second labeled probe comprises a sequence that hybridizes to a PPHLN1 gene.

Any of these compositions, alone or in combination with other compositions of the present disclosure, may be provided in the form of a kit. For example, the single labeled probe and pair of amplification oligonucleotides may be provided in a kit for the amplification and detection of fusion genes of the present disclosure. Kits may further comprise appropriate controls and/or detection reagents. The probe and antibody compositions of the present disclosure may also be provided in the form of an array.

XI. Pharmaceutical Formulations and Therapy

While it is possible to use an inhibitor or other agent (e.g., FGFR2, PPHLN1 or FGFR2-PPHL 1 fusion protein inhibitor) of the present disclosure for therapy as is, it may be preferable to administer it as a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical formulations comprise at least one active compound, or a

pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent, and/or carrier. The excipient, diluent and/or carrier must be "acceptable," as defined above.

Thus, in some embodiments, the present disclosure further provides pharmaceutical compositions comprising pharmaceutical agents that modulate (e.g., increase or decrease) the expression and/or activity of an FGFR2-PPHLN1 fusion gene or polypeptide encoded by the fusion gene (i.e., fusion protein) of the present disclosure. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial,

subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, or intratumoral administration. In certain embodiments, oral administration is the preferred route of administration, e.g., for small molecules.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal, intratumoral, or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (see, e.g., U.S. Pat. No. 5,705, 188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (see, e.g., WO 97/30731), also enhance the cellular uptake of oligonucleotides.

The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically- active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Certain embodiments of the disclosure provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents that function by a non-antisense mechanism.

Moreover, as disclosed above, in some embodiments, the present disclosure provides methods for treating ICC in a patient. In some embodiments, an effective amount of an inhibitor of an FGFR2-PPHLN1 fusion gene or polypeptide is administered to treat the ICC. In some embodiments, dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on an ECso found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The period of administration may range from a period of 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-8 days, 1-9 days, 1-10 days, 1-11 days, 1-12 days, 1-13 days, and 1-14 days, or longer, though administrations of the composition may or may not occur on each of the days within the specified period. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the composition, formulation, or agent is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, once or more weekly, once or monthly, one or more yearly, one or more every 10 years, once or more every 15 year, to once or more every 20 years.

By way of example, BGJ398 ( ovartis, Basel, Switzerland) can be administered to a subject in a dosage range of about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 40 mg to about 200 mg, about 50 mg to about 175 mg, about 75 mg to about 160 mg, about 80 mg to about 150 mg, about 100 mg to about 130 mg, about 1 10 mg to about 130 mg, or about 120 mg to about 130 mg. In other embodiments, BGJ398 can be administered in a dosage of 50 mg, 75 mg, 100 mg, 115 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 175 mg, 180 mg, 200 mg, 250 mg, or 300 mg per day. The dosage can be administered, e.g., once per day (daily), twice per day, once every other day, three times per week, four times per week, weekly, biweekly, monthly, or bimonthly. The dosage of BGJ398 can be administered over a period of about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 1 1 weeks, about 12 weeks or longer. In a specific embodiment, BGJ398 can be administered in a dosage of 125 mg per day over a period of about 21 days. In a specific embodiment, BGJ398 can be administered in a dosage of 125 mg per day over a period of about 28 days.

XII. Transgenic Animals

The present disclosure contemplates the generation of transgenic animals comprising an exogenous fusion gene/fusion protein of the present disclosure (e.g., FGFR2-PPHLN1 fusion genes or proteins) or mutants and variants thereof (e.g., other truncations of FGFR2 and/or PPHLN1 and/or or single nucleotide polymorphisms of one or both of FGF2 and PPHL 1). In some embodiments, the transgenic animal displays an altered phenotype (e.g., increased or decreased presence of markers) as compared to wild-type animals. Methods for analyzing the presence or absence of such phenotypes include but are not limited to, those disclosed herein. In some embodiments, the transgenic animals further display an increased or decreased growth of tumors or evidence of cancer (e.g., ICC).

The transgenic animals of the present disclosure find use in drug (e.g., cancer therapy) screens. In some embodiments, test compounds (e.g., a drug that is suspected of being useful to treat cancer) and control compounds (e.g., a placebo) are administered to the transgenic animals and the control animals and the effects evaluated.

The transgenic animals can be generated via a variety of methods. In some embodiments, embryonal cells at various developmental stages are used to introduce transgenes for the production of transgenic animals. Different methods are used depending on the stage of development of the embryonal cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter that allows reproducible injection of 1-2 picoliters (pi) of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host genome before the first cleavage (Brinster et al, Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. U.S. Pat. No. 4,873, 191 describes a method for the micro-injection of zygotes.

In other embodiments, retroviral infection is used to introduce transgenes into a non-human animal. In some embodiments, the retroviral vector is utilized to transfect oocytes by injecting the retroviral vector into the perivitelline space of the oocyte (see, e.g., U.S. Pat. No. 6,080,912). In other embodiments, the developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Janenich, Proc. Natl. Acad. Sci. USA 73: 1260 [1976]). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al, in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986]). The viral vector system used to introduce the transgene is typically a replication- defective retrovirus carrying the transgene (Jahner et al, Proc. Natl. Acad. Sci. USA 82:6927 [1985]). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Stewart, et al, EMBO J., 6:383 [1987]). Alternatively, infection can be performed at a later stage. Virus or virus- producing cells can be injected into the blastocoele (Jahner et al, Nature 298:623 [1982]). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of cells that form the transgenic animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germline, albeit with low efficiency, by intrauterine retroviral infection of the midgestation embryo (Jahner et al, supra [1982]).

Additional means of using retroviruses or retroviral vectors to create transgenic animals known to the art involve the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilized eggs or early embryos (PCT International Application WO 90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stem cells and the transfected stem cells are utilized to form an embryo. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans et al, Nature 292: 154 [1981]; Bradley et al, Nature 309:255 [1984]; Gossler et al, Proc. Acad. Sci. USA 83 :9065 [1986]; and Robertson et al, Nature 322:445 [1986]).

Transgenes can be efficiently introduced into the ES cells by DNA transfection by a variety of methods known to the art including calcium phosphate co-precipitation, protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated

transfection. Transgenes may also be introduced into ES cells by retrovirus-mediated transduction or by micro-injection. Such transfected ES cells can thereafter colonize an embryo following their introduction into the blastocoel of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal (for review, See, Jaenisch, Science 240: 1468 [1988]). Prior to the introduction of transfected ES cells into the blastocoel, the transfected ES cells may be subjected to various selection protocols to enrich for ES cells which have integrated the transgene assuming that the transgene provides a means for such selection. Alternatively, the polymerase chain reaction may be used to screen for ES cells that have integrated the transgene. This technique obviates the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.

In still other embodiments, homologous recombination is utilized to knock-out gene function or create deletion mutants (e.g., truncation mutants). Methods for homologous recombination are described in U.S. Pat. No. 5,614,396.

EXAMPLES

Example 1; Materials and Methods

The following are the materials and methods used in the Examples set forth below.

Parallel RNA-sequencing (RNA-seq)

RNA was fragmented and Illumina sequencing libraries were prepared according to the manufacturer's instructions. Briefly, total RNA was first degraded and converted to a library of cDNA fragments. Fragmentation sizes and final library sizes were analyzed using a Bioanalyzer (Agilent Technologies, Santa Clara, CA). Subsequently, each fragment was extended with an A' base on the 3' end, ligated with paired-end adaptors and amplified to enrich the targeted regions of the RNA (coding regions). cDNA libraries were then amplified, denaturated and loaded onto an Illumina cBot for cluster generation according to the manufacturer's recommended protocols. The primer-hybridized flow cells were subjected to Illumina sequencing on the HiSeq 2000 sequencer (single-end, depth 100X) (Illumina, San Diego, CA). Between 20 and 25 million 100-basepair reads were generated for each of 7 matched- normal samples. Actual alignment of the raw cDNA reads was carried out by tophat- fusion (Genome Biol. 2011 Aug 11 ; 12(8):R72), and for further downstream the de novo assembly of transcript level reads was carried out by Cufflinks (Trapnell et al. Nat Protoc. 2012 Mar l ;7(3):562-78; Trapnell et al, Nat Biotechnol. 2010

May;28(5):511-5). Briefly, tophat-fusion breaks up individual reads into 25 bp segments, which are mapped independently to the reference build hgl9 via bowtie (Genome Biol. 2009; 10(3):R25). Following Edgren et al (Edgreen et al. Genome

Biol. 2011; 12(1):R6), the following filtration scheme was used to identify fusion events: 1)BLAST sequence around putative breakpoint to hgl9 build to identify and remove paralogous sequences; 2) Filter fusions based on the number of reads that support the putative fusion breakpoints, setting the minimum number of such spanning reads conservatively; 3) compute scores of distributions of coverage of reads around the putative breakpoints, rejecting nonuniformly covered reads; 4) fusions between adjacent genes were rejected as read-through transcript events, with a minimum distance of 100 kb; 5) finally, a read was considered to support a fusion if it mapped to both sides of the breakpoint by at least a minimum fusion anchor length of 25 bp., or generally about a quarter of a read length. All of these parameters were essentially tuned with the constraint that the matched normal sample had no positive fusion detections.

RT-PCR and Sanger Sequencing

One microgram ^g) of RNA was retrotranscribed into cDNA using the Clontec kit (Clontech Laboratories, Inc., Mountain View, CA) following

manufacturer's instructions. The resulting cDNA was used as template for semiquantitative polymerase chain reaction (PCR) amplification using the primers reported in Table I, below:

Table I; Primers Sequences

"FFPE" means formalin fixed paraffin embedded tissue; "FW" means forward primer; and "RV" means reverse primer.

To detect the presence of the fusion product, the PCR amplifications on human tissues were performed using the following protocol: 95°C for 2 minutes, 40 cycles of 95°C 30 seconds, 55°C for 30 seconds, 72°C for 2 minutes. The PCR amplifications were performed in a volume of 25 μϊ_^ reaction mixture containing 1.5 mM MgCk, 0.2 mM of each dNTP, 0.125 mM of each primer, and 1U of Platinum Taq DNA

Polymerase (Invitrogen, Carlsbad, CA). PCR products were purified using the Qiaquick PCR purification kit (Qiagen) and sequenced using an Applied Biosystems 3700 DNA sequencer (ABI PRISM® 3730XL; Applied Biosystems, Grand Island, NY). For the amplification and subsequent cloning of the full fusion gene, 1 μg of RNA of the index case was reverse transcribed using 2 pmol of the gene specific reverse primer with Superscript III (Invitrogen) following manufacturer's instructions with the exception that 1 hour incubation was at 55°C. 2 units of RNase H (NEB) was added to the reaction to remove complementary RNA and incubated at 37°C for 20 minutes.

Cell Lines and Culture Media

293T cells were purchased from ATCC and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. Cells were maintained at 37°C in a 5% CO2 atmosphere. The FGFR2 inhibitor, BGJ398, was purchased from SelleckBio (Houston, Texas) and added at the cell culture at different concentrations (range 100 nM- 10μΜ).

Cloning Strategy and Stable Transfection

The fusion FGFR2-PPHL 1 fusion cDNA was amplified by PCR using the Pfx platinum system from Invitrogen and the primers reported in Table 1, above. Five (5) μΐ template were added to each reaction. The PCR amplifications were performed using the following protocol: 95°C for 2 minutes, 45 cycles of 95°C for 1 minute, 52°C for 1 minute, 72°C for 5 minutes. A secondary PCR was performed on 2 μΐ of primary PCR using primers designed with an attB overhang to amplify the fusion cDNA in frame for cloning into the Gateway system for mammalian expression (Invitrogen) (primers sequence is reported in Table I, above). PCR was performed using the Pfx platinum system as previously outlined. The attB PCR product was cloned first into pDONR zeo to create an entry clone, then shuttled into the mammalian expression vector pcDNA™6.2/EmGFP-Bsd/V5-DEST according to manufacturer's instructions to create pDEST_FGR2/PPHL l. Cloning reactions were incubated overnight to achieve maximum recombination efficiency. 6 well plates containing 2xl0⁵ 293T cells were stably transfected with 2.5 μg linearized pDEST_FGFR2/PPHL l or empty vector using the Lipofectamine™ transfection system (Invitrogen). Following 48 hours of transfection cells were selected with BLasticidn (10 μg/ml) for at least 2 weeks.

Co-immunoprecipitation

Proteins were extracted with RIPA buffer containing phosphatase- (78428, ThermoScientific, Maltham, MA) and protease-inhibitors (04693124001, Roche, Basel, Switzerland). 300 μg of lysate was immunoprecipitated with 2 μΐ of the anti- V5 antibody (cat#R960-25, Life Technology) for 3 hours at 4 °C. Lysates were incubated with protein A/G Agarose beads (cat# 20423, Pierce) for 1 hour at 4 °C. After washing steps, beads were resuspended in Laemmli sample buffer. Proteins were loaded on 7.5% SDS gels and transferred to PVDF membranes. Membranes were BSA-blocked and hybridized at 4°C overnight with primary antibodies anti-V5 (cat#R960-25, Life Technology), anti-FGFR2 (abeam®, Cambridge, MA), ERK, phopsho-ERK, Phospho tyrosine (Cell Signaling Technology®, Danvers, MA) and tubulin (Sigma-Aldrich, St. Louis, MO). HRP-conjugated secondary antibody was applied at room temperature for 1 hour. Blots were developed using RPN 2132 ECL plus solution (GE Healthcare, Waukesha, WI) and imaged with FUJIfilm Laser Image Analyzer (GE Healthcare Life Sciences, Waukesha, WI).

Example 2; Detection of FGFR2-PPHLN1

Fusion Gene in ICC Patients

Massive parallel RNA-sequencing (RNA-seq) using next-generation sequencing technology allows a detailed and highly comprehensive characterization of cellular transcriptomes, including the detection of intragenic fusion events, such as, e.g., in-frame fusions that may lead to oncogene activation.

Single-end RNA-seq was performed using high quality cDNA from 7 tumoral and 7 adjacent normal tissues of resected human ICC. ). By applying stringent statistical criteria based on the number of reads and computed score (see detailed online methods), a total of 13 novel inter- and intrachromosomal fusion events were identified, as shown in Table II, below:

Table II: Candidate Fusion Genes Identified by RNA-seq

Table legend: "FGFR2": fibroblast growth factor receptor 2;"FGD4": FYVE, RhoGEF and PH domain containing 4; "CHTOP": chromatin target of PRMT1; "DCDC2": doublecortin domain containing 2; "FGL1": fibrinogen-like 1; "TMEM39A": transmembrane protein 39A; "SERINC2": serine incorporator 2; "SPG7": spastic paraplegia 7; "C16oRF13": chromosome 16 open reading frame 13; "NTM": neurotrimin; "RGS12": regulator of G-protein signaling 12 ; "PPHLN1": Periphilin-1; "TM9SF3": transmembrane 9 superfamily member 3; "S100PBP": S100P binding protein; "INTS3": integrator complex subunit 3; "GPLD1": glycosylphosphatidylinositol specific phospholipase Dl; "PCM1": pericentriolar material 1); "PN P": polynucleotide kinase 3'-phosphatase; "NARFL": nuclear prelamin A recognition factor-like "METAP1": methionyl aminopeptidase 1 ; "IGLL5": immunoglobulin lambda-like polypeptide 5.

By applying stringent statistical criteria based on the number of reads and computed score, an interchromosomal fusion comprising a portion of the tyrosine kinase receptor FGFR2 (10q26) with PPHLN1 (12ql2), a gene involved in epithelial differentiation, was identified in 1 out of the 7 patient analyzed (14.3%). The FGFR2-PPHLN1 was represented by 149 single-end reads spanning or encompassing the fusion junction of exon 19 oiFGFR2 to exon 4 oiPPHLNl (Fig. 1).

Using primers spanning the breakpoint, the fusion in the index case was confirmed by RT-PCR (Fig. 2) and subsequent Sanger sequencing of the PCR product (Fig. 3). Furthermore, by performing broad range PCR using primers spanning the starting codon of FGFR2 and the 3 ^'untranslated region of PPHL 1 , the complete 5 ^' FGFR2 and the 3 ^' PPHLN 1 was characterized. As indicated in the Figure 4, two different bands around 3.5 Kb were detected. The upper band corresponds to the long isoform of FGFR2 and the lower band corresponds to the short isoform of FGFR2 (Fig. 4). It was verified that the first 19 exons oiFGFR2 were present at the 5 ^' end of the fusion gene with intact open reading frame and kinase domains, suggesting potential activity. At the 3 ' end, the fusion partner PPHL l was missing only the first 3 exons, the first two of which are usually untranslated. Both FGFR2 and PPHL l have been reported to exhibit tissue-specific splicing. RNA-seq data of the fusion case revealed that the FGFRIIIb isoform (GenBank® Accession Number NM_022970) (SEQ ID NO: 2) and PPHLNl isoform 3 (GenBank® Accession Number NM_201439.1) (SEQ ID NO: 3) were the most abundant (occurring in 48% of the samples) (Fig. 5). The resulting protein product of the FGFR2-PPHLN1 fusion gene is predicted to contain 1, 111 amino acids (122 kDa), with the amino-terminal portion (residues 1-768) identical to that of FGFR2 (accession number NP_075259.4) (SEQ ID NO: 6), whereas the carboxy-terminal portion (residues 769-1111) is identical to PPHLNl starting at residue 25 of the wild-type protein (accession number NP_958847.1) (SEQ ID NO: 7) (Fig. 6A). Fig. 6B illustrates the domains present in the FGFR2 and PPHLNl portions of the fusion gene. The FGFR2 portion contains three IgG domains and two tyrosine phosphorylation sites (TK1 and TK2).

In the normal genome, FGFR2 and PPHLNl map to chromosome 10q26 and 12ql2, respectively, and are transcribed in opposite directions. To verify the presence of a DNA rearrangement, whole-genome sequencing of the tumor and matched normal tissue was performed. An inverted translocation between chrlO and 12 was identified in the tumor, but not in the normal tissue, indicating that this translocation was the mechanism responsible for the fusion oiFGFR2 and PPHLNl on the same genomic segment and in a common direction of transcription (Fig. 7). This data allowed specific mapping of the genomic breakpoint of the FGFR2-PPHLN1 fusion gene and, using primers spanning the breakpoint and subsequent Sanger sequencing, the genomic DNA sequence around the breakpoint was confirmed (Fig. 7 and Fig.16). On the basis of these results, to determine if this fusion gene was a recurrent event in ICC patients, 108 formalin- fixed cases of ICC were screened by RT-PCR and Sanger sequencing. 21 cases out of 108 (19.4%) were found positive for the presence of the fusion event. Like the first patient sample analyzed, these cases showed an identical fusion cDNA sequence around the breakpoint (TCACAACCAATGAG - GATGGCTACAATAG) (SEQ ID NO: 10) (Fig. 8A, Fig. 8B). Example 3; Functional Analysis of

FGFR2-PPHLN1 Fusion gene Protein Product

PPHLN1 is one of several proteins that become sequentially incorporated into the cornified cell envelope during the terminal differentiation of keratinocytes. PPHL 1 over-expression has been reported to induce S-phase arrest through transcriptional repression of Cell Division Cycle 7 (Cdc-7), an essential factor for S phase progression. The C-terminus domain of the wild-type protein seems to be responsible for dimerization. The potential mechanism by which dimerization of the fusion gene and the resulting constitutive activation of the downstream signaling pathways (e.g., AKT, ERK and STAT signaling pathways) leading to proliferation, survival and angiogenesis of cancer cells, is illustrated in Fig. 9.

To determine if PPHL 1 is able to mediate dimerization and activation of the receptor in the fusion gene, 293T cells were stably transfected to express V5-tagged FGFR2-PPHL 1 fusion gene and co-immunoprecipitation was performed. As indicated in the Figure 10, both monomer (around 120 kDa) (upper arrow) and dimerized form of FGFR2 (240 KDa) (lower arrow) were identified by Western blot (Figure 10, left panel). Constitutive activation of the fusion protein was also observed, as demonstrated by tyrosine phosphorylation of the over-expressed fusion construct. Also, the downstream pathway ERK was determined to be phosphorylated in the 293T cells over-expressing the fusion gene (Fig. 10, right panel).

The complete predicted fusion gene polypeptide sequence is shown in Figure

11.

Example 4; Oncogenic Potential of FGFR2-PPHLN1 Fusion Gene

This example demonstrates that cells expressing the FGFR2-PPHL 1 fusion gene have enhanced proliferation relative to the same cells that do not express the fusion gene.

To investigate the transforming and oncogenic potential of the fusion gene, stably transfected 293T cells expressing the FGFR2-PPHLN1 fusion gene were further characterized. 293T cells over-expressing the fusion FGFR2-PPHL 1 showed an increased proliferation rate compared to their parental cell line (Fig. 12). In another experiment, stably transfected 293T cells expressing the FGFR2- PPHL 1 fusion gene and stable cell lines expressing the FGFR2 -PPHL l fusion gene are treated with the FGFR2 specific inhibitor AZD4547. It is expected that treatment with the inhibitor will significantly inhibit proliferation of the FGFR2- PPHL 1 fusion gene expressing cells relative to control cells.

In another experiment, the proliferation of HUCCT 1 cells (an ICC cell line) is measured following stable transfection of the cells with the FGFR2 -PPHLNl fusion gene. It is expected that transfection with the fusion gene will enhance proliferation of the HUCCT 1 cells relative to control cells (transfection with empty vector).

Example 5; Inhibition of the migratory capability of HUCCT1 cells overexpressing the FGFR2-PPHLN1 fusion protein

This example demonstrates the inhibition of the migratory capability of HUCCT 1 cells overexpressing the FGFR2-PPHLN1 fusion protein by the small compound inhibitor BGJ398.

As illustrated in Figure 13 A, one isoform of the FGFR2-PPHLN1 fusion polypeptide is predicted to contain 1111 amino acids (122 kDa) with the amino- terminal portion (residues 1-768) identical to that of FGFR2 whereas the carboxy- terminal portion (residues 769-1111) is identical to PPHLNl starting at residues 25 of the wild-type protein (see Fig. 11). Interestingly, the C-terminus domain of the wild- type PPHLNl protein seems to be responsible for homodimerization. To verify if PPHLNl is able to mediate dimerization and activation of the FGFR2 in the fusion gene, V5-tagged FGFR2-PPHLN1 was expressed in 293T cells. Protein

oligomerization was confirmed by immunoprecipitation. Constitutive activation of the fusion protein as shown by tyrosine phosphorylation of the fusion kinase and activation of downstream MAP kinase ERK 1/2 was observed.

In order to characterize the functional role of the identified fusion in an ICC in vitro model, a stable HUCCT 1 cell line over-expressing the FGFR2-PPHLN1 fusion protein shown in Figure 13 A was generated. Cells harboring the fusion presented increased viability, clonogenic and migratory capacity (Figs. 13B-C), demonstrating oncogenic capability of the fusion protein. Furthermore, HUCCT 1 cells over- expressing the fusion protein showed enhanced sensitivity to the selective FGFR2 inhibitor BGJ398 compared to their parental cell line transfected with the empty vector (p<0.001, Figs. 14A-B). In particular, significant inhibition of the migratory capability was observed only in the cells expressing the fusion protein (p< 0.0001, Fig. 14B).

A number of embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating intrahepatic cholangiocarcinoma (ICC) in a patient, the method comprising:

(a) providing a biological sample from the patient;

(b) detecting the presence or absence in the sample of a fusion gene having a 5' portion from an fibroblast growth factor receptor 2 (FGFR2) gene or fragment thereof and a 3' portion from a Periphilin-l (PPHL l) gene or fragment thereof or the polypeptide encoded by the fusion gene; and

(c) treating the ICC if the presence of the fusion gene or polypeptide is detected.

2. The method of claim 1, wherein the FGFR2 gene or fragment thereof comprises the first 19 exons of FGFR2.

3. The method of claim 1, wherein step (b) comprises detecting chromosomal rearrangements of genomic DNA having a 5' DNA portion from the FGFR2 gene or fragment thereof and a 3' DNA portion from the PPHLNl gene or fragment thereof.

4. The method of claim 1, wherein step (b) comprises detecting chimeric mRNA transcripts having a 5' RNA portion transcribed from the FGFR2 gene or fragment thereof and a 3' RNA portion transcribed from the PPHLNl gene or fragment thereof.

5. The method of claim 1, wherein the biological sample is selected from the group consisting of tissue, blood, plasma, serum, urine, urine supernatant, urine cell pellet, tumor cells, and liver cells.

6. The method of any of the preceding claims, wherein step (b) comprises determining the presence of the fusion gene using an automated sequencer.

7. The method of any of the preceding claims wherein the detecting in step (b) comprises detecting a polypeptide encoded by the fusion gene.

8. The method of any of the preceding claims, wherein treating ICC comprises administering to the patient one or more cancer therapies selected from the group consisting of: a chemotherapeutic agent, a biologic agent, a cytokine, radiation therapy, immunotherapy, surgery, and an inhibitor of the FGFR2-PPHL 1 fusion gene or the polypeptide encoded by the fusion gene.

9. A method for detecting the presence of a fusion gene having a 5' portion from a FGFR2 gene or fragment thereof and a 3' portion from a PPHL 1 gene or fragment thereof in a human subject, the method comprising:

(a) obtaining nucleic acid from a biological sample collected from the subject;

(b) analyzing the nucleic acid to determine its nucleotide sequence with an electronic computer sequencing device;

(c) comparing, with software programmed to perform a base-by-base comparison, the nucleotide sequence of the nucleic acid to a control sequence comprising at least 25 consecutive nucleic acids of one of the following nucleic acid sequence:

(i) if the nucleic acid comprises genomic DNA:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13);

(ii) if the nucleic acid comprises mRNA:

CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACUAGUU AA (SEQ ID NO: 12); and

(iii) if the nucleic acid sequence comprises cDNA:

CGAATTCTCACTCTCACAACCAATGAGGATGGCTACAATAGACTAGTTAA

(SEQ ID NO: 14) or its complement:

TTAACTAGTCTATTGTAGCCATCCTCATTGGTTGTGAGAGTGAGAATTCG

(SEQ ID NO: 15); (d) displaying the comparison between the nucleotide sequence of the nucleic acid and the control sequence on an electronically operated (computer monitor) screen; and

(e) determining that the fusion gene is present in the subject when the compared sequences are substantially identical; or

(f) determining that the fusion gene is not present in the sample when the compared sequences are the not substantially identical.

10. The method of claim 9, wherein the biological sample is obtained from a tumor biopsy.

11. The method of claim 9 or 10, wherein the treating comprises administering an inhibitor of the FGFR2-PPHL 1 fusion gene.

12. A method for determining the susceptibility of a cancer in a patient to a cancer therapy comprising administration of an inhibitor of the FGF pathway, the method comprising,

(a) providing a biological sample from the patient;

(b) detecting the presence or absence in the sample of a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL l gene or fragment thereof or the polypeptide encoded by the fusion gene; and

(c) identifying the cancer as likely to be susceptible to the cancer therapy if the fusion gene or polypeptide is detected in the sample.

13. The method of claim 12, wherein the cancer is ICC.

14. The method of claim 12 or 13, further comprising prescribing or

administering the therapy comprising administration of an FGF pathway inhibitor to the patient when the fusion gene is detected in the sample.

15. The method of any of the preceding claims, wherein the PPHL l gene or fragment thereof encodes a polypeptide that is capable of inducing the

oligomerization of FGFR2.

16. The method of any of the preceding claims, wherein the FGFR2 gene or fragment thereof encodes one or more kinase domains.

17. The method of any of the preceding claims, wherein the fusion gene encodes one or more tyrosine phosphorylation sites.

18. The method of any of the preceding claims, wherein the FGFR2 gene has a nucleic acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence variant thereof.

19. The method of any of the preceding claims, wherein the PPHLN1 gene has a nucleic acid sequence comprising SEQ ID NO: 3 or a sequence variant thereof.

20. The method of claim 19, wherein the fragment of the PPHLN1 gene has a nucleic acid sequence comprising residues 178-1663 of SEQ ID NO: 3.

21. The method of any of the preceding claims, wherein the amino terminal portion of the polypeptide encoded by the fusion gene comprises a carboxy -terminally truncated portion of an FGFR2 polypeptide having the amino acid sequence identified by SEQ ID NO: 6 or a sequence variant thereof.

22. The method of any of the preceding claims, wherein the carboxy terminal portion of the polypeptide encoded by the fusion gene comprises an amino-terminally truncation portion of the PPHLN1 polypeptide having an amino acid sequence identified by SEQ ID NO: 7 or a sequence variant thereof.

23. The method of claim 8, wherein the therapy for treating ICC comprise administering a therapy that targets the fusion gene or polypeptide encoded by the fusion gene.

24. The method of claim 23, wherein the therapy for treating ICC is an antisense therapy, a gene therapy, or an antibody therapy.

25. A method for treating ICC in a subject in need thereof, the method comprising targeting in the subject a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL l gene or fragment thereof, or targeting a polypeptide encoded by the fusion gene.

26. The method of claim 25, wherein the targeting comprises administering to the subject an inhibitor of the fusion gene or polypeptide encoded by the fusion gene.

27. The method of claim 26, wherein the inhibitor is an antisense molecule specific for the fusion gene.

28. The method of claim 26, wherein the inhibitor is an antibody specific for the polypeptide encoded by the fusion gene.

29. The method of claim 25, wherein the targeting comprises administering gene therapy to the subject.

30. The method of claim 29, wherein the gene therapy is targeted to the FGFR2- PPHL 1 fusion gene.

31. A method for inhibiting the development or progression of ICC in a subject in need thereof, the method comprising targeting in the subject a fusion gene having a 5' portion from an FGFR2 gene or fragment thereof and a 3' portion from a PPHL l gene or fragment thereof, or targeting a polypeptide encoded by the fusion gene.

32. The method of claim 31, wherein the targeting comprises administering to the subject an inhibitor of the fusion gene or polypeptide encoded by the fusion gene.

33. The method of claim 32, wherein the inhibitor is an antisense molecule specific for the fusion gene.

34. The method of claim 32, wherein the inhibitor is an antibody specific for the polypeptide encoded by the fusion gene.

35. The method of claim 31, wherein the targeting comprises administering gene therapy to the subject.

36. The method of claim 35, wherein the gene therapy is targeted to the FGFR2- PPHL 1 fusion gene.

37. The method of any of the preceding claims, wherein the fusion gene comprises a fusion junction genomic nucleic acid sequence comprising:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13).

38. A composition comprising at least one of the following: (a) an

oligonucleotide probe comprising a sequence that hybridizes to a junction of a chimeric genomic DNA or chimeric mRNA in which a 5' portion of the chimeric genomic DNA or chimeric mRNA is from an FGFR2 gene or fragment thereof and a 3' portion of the chimeric genomic DNA or chimeric mRNA is from a PPHLNl gene or fragment thereof; (b) a first oligonucleotide probe comprising a sequence that hybridizes to a 5' portion of a chimeric genomic DNA or chimeric mRNA from an FGFR2 gene or fragment thereof and a second oligonucleotide probe comprising a sequence that hybridizes to a 3' portion of the chimeric genomic DNA or chimeric mRNA from a PPHLNl gene or fragment thereof; and (c) a first amplification oligonucleotide comprising a sequence that hybridizes to a 5' portion of a chimeric genomic DNA or chimeric mRNA from an FGFR2 gene or fragment thereof and a second amplification oligonucleotide comprising a sequence that hybridizes to a 3' portion of the chimeric genomic DNA or chimeric mRNA from a PPHLNl gene or fragment thereof.

39. The composition of claim 38, wherein the junction of a chimeric genomic DNA comprises the sequence:

TGCCTTGCAGCCTCTTTCTAGGTGTATATAAACATACACTGTTTTCAGGTC TACTTAATTTAGTAATTCAGTTATATACATATTCCATAT (SEQ ID NO: 9), or its complement:

ATATGGAATATGTATATAACTGAATTACTAAATTAAGTAGACCTGAAAAC AGTGTATGTTTATATACACCTAGAAAGAGGCTGCAAGGCA (SEQ ID NO: 13).

40. The composition of claim 38, wherein the junction of a chimeric mRNA comprises the sequence:

CGAAUUCUCACUCUCACAACCAAUGAGGAUGGCUACAAUAGACUAGUU AA (SEQ ID NO: 12).

41. The composition of claim 38, wherein the FGFR2 gene has a nucleic acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence variant thereof.

42. The composition of claim 38, wherein the PPHLN1 gene has a nucleic acid sequence comprising SEQ ID NO: 3, or a sequence variant thereof.