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WO2013119950A2 - Procédés et compositions concernant des fusions de ros1 pour diagnostiquer et traiter le cancer - Google Patents

Procédés et compositions concernant des fusions de ros1 pour diagnostiquer et traiter le cancer Download PDF

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WO2013119950A2
WO2013119950A2 PCT/US2013/025345 US2013025345W WO2013119950A2 WO 2013119950 A2 WO2013119950 A2 WO 2013119950A2 US 2013025345 W US2013025345 W US 2013025345W WO 2013119950 A2 WO2013119950 A2 WO 2013119950A2
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Prior art keywords
rosl
primer
cancer
fusion
pcr
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PCT/US2013/025345
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English (en)
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WO2013119950A3 (fr
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David HOUT
John HANDSHOE
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Insight Genetics, Inc.
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Priority to CN201380013401.4A priority Critical patent/CN104364391A/zh
Priority to AU2013216904A priority patent/AU2013216904A1/en
Priority to JP2014556726A priority patent/JP2015508644A/ja
Priority to EP13746869.0A priority patent/EP2812451A4/fr
Publication of WO2013119950A2 publication Critical patent/WO2013119950A2/fr
Publication of WO2013119950A3 publication Critical patent/WO2013119950A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • ROS 1 tyrosine kinase Oncogenic fusions of the ROS 1 tyrosine kinase have recently been reported to occur in subsets of several human cancers including non-small cell lung carcinoma (NSCLC), glioblastoma multiforme (GBM) brain tumors, and cholangiocarcinomas (biliary tract tumors). Cancer cells that express ROS1 fusions are "addicted" to the aberrant signaling associated with the constitutively active chimeric forms of the kinase for their proliferation and survival. In keeping with this dependence upon abnormal ROS1 signaling, preclinical studies have demonstrated ROS1 -driven cancers to beakily sensitive to
  • the methods and compositions disclosed herein relate to the field of detection or diagnosis of a disease or condition such as cancer; assessing the susceptibility or risk for a disease or condition associated with a nucleic acid variation, truncation, or gene fusion; the monitoring disease progression; and the determination of susceptibility or resistance to therapeutic treatment.
  • the method of detection and diagnosis disclosed herein relate to the detection and/or diagnosis of ROS l-fusion related cancers.
  • this invention in one aspect, relates to methods of detecting the presence of a ROS 1 related cancer by detecting a nucleotide variation, such as a fusion, within a nucleic acid of interest comprising conducting, real-time polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), and or real-time RT-PCR on extracted from a tissue sample mRNA from a subject with a cancer or PCR on cDNA synthesized from the RNA extracted from a subject with a cancer; wherein the presence of amplification product or an increase in amplification product relative to a control indicates the presence of nucleotide variation, truncation, or excessive expression, thereby detecting the presence of a cancer.
  • RT-PCR reverse transcription polymerase chain reaction
  • methods of methods of diagnosing a subject with a cancer as having a ROSl related cancer further comprising determining the cycle thresholds (Ct) values for wild-type ROSl and wild-type ROSl kinase; wherein a high (Ct) value for wild-type ROSl relative to ROSl kinase indicates the presence of a fusion.
  • Ct cycle thresholds
  • a forward primer which binds to a ROS l fusion partner 5' to the fusion breakpoint and a reverse primer which binds to ROSl 3' to the fusion breakpoint, wherein said primers extend through the fusion, wherein detection of the presence of an amplicon having both ROSl and fusion partner nucleic acids indicates the presence of a fusion.
  • methods of methods of diagnosing a subject with a cancer as having a ROSl related cancer comprising a) a first amplification reaction using a forward primer which binds to a ROSl fusion partner; wherein the amplicon from the first reaction is used as a template for a second amplification reaction; b) a second amplification reaction following the first reaction, wherein primers specific for a ROSl sequence 3' of the fusion breakpoint are used in the second amplification reaction; and c) detecting the presence of ROS l in the amplicon from the second reaction; wherein detection of ROSl in the amplicon from the second reaction indicates the presence of a ROSl fusion.
  • a nucleotide variation such as a fusion
  • a nucleic acid of interest comprising conducting fluorescence in situ hybridization (FISH) on a tissue sample from a subject with cancer, wherein the probes used for the hybridization flank the breakpoint for ROS l fusions, wherein the probes are differently labeled, and wherein the separation of the probes indicates the presence of a ROS 1 related cancer.
  • FISH fluorescence in situ hybridization
  • closely placed hybridized probes indicate wild-type RTK, such as, for example, ROS l.
  • kits for diagnosing an ROS l related cancer comprising (a) a first primer labeled with a first detection reagent, wherein said first primer is a reverse primer, wherein said reverse primer is one or more polynucleotide(s) that hybridizes, to a first polynucleotide encoding the amino acid sequence of SEQ ID NO 1 or the complement thereof; and (b) at least one second primer, wherein said second primer is a forward primer, wherein said forward primer is one or more polynucleotide(s) that hybridizes to a second polynucleotide encoding wild-type ROS1.
  • FIG. 1 shows a schematic representation of receptor tyrosine kinases (RTKs) that are involved in oncogenesis due to the generation of fusion kinases.
  • RTKs receptor tyrosine kinases
  • the drawings illustrate the normal, membrane-spanning RTKs.
  • the proteins shown in red underneath each RTK form constitutively active, oncogenic fusions with the kinase.
  • Figure 2 shows representative ROS1 fusion kinase, the CD74-ROS1 fusion kinase.
  • Figure 3 shows ROS 1 fusions in NSCLC. Schematic of transcripts created by chromosomal translocations with the ROS1 gene. Exon locations are indicated to demonstrate diversity of fusion configurations.
  • Figure 4 shows a ROS1 breakapart FISH assay. Genomic DNA clones flanking the breakpoint location within the ROS1 gene at which chromosomal rearrangements occur to create ROS1 fusion genes in human cancers (upper schematic) were differentially labeled with green or red fluorochromes, then hybridized to interphase nuclei and metaphase chromosomes from normal peripheral blood lymphocytes (lower left panel) or an
  • FIG. 5 shows the Insight ROS 1 Fusion Screen Methodology.
  • the assay uses fusion-specific reverse transcription (FS-RT) at high temperature to prevent promiscuity of the reverse transcriptase to prime cDNA synthesis from stem-loop structures located within the RNA transcripts. By performing the assay at high temperatures these structures are minimized during the elongation process and restrict production of the first-strand cDNA to ROS1 fusions only.
  • the first-strand reaction is then subject to RNase digestion in order to remove all RNA which may allow for non-specific amplification in the downstream PCR detection phase.
  • the fusion-specific cDNA is then used as a template for the universal downstream ROS1 kinase quantitative PCR (qPCR) assay.
  • qPCR quantitative PCR
  • Multiple ROS1 fusions can be targeted by multiplexing numerous FS-RT primers during the reverse transcription phase.
  • This FS-RT primer cocktail can be readily modified to capture newly identified oncogenic fusions without increasing the number of primers used for detection maintaining specificity of the reaction and high-throughput capability.
  • Figure 6 shows the Insight ROS1 Fusion ScreenTM. Schematic representation of the ROS1 cDNA, the location of the breakpoints in cancer-associated ROS 1 fusions, and the approximate locations of the PCR primer sets employed in the real-time qPCR assay. The Ct values indicative of normal levels of wild-type ROS1 expression, wild-type ROS1 overexpression, or the presence of a ROS 1 fusion are shown in the lower panels.
  • Figure 7 shows the Insight ROS1 ScreenTM v2. Schematic representation of the ROS1 RNA, the location of the breakpoints in cancer-associated ROS1 fusions, and the approximate locations of the primers used for cDNA synthesis. The position of the ROS1 template specific kinase reaction is also shown for the post cDNA synthesis qPCR detection phase of the reaction. Blue arrows indicate the relative position of the SLC34A2 cDNA primer and the red arrow indicates the relative position the CD74 specific cDNA primer in approximation to the actual ROS1 translocation.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • an “increase” can refer to any change that results in a larger amount of a composition or compound, such as an amplification product relative to a control.
  • an increase in the amount in amplification products can include but is not limited to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase.
  • the detection an increase in expression or abundance of a DNA, mR A, or protein relative to a control necessarily includes detection of the presence of the DNA, mRNA, or protein in situations where the DNA, mRNA, or protein is not present in the control.
  • tissue samples can be obtained by any means known in the art including invasive and non-invasive techniques. It is also understood that methods of measurement can be direct or indirect. Examples of methods of obtaining or measuring a tissue sample can include but are not limited to tissue biopsy, tissue lavage, aspiration, tissue swab, spinal tap, magnetic resonance imaging (MRI), Computed Tomography (CT) scan, Positron Emission Tomography (PET) scan, and X-ray (with and without contrast media).
  • MRI magnetic resonance imaging
  • CT Computed Tomography
  • PET Positron Emission Tomography
  • Allele specific primers can be designed to target a mutation at a known location such that its signal can be preferentially amplified over wild-type DNA. Unfortunately, this is not possible with unknown mutations that may occur at any position (base) in the target sequence.
  • tissue sample can come from any tissue in a body.
  • tissue refers to blood, neural tissue (e.g., brain tissue or spinal cord tissue), lymphatic tissue, hepatic tissue, splenic tissue, pulmonary tissue, cardiac tissue, gastric tissue, intestinal tissue, pancreatic tissue, tissue from the thyroid gland, salivary glands, joints, and the skin.
  • neural tissue e.g., brain tissue or spinal cord tissue
  • lymphatic tissue e.g
  • Erythrocyte Platelet or other blood cell.
  • the cell could be an epithelial cell, hypatocyte, neuron, or other cell.
  • the disclosed methods in one aspect related to methods of detection or diagnosis of the presence of a disease or condition such as a ROS 1 related cancer (such as, for example, Non-small cell lung carcinoma (NSCLC), glioblastoma or cholangiocarcinomas), assessing the susceptibility or risk for a disease or condition such as a ROS1 related cancer (such as, for example, NSCLC, glioblastoma or cholangiocarcinomas), the monitoring of the progression of a disease or condition such as a ROS1 related cancer (such as, for example, NSCLC, glioblastoma or cholangiocarcinomas), and the determination of susceptibility or resistance to therapeutic treatment for a disease or condition such as a ROS 1 related cancer (such as, for example, NSCLC, glioblastoma or cholangiocarcinomas) in a subject previously diagnosed with a cancer comprising obtaining a tissue sample, detecting the presence or measuring the expression level of ROS 1 mRNA from a
  • the detection or diagnosis of the presence of a disease or condition such as a ROS 1 related cancer can be accomplished using in situ hybridization, wherein the separation of probes flanking the breakpoint of a gene that is site of fusion to another gene in cancers indicates the presence of a ROS 1 related cancer.
  • a disease or condition such as a ROS 1 related cancer
  • ROS1 Proto-oncogene Tyrosine Protein Kinase ROS Precursor
  • RTKs Receptor tyrosine kinases
  • ROS1 is a distinct receptor which is distantly related to the Anaplastic Lymphoma Kinase/Leukocyte Tyrosine Kinase (ALK/LTK) and Insulin Receptor (INSR) families.
  • ROS 1 is the orphan (i.e., for which a ligand has yet to be determined) vertebrate counterpart of the Drosophila sevenless receptor tyrosine kinase (receptor of sevenless a.k.a., ROS), which when activated by its ligand BOSS (bride of sevenless) is responsible for the differentiation of the R7 photoreceptors in the developing fly compound eye.
  • ROS leukocyte tyrosine kinase
  • FIG-ROS1 an interstitial micro-deletion on chromosome 6q21 that results in a fusion event between a novel gene called FIG (/used m glioblastoma) and sequences coding for the intracellular portion of ROS 1 was discovered in human glioblastoma cell lines.
  • FIG-ROS1 The experimentally enforced expression of FIG-ROS1 in the CNS of genetically engineered adult mice results in the formation of glioblastoma multiforme tumors, confirming the oncogenic activity of this fused kinase.
  • FIG-ROS 1 fusion in glioblastoma brain tumors.
  • FIG- ROS1 fusions are not restricted only to glioblastoma; in 201 1, this chimeric kinase was shown to be expressed in cholangiocarcinomas (biliary tract cancers) as well, being present in 8.7% (2 of 23) primary tumor specimens. Like glioblastoma and NSCLC, the prognosis of patients with cholangiocarcinoma - which is the second most common primary hepatic carcinoma - is quite poor, the median survival being less than two years.
  • FIG-ROS1 expression experimentally rendered tumorigenic by FIG-ROS1 expression with a ROSl small-molecule inhibitor is associated with robust tumor cell killing.
  • ROS l signaling for instance, high levels of ROS l expression are found in 30-40% of glioblastoma surgical tumors and ROSl mutations have been identified in colorectal and renal carcinoma cell lines.
  • Commercial availability of efficient and reliable diagnostic tests to detect the presence of ROSl fusions in tumors would both greatly facilitate translational research to profile other cancers for these mutations while also providing for a more effective and less costly diagnosis of a particular cancer and enabling more effective and less costly personalized therapy of patients with ROSl fusion-positive tumors using inhibitors of this mutant kinase.
  • ROSl fusions do not represent the most common mutation of this tyrosine kinase they remain a significant proportion of tyrosine kinase fusion related cancers the significance of which increases with the identification of additional fusion partners.
  • Such fusions include but are not limited to Fused in
  • glioblastoma (FIG)-ROS l SLC34A2-ROS 1, , TPM3-ROS1, SDC4-ROS1, EZR-ROS 1, LRIG3-ROS1, and CD74-ROS1. All three fusions have been found in cholangiocarcinoma, non-small cell lung carcinoma ( SCLC), and glioblastomas.
  • SCLC non-small cell lung carcinoma
  • a disease or condition such as ROSl -related cancer
  • assessing the susceptibility or risk for developing a disease or condition such as ROSl-related cancer
  • a "ROS-1 related cancer” refers to any cancer where ROSl is dysregulated through the presence of a ROSl fusion, overexpression, mutation, or other mechanism.
  • the disclosed methods can be accomplished through quantitative PCR
  • qPCR qPCR assays.
  • methods of detecting the presence (i.e., diagnosing the presence) of an ROS l related fusion in a subject with a cancer comprising obtaining a tissue sample, isolating nucleic acid form the sample, performing PCR on the nucleic acid isolated form the tissue sample from a subject, wherein the primers for the qPCR assay comprise a primer pair specific for a ROSl kinase and a primer pair specific for wt ROSl 5' of the fusion breakpoint; and wherein cycle thresholds (Ct) values are determined; and wherein a high (Ct) value for wild-type ROSl (e.g., the extracellular domain (ECD) of ROS l) relative to ROSl kinase indicates the presence of a fusion and therefore a ROSl related-cancer.
  • Ct cycle thresholds
  • qPCR qPCR fusion-specific telomere sequencing a reverse primer which binds to ROSl 3' to the fusion breakpoint, wherein said primers extend through the fusion, wherein detection of the presence of an amplicon having both ROSl and fusion partner nucleic acids indicates the presence of a fusion and therefore a ROSl related-cancer.
  • the disclosed method can be used to diagnose a ROSl related cancer.
  • nucleic acid e.g., DNA or RNA such as mRNA
  • the disclosed methods comprise obtaining a tissue sample and isolating nucleic acid from the tissue sample.
  • the methods can comprise taking a pulmonary tissue biopsy or sputum sample and isolating mRNA from the sample.
  • cDNA can be synthesized from the mRNA and PCR performed on the cDNA (for example, as part of an RT-PCR reaction).
  • RNA-based reverse primer pair that specifically hybridizes to a wild- type ROS1 sequence (such as, for example, primers that hybridize to the extracellular domain (ECD) oiROSl, such as SEQ ID NOs: 13, 14, 20, and 21) and/or a forward and reverse primer pair that specifically hybridizes to a wild- type ROS1 sequence (such as, for example, primers that hybridize to the extracellular domain (ECD) oiROSl, such as SEQ ID NOs: 13, 14, 20, and 21) and/or a forward and reverse primer pair that specifically hybridizes to a wild- type ROS1 sequence (such as, for example, primers that hybridize to the extracellular domain (ECD) oiROSl, such as SEQ ID NOs: 13, 14, 20, and 21) and/or a forward and reverse primer pair that specifically hybridizes to a wild- type ROS1 sequence (such as, for example, primers that hybridize to the extracellular domain (ECD) oiROSl, such as S
  • Also disclosed are methods of diagnosing a ROS1 related cancer in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, wherein the nucleic acid from the tissue sample is RNA, wherein the method further comprises synthesizing cDNA from the RNA sample, conducting PCR on the cDNA; and detecting the presence of or measuring the amount of nucleic acid associated with one or a combination of both wild-type ROS1 (such as , for example the ECD oiROSl) and ROS1 kinase domain in the tissue sample, wherein an increase in amplicon relative to a normal control or the presence of an amplicon indicates the that the subject has a ROS 1 related cancer.
  • wild-type ROS1 such as , for example the ECD oiROSl
  • ROS1 kinase domain in the tissue sample
  • the disclosed methods can further comprise determining cycle thresholds (Ct) values, wherein a high (Ct) value for wild-type ROS1 relative to ROS1 kinase indicates the presence of a fusion and therefore a ROS1 related-cancer or contacting the amplicon with a labeled probe that is complementary to a sequence of the amplicon for detecting and measuring the amount of amplicon.
  • Ct cycle thresholds
  • ROS 1 fusions are detected by qPCR methodology using a two-step detection where the wherein only a forward primer which binds to a ROS 1 fusion partner is used in a first reaction and wherein in a second reaction following the first reaction the amplicon from the first reaction is used as a template for a second
  • methods of diagnosing a ROS l related cancer in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, conducting a first amplification reaction using a forward primer which binds to a ROSl fusion partner; wherein the amplicon from the first reaction is used as a template for a second amplification reaction; conducting a second amplification reaction following the first reaction, wherein primers specific for a ROSl sequence 3 ' of the fusion breakpoint are used in the second amplification reaction; and c) detecting the presence of ROSl in the amplicon from the second reaction; wherein detection of ROSl in the amplicon from the second reaction indicates the presence of a ROSl fusion; and detecting the presence of nucleic acid associated with ROSl kinase domain in the tissue sample, wherein the presence of an amplicon indicates the that the subject has a ROSl related cancer.
  • an allele specific method of detecting the presence of an ROSl related fusion comprising performing qPCR on a tissue sample from a subject, wherein the primers for the qPCR assay comprise a reverse primer specific for a ROSl kinase and a forward primer which binds 5' to the fusion breakpoint of a ROSl fusion partner; and wherein the presence of amplicon that reads across the fusion break point indicates the presence of a fusion and therefore indicates a ROS l related cancer. It is understood that in such a method amplicon resulting from the reverse primer or forward primer will be present, but as such
  • amplifications will only result from a single directional primer, the signal will be significantly less than the signal from a fusion event. Moreover, the size of such amplicons would be different from the size of fusion amplicon as the forward primer would only amplify the remaining portion of the fusion partner and the reverse primer would only amplify the portion oiROSl 5' of the reverse ROSl kinase primer being used.
  • a nucleotide variation such as a fusion
  • a nucleic acid of interest comprising conducting fluorescence in situ hybridization (FISH) on a tissue sample from a subject with cancer, wherein the probes used for the hybridization flank the breakpoint for ROSl fusions, wherein the probes are differently labeled, and wherein the separation of the probes indicates the presence of a ROS 1 related cancer.
  • FISH fluorescence in situ hybridization
  • closely placed hybridized probes indicate wild-type RTK, such as, for example, ROS l.
  • ROSl fusions are associated with several known cancer types. It is understood that one or more ROSl fusions can be associated with a particular cancer. It is further understood that there are several types of cancer associated with ROSl fusions including but not limited to anaplastic large-cell lymphoma (ALCL), neuroblastoma, breast cancer, ovarian cancer, colorectal carcinoma, renal carcinoma, hepatic carcinoma,
  • ACL anaplastic large-cell lymphoma
  • neuroblastoma neuroblastoma
  • breast cancer ovarian cancer
  • colorectal carcinoma renal carcinoma
  • renal carcinoma hepatic carcinoma
  • cholangiocarcinomas non-small cell lung carcinoma (NSCLC), diffuse large B-cell lymphoma, esophageal squamous cell carcinoma, anaplastic large-cell lymphoma, neuroblastoma, inflammatory myofibroblastic tumors, malignant histiocytosis, and glioblastomas.
  • NSCLC non-small cell lung carcinoma
  • esophageal squamous cell carcinoma anaplastic large-cell lymphoma
  • neuroblastoma inflammatory myofibroblastic tumors
  • malignant histiocytosis malignant histiocytosis
  • the subject it is understood to have been previously diagnosed with a cancer such as, for example, a NSCLC, a glioblastoma, or cholangiocarcinoma.
  • RTK receptor tyrosine kinases
  • a disease or condition such as cancer, for example an ROSl related cancer
  • corresponding ROSl wild-type sequence or an abundance relative to the ROSl wild-type sequence indicates the presence of a ROSl fusion sequences and therefore the presence of a cancer. Therefore, disclosed herein are methods of diagnosing an ROS l related cancer in a subject comprising detecting the presence or measuring the expression level of mRNA from a tissue sample from the subject; wherein the mRNA is specific to an ROSl fusion; and wherein an increase in the amount of mRNA relative to a control indicates the presence of an ROSl related cancer.
  • the disclosed methods of diagnosis and determination of susceptibility or resistance to ROS 1 inhibitor treatment can be used not only on subjects that have not previously been diagnosed with a cancer to identify that the subject has cancer, but specifically on subjects having been previously diagnosed with a cancer and the method used to diagnose that the cancer is specifically ROs 1 related or susceptible to treatment and thus not to diagnose a cancer but to determine if a known cancer in a subject is ROS l related or susceptible to treatment with a ROSl inhibitor.
  • ROSl related cancer can be due not only dysregulation of wild-type ROSl or known ROSl fusions, but one or more unidentified ROS l fusions. Methods that are only able to detect known fusions would be unable to detect previously unknown fusions or mutations of ROSl .
  • ROSl kinase activity or the presence of ROSl kinase amplicon By detecting not only the presence of a truncation, nucleic acid variation of ROSl, an ROSl fusion, and/or wild-type ROSl, but also detecting ROSl kinase activity or the presence of ROSl kinase amplicon, the skilled artisan can determine if the cancer is due to dysregulated wild- type ROSl, a known ROSl fusion, or a previously unidentified ROS l fusion or mutation of ROSl .
  • disclosed herein are methods for diagnosing a ROSl related cancer, assessing the susceptibility or risk of a cancer, or detecting the presence of dysregulation of ROSl and/or presence of wild-type ROSl and further comprising detecting the presence of ROSl kinase activity.
  • methods of diagnosing an ROSl related cancer in a subject with a cancer comprising detecting the presence of nucleic acid associated with an ROSl fusion, wild-type ROS l and/or a ROSl kinase domain from a tissue sample in a subject.
  • the presence or increase of wild-type ROSl and ROSl kinase alone can be tested.
  • the presence of the wild-type ROS l and ROSl kinase or increase in amplification thereof relative to a control can indicate dysregulation of ROSl which can be involved in ROS-1 related cancers not due to a fusion event. No change relative to the control indicates that ROSl is not involved in the cancer.
  • the presence or amplification relative to a control of only the ROSl kinase indicates a ROS l fusion.
  • cycle threshold is a relative value based on internal controls and a high Ct indicates a low expression level.
  • Ct cycle threshold
  • both the wild-type and ROSl kinase Ct values are low (i.e., expression level is high), but there is no statistically significant difference between the kinase and wild-type primer pairs, ROSl is being overexpressed.
  • ROSl Ct values are high (i.e., low expression) relative to ROSl kinase Ct values, a ROS l fusion is present.
  • the method can comprise a first amplification reaction wherein only a forward primer that binds to a ROSl fusion partner 5' to the fusion break point is used generating an amplicon.
  • the amplicon is used as a template for sequencing, amplification, or probe-based detection using primers or probes specific for ROSl 3 ' of the fusion breakpoint.
  • the presence of ROSl in an amplicon indicates the presence of a ROSl fusion, and therefore a ROSl related cancer.
  • the primer from the first PCR reaction is a forward primer which binds to a ROSl fusion partner 5' of the fusion breakpoint
  • any primers or probes used in the second PCR reaction or detection are specific for RO
  • RNA samples comprising performing a PCR based reaction on nucleic acid from the subject wherein the forward primer is specific for a ROSl fusion partner and binds 5' to the fusion breakpoint and the reverse primer is specific for ROSl and binds 3 ' to the fusion breakpoint, wherein detection of amplicon containing ROSl and ROS 1 fusion partner indicates the presence of a ROSl related cancer.
  • the presence of separately spaced probes which hybridize to sequences flanking the breakpoint of ROSl indicates the presence of a fusion event and therefore a cancer.
  • the methods may then further comprise administering to the subject with an ROSl related cancer a ROSl inhibitor.
  • disclosed herein are methods for diagnosing a ROSl -related cancer in a subject with a cancer comprising detecting the presence of ROSl kinase activity.
  • methods of diagnosing a ROSl related cancer in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, conducting RT-PCR, real-time PCR, or real-time RT-PCR on the nucleic acid, and detecting the presence of or measuring the amount of nucleic acid associated with wild-type ROSl and ROSl kinase domain in the tissue sample, wherein the RT-PCR or real-time PCR reaction comprises the use of one or a combination of a forward and reverse primer pair that specifically hybridizes to a wild-type ROSl sequence (e.g., SEQ ID NOs: 13, 14, 20 and 21) and a forward and reverse primer pair that specifically hybridizes to a wild-type ROSl kinase
  • a forward and reverse primer pair that
  • Absence of amplicon or amplicon levels equivalent to normal controls indicates that the cancer is a ROSl related cancer.
  • methods for diagnosing a ROS l -related cancer in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, wherein the nucleic acid from the tissue sample is RNA, wherein the method further comprises synthesizing cDNA from the RNA sample, conducting PCR on the cDNA; and detecting the presence of or measuring the amount of nucleic acid associated with one or a combination of both wild-type ROSl and ROSl kinase domain in the tissue sample, wherein an increase in amplicon relative to a normal control or the presence of an amplicon indicates the that the subject has a ROS l related cancer.
  • the disclosed methods can utilize a probe that is complementary to a sequence with the product of the real-time RT-PCR (e.g., SEQ DI NOs: 6, 8, 19, and 22) or the method can comprise determining the cycle thresholds (Ct) values for wild-type ROSl and wild-type ROSl kinase; wherein a high (Ct) value for wild-type ROSl relative to ROSl kinase indicates the presence of a fusion.
  • a cancer is determined to be a ROS l related cancer
  • methods further comprising administering to a subject with a cancer susceptible to ROSl inhibitor treatment, a ROSl inhibitor.
  • the method can further comprise treating the subject with the cancer using a form of treatment other than a ROS l inhibitor.
  • ROSl fusions are detected by qPCR methodology using a two-step detection where the wherein only a forward primer which binds to a ROSl fusion partner is used in a first reaction and wherein in a second reaction following the first reaction the amplicon from the first reaction is used as a template for a second amplification, a probe based detection, or a sequencing reaction, wherein the probes or primers used are specific for ROSl 3 ' to a fusion breakpoint, and wherein detection of ROSl in the amplicon from the second reaction indicates a fusion and thus a ROSl related cancer.
  • a cancer for diagnosing a ROSl-related cancer in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, conducting a first amplification reaction using a forward primer which binds to a ROSl fusion partner; wherein the amplicon from the first reaction is used as a template for a second amplification reaction; conducting a second amplification reaction following the first reaction, wherein primers specific for a ROSl sequence 3 ' of the fusion breakpoint are used in the second amplification reaction (e.g., SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18); and detecting the presence of ROSl in the amplicon from the second reaction; wherein detection of ROS l in the amplicon from the second reaction indicates the presence of a ROSl fusion and therefore a ROSl related cancer.
  • a cancer is determined to be a ROSl related cancer also disclosed are methods further comprising administering to a subject with a cancer
  • the method can further comprise treating the subject with the cancer using a form of treatment other than a ROS l inhibitor.
  • methods of diagnosing a ROSl related cancer in a subject comprising contacting nucleic acid in a cell with a first probe that hybridizes to a ROSl kinase and a second probe that hybridizes to a ROSl sequence 3' to the fusion breakpoint of ROSl ; wherein the probes a differently labeled; wherein detection of a disrupted gene locus indicated by separated probes indicates the presence of an ROSl fusion which indicates the presence of a ROSl related cancer.
  • probe comprises a sequence complementary to a sequence with the product of the real-time RT- PCR, and wherein the probe has a reporter dye on the end thereof and a quencher dye on the another end thereof. It is further understood that the probe can be selected from any of the probes in Table 7 including, but not limited to SEQ ID NOs: 6, 8, 19, and 22.
  • the methods disclosed herein relate to the detection of nucleic acid variation in the form of, for example, point mutations and truncations, or the detection of expression of ROSl fusions, aberrant wild-type ROSl expression, wild-type ROSl expression, or expression of ROSl truncation mutants.
  • the methods comprise detecting either the abundance or presence of mRNA, or both.
  • methods and compositions for diagnosing an ROSl related cancer in a subject comprising measuring the presence or level of mRNA from a tissue sample from the subject; wherein an increase in the amount of mRNA relative to a control indicates the presence of an ROS 1 related cancer.
  • specific mRNAs can be detected using Northern blot analysis, nuclease protection assays (NPA), in situ hybridization (e.g., fluorescence in situ hybridization), real-time PCR reaction, or reverse transcription-polymerase chain reaction (RT-PCR), and microarray.
  • NPA nuclease protection assays
  • in situ hybridization e.g., fluorescence in situ hybridization
  • real-time PCR reaction e.g., real-time PCR reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • RNAs can be used to detect specific RNAs and to precisely determine their expression level.
  • Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to
  • In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression.
  • RNA transcript of any gene regardless of the scarcity of the starting material or relative abundance of the specific mRNA.
  • RT-PCR an RNA template is copied into a complementary DNA (cDNA) using a retroviral reverse transcriptase.
  • the cDNA is then amplified exponentially by PCR using a DNA polymerase.
  • the reverse transcription and PCR reactions can occur in the same or difference tubes.
  • RT-PCR is somewhat tolerant of degraded RNA. As long as the RNA is intact within the region spanned by the primers, the target will be amplified.
  • Relative quantitative RT-PCR involves amplifying an internal control
  • the internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples. It is crucial to choose an internal control with a constant level of expression across all experimental samples (i.e., not affected by experimental treatment). Commonly used internal controls (e.g., GAPDH, ⁇ -actin, cyclophilin) often vary in expression and, therefore, may not be appropriate internal controls. Additionally, most common internal controls are expressed at much higher levels than the mRNA being studied. For relative RT-PCR results to be meaningful, all products of the PCR reaction must be analyzed in the linear range of amplification. This becomes difficult for transcripts of widely different levels of abundance.
  • RT-PCR is used for absolute quantitation. This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence. Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample.
  • Disclosed herein in one aspect are methods of diagnosing an ROSl related cancer in a subject comprising conducting real-time PCR, RT-PCR, or other PCR reaction on nucleic acid such as, for example, mRNA or DNA from a tissue sample from the subject; wherein the polymerase chain reaction comprises a reverse primer capable of specifically hybridizing to one or more ROSl sequences and at least one forward primer; and wherein an increase in the amount of amplification product relative to a control indicates the presence of an ROS l related cancer.
  • Also disclosed herein are methods of diagnosing an ROSl related cancer in a subject comprising conducting FISH on a tissue sample from the subject; wherein the polymerase chain reaction comprises probes capable of specifically hybridizing to one or more ROSl sequences on separate sides of a ROS l fusion breakpoint; and wherein a disrupted gene locus indicated by separated probes indicates the presence of an ROSl related cancer.
  • probes for use in this assay include those found on Table 7.
  • the disclosed methods can be used to detect wild-type ROS l, ROSl fusions, and ROSl kinase domain activity
  • methods of diagnosing an ROSl related cancer or detecting the dysregulation of an ROSl kinase in a subject comprising conducting a first RT-PCR reaction on mRNA from a tissue sample from the subject;
  • RT-PCR reverse transcription polymerase chain reaction
  • a ROSl kinase sequences such as, for example SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18
  • at least one primer pair capable of specifically hybridizing to ROSl 5' of any fusion breakpoint i.e., an external wild-type
  • ROSl site such as, for example, SEQ ID NOs: 13, 14, 20, and 21
  • determining the cycle threshold for the amplicons from each primer pair and wherein a cycle threshold of the wild-type primer pair amplicon is higher than the cycle threshold for the ROSl kinase by a statistically significant amount indicates the presence of a fusion or mutated ROSl.
  • Also disclosed are methods of diagnosing an ROSl related cancer or detecting the dysregulation of an ROSl kinase in a subject comprising conducting a first RT-PCR reaction on mRNA from a tissue sample from the subject; wherein the reverse transcription polymerase chain reaction (RT-PCR) comprises one primer pair capable of specifically hybridizing to a ROSl fusion partner 5' of any fusion breakpoint and amplifying only in the forward direction; wherein the method further comprises detecting the presence of or amplifying the amplicon from the first reaction using one or more primers that specifically hybridize to ROSl sequences 3 ' of the fusion breakpoint, wherein the presence of ROSl sequences in the amplicon from the first reaction indicates that presence of a ROSl fusion.
  • RT-PCR reverse transcription polymerase chain reaction
  • Northern analysis is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. It can also be used to directly compare the relative abundance of a given message between all the samples on a blot.
  • the Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., intactness of the RNA sample and how efficiently it has transferred to the membrane).
  • RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe.
  • Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick-translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an exon) may be used as probes.
  • the Nuclease Protection Assay (including both ribonuclease protection assays and SI nuclease assays) is a sensitive method for the detection and quantitation of specific mRNAs.
  • the basis of the NPA is solution hybridization of an antisense probe (radiolabeled or nonisotopic) to an RNA sample. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The remaining protected fragments are separated on an acrylamide gel. Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 ⁇ g of sample RNA, compared with the 20-30 ⁇ g maximum of blot hybridizations. NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length).
  • NPAs are the method of choice for the simultaneous detection of several RNA species. During solution hybridization and subsequent analysis, individual probe/target interactions are completely independent of one another. Thus, several RNA targets and appropriate controls can be assayed simultaneously (up to twelve have been used in the same reaction), provided that the individual probes are of different lengths. NPAs are also commonly used to precisely map mRNA termini and intron/exon junctions.
  • ISH In situ hybridization
  • ISH fluorescence in situ hybridization
  • the procedure begins by fixing samples in neutral-buffered formalin, and embedding the tissue in paraffin. The samples are then sliced into thin sections and mounted onto microscope slides. (Alternatively, tissue can be sectioned frozen and post-fixed in paraformaldehyde.) After a series of washes to de-wax and rehydrate the sections, a
  • Proteinase K digestion is performed to increase probe accessibility, and a labeled probe is then hybridized to the sample sections. Radiolabeled probes are visualized with liquid film dried onto the slides, while nonisotopically labeled probes are conveniently detected with colorimetric or fluorescent reagents.
  • the methods disclosed herein relate to the detection of nucleic acid variation in the form of, for example, point mutations and truncations, or the detection of expression of ROS1 fusions, aberrant wild-type ROS 1 expression, or expression of ROS1 truncation mutants.
  • the methods comprise detecting either the abundance or presence of mRNA, or both.
  • detection can be directed to the abundance or presence of DNA, for example, cDNA.
  • methods and compositions for diagnosing an ROS 1 related cancer in a subject comprising measuring the presence or level of DNA from a tissue sample from the subject; wherein an increase in the amount of DNA relative to a control indicates the presence of an ROS1 related cancer.
  • PCR a number of widely used procedures exist for detecting and determining the abundance of a particular DNA in a sample.
  • the technology of PCR permits amplification and subsequent detection of minute quantities of a target nucleic acid. Details of PCR are well described in the art, including, for example, U.S. Pat. Nos. 4,683, 195 to Mullis et al., 4,683,202 to Mullis and 4,965,188 to Mullis et al.
  • oligonucleotide primers are annealed to the denatured strands of a target nucleic acid, and primer extension products are formed by the polymerization of deoxynucleoside triphosphates by a polymerase.
  • a typical PCR method involves repetitive cycles of template nucleic acid denaturation, primer annealing and extension of the annealed primers by the action of a thermostable polymerase. The process results in exponential amplification of the target nucleic acid, and thus allows the detection of targets existing in very low concentrations in a sample. It is understood and herein contemplated that there are variant PCR methods known in the art that may also be utilized in the disclosed methods, for example,
  • QPCR Quantitative PCR
  • microarrays real-time PCT; hot start PCR; nested PCR; allele-specific PCR; and Touchdown PCR.
  • An array is an orderly arrangement of samples, providing a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns.
  • An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample.
  • arrays are described as macroarrays or microarrays, the difference being the size of the sample spots.
  • Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners.
  • the sample spot sizes in microarray can be 300 microns or less, but typically less than 200 microns in diameter and these arrays usually contains thousands of spots.
  • Microarrays require specialized robotics and/or imaging equipment that generally are not commercially available as a complete system. Terminologies that have been used in the literature to describe this technology include, but not limited to: biochip, DNA chip, DNA microarray, GENECHIP® (Affymetrix, Inc. which refers to its high density,
  • oligonucleotide-based DNA arrays oligonucleotide-based DNA arrays
  • gene array oligonucleotide-based DNA arrays
  • DNA microarrays or DNA chips are fabricated by high-speed robotics, generally on glass or nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. It is herein contemplated that the disclosed microarrays can be used to monitor gene expression, disease diagnosis, gene discovery, drug discovery (pharmacogenomics), and toxicological research or toxicogenomics.
  • Type I microarrays comprise a probe cDNA (500-5,000 bases long) that is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method is traditionally referred to as DNA microarray.
  • Type I microarrays localized multiple copies of one or more polynucleotide sequences, preferably copies of a single
  • polynucleotide sequence are immobilized on a plurality of defined regions of the substrate's surface.
  • a polynucleotide refers to a chain of nucleotides ranging from 5 to 10,000 nucleotides. These immobilized copies of a polynucleotide sequence are suitable for use as probes in hybridization experiments.
  • Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously.
  • a microarray is formed by using ink-jet technology based on the piezoelectric effect, whereby a narrow tube containing a liquid of interest, such as oligonucleotide synthesis reagents, is encircled by an adapter. An electric charge sent across the adapter causes the adapter to expand at a different rate than the tube and forces a small drop of liquid onto a substrate.
  • a liquid of interest such as oligonucleotide synthesis reagents
  • Samples may be any sample containing polynucleotides (polynucleotide targets) of interest and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • DNA or R A can be isolated from the sample according to any of a number of methods well known to those of skill in the art.
  • total RNA is isolated using the TRIzol total RNA isolation reagent (Life Technologies, Inc., Rockville, Md.) and RNA is isolated using oligo d(T) column chromatography or glass beads. After hybridization and processing, the
  • hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.
  • the plurality of defined regions on the substrate can be arranged in a variety of formats.
  • the regions may be arranged perpendicular or in parallel to the length of the casing.
  • the targets do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group.
  • the linker groups may typically vary from about 6 to 50 atoms long. Linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the probes.
  • Sample polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes.
  • the labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means.
  • the labeling moieties include radioisotopes, such as P, P or S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, biotin, and the like.
  • Labeling can be carried out during an amplification reaction, such as polymerase chain reaction and in vitro or in vivo transcription reactions.
  • the labeling moiety can be incorporated after hybridization once a probe-target complex his formed.
  • biotin is first incorporated during an amplification step as described above. After the hybridization reaction, unbound nucleic acids are rinsed away so that the only biotin remaining bound to the substrate is that attached to target polynucleotides that are hybridized to the polynucleotide probes. Then, an avidin-conjugated fluorophore, such as avidin-phycoerythrin, that binds with high affinity to biotin is added.
  • avidin-conjugated fluorophore such as avidin-phycoerythrin
  • Hybridization causes a polynucleotide probe and a complementary target to form a stable duplex through base pairing.
  • Hybridization methods are well known to those skilled in the art
  • Stringent conditions for hybridization can be defined by salt concentration, temperature, and other chemicals and conditions. Varying additional parameters, such as hybridization time, the concentration of detergent (sodium dodecyl sulfate, SDS) or solvent (formamide), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy.
  • An argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier and the amount of emitted light detected and quantitated.
  • the detected signal should be proportional to the amount of probe/target polynucleotide complex at each position of the microarray.
  • the fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensities. The scanned image is examined to determine the abundance/expression level of each hybridized target polynucleotide.
  • polynucleotide targets from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the target polynucleotides in two or more samples is obtained.
  • microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions.
  • individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
  • Type II microarrays comprise an array of oligonucleotides (20 ⁇ 80-mer oligos) or peptide nucleic acid (PNA) probes that is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined.
  • This method "historically” called DNA chips, was developed at Affymetrix, Inc. , which sells its photolithographically fabricated products under the GENECHIP® trademark.
  • Type II arrays for gene expression are simple: labeled cDNA or cRNA targets derived from the mRNA of an experimental sample are hybridized to nucleic acid probes attached to the solid support. By monitoring the amount of label associated with each DNA location, it is possible to infer the abundance of each mRNA species represented.
  • hybridization has been used for decades to detect and quantify nucleic acids, the combination of the miniaturization of the technology and the large and growing amounts of sequence information, have enormously expanded the scale at which gene expression can be studied.
  • Microarray manufacturing can begin with a 5 -inch square quartz wafer. Initially the quartz is washed to ensure uniform hydroxylation across its surface. Because quartz is naturally hydroxylated, it provides an excellent substrate for the attachment of chemicals, such as linker molecules, that are later used to position the probes on the arrays.
  • chemicals such as linker molecules
  • the wafer is placed in a bath of silane, which reacts with the hydroxyl groups of the quartz, and forms a matrix of covalently linked molecules.
  • the distance between these silane molecules determines the probes' packing density, allowing arrays to hold over 500,000 probe locations, or features, within a mere 1.28 square centimeters. Each of these features harbors millions of identical DNA molecules.
  • the silane film provides a uniform hydroxyl density to initiate probe assembly.
  • Linker molecules, attached to the silane matrix provide a surface that may be spatially activated by light.
  • Probe synthesis occurs in parallel, resulting in the addition of an A, C, T, or G nucleotide to multiple growing chains simultaneously.
  • photolithographic masks carrying 18 to 20 square micron windows that correspond to the dimensions of individual features, are placed over the coated wafer. The windows are distributed over the mask based on the desired sequence of each probe.
  • ultraviolet light is shone over the mask in the first step of synthesis, the exposed linkers become deprotected and are available for nucleotide coupling.
  • a solution containing a single type of deoxynucleotide with a removable protection group is flushed over the wafer's surface.
  • the nucleotide attaches to the activated linkers, initiating the synthesis process.
  • each position in the sequence of an oligonucleotide can be occupied by lof Nucleotides, resulting in an apparent need for 25 x 4, or 100, different masks per wafer, the synthesis process can be designed to significantly reduce this requirement.
  • Algorithms that help minimize mask usage calculate how to best coordinate probe growth by adjusting synthesis rates of individual probes and identifying situations when the same mask can be used multiple times.
  • Hybridization under particular pH, salt, and temperature conditions can be optimized by taking into account melting temperatures and using empirical rules that correlate with desired hybridization behaviors.
  • probes are selected from regions shared by multiple splice or polyadenylation variants. In other cases, unique probes that distinguish between variants are favored. Inter-probe distance is also factored into the selection process.
  • a different set of strategies is used to select probes for genotyping arrays that rely on multiple probes to interrogate individual nucleotides in a sequence.
  • the identity of a target base can be deduced using four identical probes that vary only in the target position, each containing one of the four possible bases.
  • the presence of a consensus sequence can be tested using one or two probes representing specific alleles.
  • arrays with many probes can be created to provide redundant information, resulting in unequivocal genotyping.
  • generic probes can be used in some applications to maximize flexibility.
  • Some probe arrays allow the separation and analysis of individual reaction products from complex mixtures, such as those used in some protocols to identify single nucleotide polymorphisms (SNPs).
  • the disclosed diagnostic methods can be performed by Real-time PCR methods.
  • methods of detecting the presence of an ROS 1 related fusion or methods of diagnosing an ROS1 related cancer or detecting the dysregulation of an ROS 1 kinase in a subject comprising conducting a first RT-PCR reaction on mRNA from a tissue sample from the subject; wherein the reverse transcription polymerase chain reaction (RT-PCR) comprises one primer pair capable of specifically hybridizing to a ROS1 kinase sequences and at least one primer pair capable of specifically hybridizing to ROS1 5' of any fusion breakpoint (i.e., an external wild-type ROS1 site) and determining the cycle threshold for the amplicons from each primer pair; and wherein a cycle threshold of the wild-type primer pair amplicon is higher than the cycle threshold for the ROS1 kinase by a statistically significant amount indicates the presence of a fusion or mutated ROS1.
  • RT-PCR reverse transcription polymerase chain reaction
  • Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time) as opposed to the endpoint detection.
  • the real-time progress of the reaction can be viewed in some systems.
  • Real-time PCR does not detect the size of the amplicon and thus does not allow the differentiation between DNA and cDNA amplification, however, it is not influenced by non-specific amplification unless SYBR Green is used.
  • Real-time PCR quantitation eliminates post-PCR processing of PCR products. This helps to increase throughput and reduce the chances of carryover contamination.
  • Real-time PCR also offers a wide dynamic range of up to 10 7 -fold.
  • Dynamic range of any assay determines how much target concentration can vary and still be quantified.
  • a wide dynamic range means that a wide range of ratios of target and normaliser can be assayed with equal sensitivity and specificity. It follows that the broader the dynamic range, the more accurate the quantitation.
  • a real-time RT- PCR reaction reduces the time needed for measuring the amount of amplicon by providing for the visualization of the amplicon as the amplification process is progressing.
  • the real-time PCR system is based on the detection and quantitation of a fluorescent reporter. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. A significant increase in fluorescence above the baseline value measured during the 3-15 cycles can indicate the detection of accumulated PCR product.
  • a fixed fluorescence threshold is set significantly above the baseline that can be altered by the operator.
  • the parameter CT threshold cycle is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold.
  • hydrolysis probes include TaqMan probes, molecular beacons and scorpions. They use the fluorogenic 5' exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA samples.
  • TaqMan probes are designed to anneal to an internal region of a PCR product.
  • Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL) at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur. They have two arms with complementary sequences that form a very stable hybrid or stem. The close proximity of the reporter and the quencher in this hairpin configuration suppresses reporter fluorescence. When the beacon hybridises to the target during the annealing step, the reporter dye is separated from the quencher and the reporter fluoresces (FRET does not occur). Molecular beacons remain intact during PCR and must rebind to target every cycle for fluorescence emission. This will correlate to the amount of PCR product available.
  • All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair as long as the platform is suitable for melting curve analysis if SYBR green is used.
  • multiplexing the target(s) and endogenous control can be amplified in single tube.
  • Scorpion probes sequence-specific priming and PCR product detection is achieved using a single oligonucleotide.
  • the Scorpion probe maintains a stem-loop configuration in the unhybridised state.
  • the fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end.
  • the 3' portion of the stem also contains sequence that is complementary to the extension product of the primer.
  • This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed.
  • SYBR- green I or ethidium bromide a non-sequence specific fluorescent intercalating agent
  • SYBR green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA.
  • Disadvantages of SYBR green-based real-time PCR include the requirement for extensive optimisation.
  • non-specific amplifications require follow-up assays (melting point curve or dissociation analysis) for amplicon identification.
  • the threshold cycle or the CT value is the cycle at which a significant increase in ARn is first detected (for definition of ARn, see below).
  • the threshold cycle is when the system begins to detect the increase in the signal associated with an exponential growth of PCR product during the log-linear phase. This phase provides the most useful information about the reaction (certainly more important than the end-point).
  • the slope of the log-linear phase is a reflection of the amplification efficiency.
  • the efficiency of the PCR should be 90 - 100% (3.6 > slope > 3.1).
  • a number of variables can affect the efficiency of the PCR. These factors include length of the amplicon, secondary structure and primer quality.
  • the qRT-PCR should be further optimised or alternative amplicons designed.
  • the slope to be an indicator of real amplification (rather than signal drift)
  • the important parameter for quantitation is the Or. The higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the CT value.
  • the threshold should be placed above any baseline activity and within the exponential increase phase (which looks linear in the log transformation).
  • CT cycle threshold
  • Multiplex TaqMan assays can be performed using multiple dyes with distinct emission wavelengths.
  • Available dyes for this purpose are FAM, TET, VIC and JOE (the most expensive).
  • TAMRA is reserved as the quencher on the probe and ROX as the passive reference.
  • FAM target
  • VIC endogenous control
  • JOE endogenous control
  • VIC endogenous control
  • the spectral compensation for the post run analysis should be turned on (on ABI 7700: Instrument/Diagnostics/Advanced Options/Miscellaneous). Activating spectral compensation improves dye spectral resolution.
  • the disclosed methods can further utilize nested PCR.
  • Nested PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA.
  • Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments.
  • the product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction.
  • Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
  • methods of diagnosing an ROS1 related cancer in a subject comprising conducting a PCR reaction on DNA from a tissue sample from the subject; wherein the PCR reaction comprises a reverse primer capable of specifically hybridizing to one or more ROS1 sequences and at least one forward primer; and wherein an increase in the amount of amplification product relative to a control indicates the presence of an ROS1 related cancer.
  • primers are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. AS used herein, the probes can comprise a reporter dye on the end thereof and a quencher dye on the another end thereof.
  • compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids such as SEQ ID NO: 1 or its complement such as those listed in Table 7 (e.g., SEQ ID NOs: 4, 5, 7, 8, 12, 13, 14, 15, 16, 17, 18, 20, and 21 which are primers that interact with SEQ ID NO: 1 and SEQ ID NOs: 6, 8, 19, and 22 which are probes that interact with SEQ ID NO: 1).
  • the disclosed primers and probes can be used in any of the disclosed methods for diagnosing a ROS1 related cancer disclosed herein as well as the methods for determining whether a cancer is susceptible or resistant to treatment with a ROS1 inhibitor.
  • the primers are used to support nucleic acid extension reactions, nucleic acid replication reactions, and/or nucleic acid amplification reactions.
  • the primers will be capable of being extended in a sequence specific manner.
  • Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
  • Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are disclosed.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.
  • the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids.
  • one or more primers can be used to create extension products from and templated by a first nucleic acid.
  • the size of the primers or probes for interaction with the nucleic acids can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer.
  • a typical primer or probe would be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
  • a primer or probe can be less than or equal to 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550,
  • the primers for the nucleic acid of interest typically will be used to produce extension products and/or other replicated or amplified products that contain a region of the nucleic acid of interest.
  • the size of the product can be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides.
  • the product can be, for example, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,
  • the product can be, for example, less than or equal to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,
  • the disclosed RT-PCR and PCR reactions require at least one forward primer and/or at least one reverse primer to amplify target nucleic acid.
  • the forward primer can be, for example, an intracellular (i.e., 3' to the ROSl breakpoint) ROSl primer or a ROSl kinase forward primer such as, for example, SEQ ID NO: 4, 7, 15, 17, or 20.
  • the reverse primer can be, for example, ROSl kinase reverse primer such as, for example, SEQ ID NO: 5, 8, 12, 16, 18, or 21. It is understood that the methods can comprise at least one forward primer and a reverse primer.
  • RT-PCR polymerase chain reaction
  • the polymerase chain reaction comprises one or more reverse primers capable of specifically hybridizing to one or more ROSl sequences and at least one forward primer
  • the forward primer can be, for example, a CD74-ROS1 primer (such as, for example, SEQ ID NO: 2 and 10), a FIG-ROS1 primer, a SLC34A2-ROS (short) primer, a SLC34A2-ROS (short and long) primer (such as, for example, SEQ ID NOs: 3 and 11), a SLC34A2-ROS (long) primer, a ROSl kinase primer (such as, for example, SEQ ID NOs: 4, 7, 15, or 17), and/or a wild-type ROSl primer including wild
  • amplification product of the ROSl kinase and an increase in amplification of the fusion partner relative to a control can occur through the use of a primer pair comprising reverse primer that hybridizes ROS1 3 ' to the break point and a forward primer the hybridizes to the fusion partner 5' of the breakpoint or a forward primer that hybridizes to the fusion partner only.
  • a primer pair consisting of a forward primer that binds to the fusion partner and a reverse primer that binds to ROS 1 3' of the fusion breakpoint
  • the detection of both ROS1 and the fusion partner in the amplicon the detection of an amplicon of the appropriate size for a amplicon comprising a fusion between the primers, or detection of amplicon in an amount greater than controls using only the forward or reverse primer indicates a ROS1 fusion.
  • a primer pair with a forward primer binding to the fusion partner a primer pair that hybridizes to ROS1 5' to the breakpoint serves as a control against
  • the methods disclosed herein can comprise the use of only a forward primer that binds to the fusion partner to amplify a nucleic acid from the subject in a first reaction and a primer pair that binds to ROS1 3 ' of the fusion breakpoint to amplify the amplicon of the first reaction, where amplicon containing ROS1 sequences indicates the presence of an ROS1 related cancer.
  • methods of diagnosing a ROS1 related cancer in a subject comprising conducting a real-time PCR, RT-PCR, or other PCR reaction on nucleic acid such as mRNA or DNA from a tissue sample from the subject; wherein the polymerase chain reaction comprises one or more reverse primers capable of specifically hybridizing to one or more ROS1 sequences and at least one forward primer; wherein the forward primer is an external wild-type ROS1 primer (i.e., 5' to the fusion breakpoint), and wherein an increase in the amount of amplification product relative to a control indicates the presence of an ROS1 related cancer.
  • the reaction can comprise a wild-type ROS1 primer paired with a reverse primer, as well as, an ROS1 kinase primer paired with the same reverse primer.
  • methods of diagnosing a ROSl related cancer in a subject comprising conducting an realtime or RT-PCR reaction on mRNA from a tissue sample from the subject; wherein the polymerase chain reaction comprises a reverse primer capable of specifically hybridizing to one or more ROS1 sequences and at least two forward primers; and wherein an increase in the amount of amplification product relative to a control for the ROS 1 kinase, but not both primers indicates the presence of an ROS1 related cancer.
  • methods comprising at least three forward primers. It is understood and herein contemplated that any combination of two or more or thee or more the forward primers disclosed herein can be used in the multiplex reaction.
  • the forward primers are a ROS1 fusion primer (such as, for example, CD74-ROS1, FIG-ROS1, or SLC34-ROS 1), a ROS1 kinase primer and a wild-type ROS1 primer.
  • a ROS1 fusion primer such as, for example, CD74-ROS1, FIG-ROS1, or SLC34-ROS 1
  • ROS1 kinase primer and a wild-type ROS1 primer.
  • Also disclosed herein are methods of diagnosing a ROS1 related cancer in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, conducting a nucleic acid amplification process on the nucleic acid, and detecting the presence of or measuring the amount of nucleic acid associated with one or a combination of both wild-type ROS1 and ROS1 kinase domain in the tissue sample, wherein the amplification process is PCR on cDNA or real-time PCR, RT-PCR, or real-time RT-PCR on mRNA, wherein the PCR, RT-PCR, real-time PCR, or real-time RT-PCR reaction comprises the use of a forward and reverse primer pair that specifically hybridizes to a wild-type ROS1 sequence (such as, for example, primers that bind to the extracellular domain of ROS1, such as the forward primers SEQ ID NOs: 13 and 20 and the reverse primers SEQ ID NOs: 14 and 21)
  • forward and reverse primer pair for the wild-type ROS 1 e.g., the ECD of ROS 1
  • the forward and reverse primer pair for the wild-type ROS 1 can be SEQ ID NO: 1
  • the primers specific for the kinase domain of ROS 1 can be any combination of forward and reverse primers for the kinase domain such as, for example SEQ ID NOs: 4 and 5; SEQ ID NOs: 4 and 8; SEQ ID NOs: 4 and 14; SEQ ID NOs: 4 and 16; SEQ ID NOs: 4 and 18; SEQ ID NOs: 4 and 21 ; SEQ ID NOs: 7 and 5; SEQ ID NOs: 7 and 8; SEQ ID NOs: 7 and 14; SEQ ID NOs: 7 and 16; SEQ ID NOs: 7 and 18; SEQ ID NOs: 7 and 21; SEQ ID NOs: 13 and 5; SEQ ID NOs: 13 and 8; SEQ ID NOs: 13 and 14; SEQ ID NOs: 13 and 16; SEQ ID NOs: 13 and 18; SEQ ID NOs: 13 and 18; SEQ ID NOs: SEQ ID NOs: 13 and 18; SEQ ID NOs: 4 and 18; SEQ ID NOs: 7 and
  • Fluorescent change probes and fluorescent change primers refer to all probes and primers that involve a change in fluorescence intensity or wavelength based on a change in the form or conformation of the probe or primer and nucleic acid to be detected, assayed or replicated.
  • fluorescent change probes and primers include molecular beacons, Amplifluors, FRET probes, cleavable FRET probes, TaqMan probes, scorpion primers, fluorescent triplex oligos including but not limited to triplex molecular beacons or triplex FRET probes, fluorescent water-soluble conjugated polymers, PNA probes and QPNA probes.
  • Fluorescent change probes and primers can be classified according to their structure and/or function.
  • Fluorescent change probes include hairpin quenched probes, cleavage quenched probes, cleavage activated probes, and fluorescent activated probes.
  • Fluorescent change primers include stem quenched primers and hairpin quenched primers.
  • Hairpin quenched probes are probes that when not bound to a target sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the probe binds to a target sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases.
  • hairpin quenched probes are molecular beacons, fluorescent triplex oligos, triplex molecular beacons, triplex FRET probes, and QPNA probes.
  • Cleavage activated probes are probes where fluorescence is increased by cleavage of the probe.
  • Cleavage activated probes can include a fluorescent label and a quenching moiety in proximity such that fluorescence from the label is quenched.
  • the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases.
  • TaqMan probes are an example of cleavage activated probes.
  • Cleavage quenched probes are probes where fluorescence is decreased or altered by cleavage of the probe.
  • Cleavage quenched probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity, fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce.
  • the probes are thus fluorescent, for example, when hybridized to a target sequence.
  • the donor moiety is no longer in proximity to the acceptor fluorescent label and fluorescence from the acceptor decreases.
  • the donor moiety is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor.
  • the overall effect would then be a reduction of acceptor fluorescence and an increase in donor fluorescence.
  • Donor fluorescence in the case of cleavage quenched probes is equivalent to fluorescence generated by cleavage activated probes with the acceptor being the quenching moiety and the donor being the fluorescent label.
  • Cleavable FRET (fluorescence resonance energy transfer) probes are an example of cleavage quenched probes.
  • Fluorescent activated probes are probes or pairs of probes where fluorescence is increased or altered by hybridization of the probe to a target sequence.
  • Fluorescent activated probes can include an acceptor fluorescent label and a donor moiety such that, when the acceptor and donor are in proximity (when the probes are hybridized to a target sequence), fluorescence resonance energy transfer from the donor to the acceptor causes the acceptor to fluoresce.
  • Fluorescent activated probes are typically pairs of probes designed to hybridize to adjacent sequences such that the acceptor and donor are brought into proximity.
  • Fluorescent activated probes can also be single probes containing both a donor and acceptor where, when the probe is not hybridized to a target sequence, the donor and acceptor are not in proximity but where the donor and acceptor are brought into proximity when the probe hybridized to a target sequence. This can be accomplished, for example, by placing the donor and acceptor on opposite ends of the probe and placing target complement sequences at each end of the probe where the target complement sequences are complementary to adjacent sequences in a target sequence. If the donor moiety of a fluorescent activated probe is itself a fluorescent label, it can release energy as fluorescence (typically at a different wavelength than the fluorescence of the acceptor) when not in proximity to an acceptor (that is, when the probes are not hybridized to the target sequence). When the probes hybridize to a target sequence, the overall effect would then be a reduction of donor fluorescence and an increase in acceptor fluorescence.
  • FRET probes are an example of fluorescent activated probes.
  • Stem quenched primers are primers that when not hybridized to a complementary sequence form a stem structure (either an intramolecular stem structure or an intermolecular stem structure) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched.
  • stem quenched primers are used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid.
  • Examples of stem quenched primers are peptide nucleic acid quenched primers and hairpin quenched primers.
  • Peptide nucleic acid quenched primers are primers associated with a peptide nucleic acid quencher or a peptide nucleic acid fluor to form a stem structure.
  • the primer contains a fluorescent label or a quenching moiety and is associated with either a peptide nucleic acid quencher or a peptide nucleic acid fluor, respectively. This puts the fluorescent label in proximity to the quenching moiety. When the primer is replicated, the peptide nucleic acid is displaced, thus allowing the fluorescent label to produce a fluorescent signal.
  • Hairpin quenched primers are primers that when not hybridized to a complementary sequence form a hairpin structure (and, typically, a loop) that brings a fluorescent label and a quenching moiety into proximity such that fluorescence from the label is quenched. When the primer binds to a complementary sequence, the stem is disrupted, the quenching moiety is no longer in proximity to the fluorescent label and fluorescence increases. Hairpin quenched primers are typically used as primers for nucleic acid synthesis and thus become incorporated into the synthesized or amplified nucleic acid. Examples of hairpin quenched primers are Amplifluor primers and scorpion primers.
  • Cleavage activated primers are similar to cleavage activated probes except that they are primers that are incorporated into replicated strands and are then subsequently cleaved.
  • labels can be directly incorporated into nucleotides and nucleic acids or can be coupled to detection molecules such as probes and primers.
  • a label is any molecule that can be associated with a nucleotide or nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly.
  • labels for incorporation into nucleotides and nucleic acids or coupling to nucleic acid probes are known to those of skill in the art.
  • Examples of labels suitable for use in the disclosed method are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands.
  • Fluorescent labels are useful for real-time detection of amplification.
  • suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl ( BD), coumarin, dansyl chloride, rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY ® , CASCADE BLUE ® , OREGON GREEN ® , pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such as quantum dyeTM, fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • FITC fluorescein isothio
  • fluorescent labels examples include 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B,
  • Leucophor WS Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene), Mithramycin, NBD Amine,
  • Nitrobenzoxadidole Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, Phthalocyanine,
  • Phycoerythrin R Phycoerythrin B, Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine G Extra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN, Thioflavin
  • FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing their simultaneous detection.
  • fluorescein dyes include 6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), 2',7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'- fused phenyl- l,4-dichloro-6-carboxyfluorescein (NED), and 2'-chloro-7'-phenyl-l,4- dichloro-6-carboxyfluorescein (VIC).
  • Fluorescent labels can be obtained from a variety of commercial sources, including Amersham Pharmacia Biotech, Piscataway, NJ; Molecular Probes, Eugene, OR; and Research Organics, Cleveland, Ohio.
  • Additional labels of interest include those that provide for signal only when the probe with which they are associated is specifically bound to a target molecule, where such labels include: "molecular beacons" as described in Tyagi & Kramer, Nature Biotechnology (1996) 14:303 and EP 0 070 685 B l.
  • Other labels of interest include those described in U.S. Pat. No. 5,563,037 which is incorporated herein by reference.
  • Labeled nucleotides are a form of label that can be directly incorporated into the amplification products during synthesis.
  • labels that can be incorporated into amplified nucleic acids include nucleotide analogs such as BrdUrd, aminoallyldeoxyuridine, 5-methylcytosine, bromouridine, and nucleotides modified with biotin or with suitable haptens such as digoxygenin.
  • Suitable fluorescence-labeled nucleotides are Fluorescein- isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP.
  • nucleotide analog label for DNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma- Aldrich Co).
  • nucleotide analogs for incorporation of label into DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.), and 5-methyl-dCTP (Roche Molecular Biochemicals).
  • AA-dUTP aminoallyl-deoxyuridine triphosphate
  • 5-methyl-dCTP Roche Molecular Biochemicals
  • nucleotide analog for incorporation of label into RNA is biotin- 16-UTP (biotin- 16-uridine-5'-triphosphate, Roche Molecular Biochemicals). Fluorescein, Cy3, and Cy5 can be linked to dUTP for direct labeling.
  • Cy3.5 and Cy7 are available as avidin or anti-digoxygenin conjugates for secondary detection of biotin- or digoxygenin-labeled probes. Labels that are incorporated into amplified nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art.
  • biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[l,2,- dioxetane-3-2'-(5'-chloro)tricyclo [3.3.1.1 3 ' 7 ]decane]-4-yl) phenyl phosphate; Tropix, Inc.).
  • suitable substrates for example, chemiluminescent substrate CSPD: disodium, 3-(4-methoxyspiro-[l,2,- dioxetane-3-2'-(5'-chloro)tricyclo [3.3.1.1 3 ' 7 ]decane]-4-yl
  • Labels can also be enzymes, such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases, that can be detected, for example, with chemical signal amplification or by using a substrate to the enzyme which produces light (for example, a chemiluminescent 1 ,2-dioxetane substrate) or fluorescent signal.
  • enzymes such as alkaline phosphatase, soybean peroxidase, horseradish peroxidase and polymerases
  • a substrate to the enzyme which produces light for example, a chemiluminescent 1 ,2-dioxetane substrate
  • fluorescent signal for example, a chemiluminescent 1 ,2-dioxetane substrate
  • Molecules that combine two or more of these labels are also considered labels. Any of the known labels can be used with the disclosed probes, tags, and method to label and detect nucleic acid amplified using the disclosed method. Methods for detecting and measuring signals generated by labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary label coupled to the antibody. As used herein, detection molecules are molecules which interact with amplified nucleic acid and to which one or more labels are coupled.
  • contemplated herein are methods of diagnosing a cancer in a subject comprising conducting a real-time PCR, RT-
  • RT-PCR polymerase chain reaction
  • the polymerase chain reaction comprises at least one reverse primer capable of specifically hybridizing to one or more ROS1 sequences and at least one forward primer; wherein an increase in the amount of amplification product relative to a control indicates the presence of an ROS1 related cancer; and wherein the control tissue is obtained is a noncancerous tissue.
  • the use of a non-cancerous tissue control can be utilized but is not necessary as cancerous tissue from a non-ROSl related cancer may also be used.
  • RNA samples from the subject comprising conducting an real-time PCR or reverse transcription-PCR (RT-PCR) reaction on mRNA from a tissue sample from the subject; wherein the polymerase chain reaction comprises a reverse primer capable of specifically hybridizing to one or more ROSl sequences and at least one forward primer; and wherein an increase in the amount of amplification product relative to a control indicates the presence of an ROSl related cancer; and wherein the control tissue is obtained from non- ROS1 related cancerous tissue.
  • RT-PCR reverse transcription-PCR
  • the disclosed methods can be used to diagnose any disease where uncontrolled cellular proliferation occurs herein referred to as "cancer".
  • cancer any disease where uncontrolled cellular proliferation occurs herein referred to as "cancer”.
  • lymphomas Hodgkins and non-Hodgkins
  • leukemias carcinomas
  • carcinomas of solid tissues squamous cell carcinomas
  • adenocarcinomas adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, AIDS- related lymphomas or sarcomas, metastatic cancers, or cancers in general.
  • a representative but non-limiting list of cancers that the disclosed methods can be used to diagnose is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung carcinoma, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cholangiocarcinoma, colorectal carcinoma, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer;
  • cancer selected from the group consisting of non-small cell lung carcinoma, diffuse large B-cell lymphoma, , systemic histiocytosis, breast cancer, colorectal carcinoma, esophageal squamous cell carcinoma, anaplastic large-cell lymphoma, neuroblastoma,
  • cholangiocarcinoma cholangiocarcinoma, renal carcinoma, colorectal carcinoma, glioblastoma, and
  • IMTs inflammatory myofibroblastic tumors
  • methods of diagnosing an ROSl related cancer in a subject comprising conducting a real-time PCR, RT-PCR, or other PCR reaction on mRNA from a tissue sample from the subject; wherein the polymerase chain reaction comprises a reverse primer capable of specifically hybridizing to one or more ROSl sequences and at least one forward primer; wherein an increase in the amount of amplification product relative to a control indicates the presence of an ROSl related cancer, and wherein the cancer is selected from the group consisting of non-small cell lung carcinoma, diffuse large B-cell lymphoma, systemic histiocytosis, breast cancer, colorectal carcinoma, esophageal squamous cell carcinoma, anaplastic large-cell lymphoma, neuroblastoma, cholangiocarcinoma, renal carcinoma, colorectal carcinoma, glioblastoma, and inflammatory myofibroblastic tumors (IMTs).
  • non-small cell lung carcinoma diffuse
  • any of the disclosed mRNA measuring techniques disclosed herein can be used in these methods.
  • methods of assessing the suitability of an ROS 1 inhibitor treatment for a cancer in a subject comprising conducting a real-time PCR, RT-PCR, or other PCR reaction with mRNA or DNA from a tissue sample from the subject; wherein the PCR reaction comprises a reverse primer capable of specifically hybridizing to one or more ROSl sequences and at least one forward primer; and wherein an increase in the amount of amplification product relative to a control indicates a cancer that can be treated with an ROSl inhibitor.
  • the disclosed methods can further comprise any of the primers disclosed herein and utilize the multiplexing PCR techniques disclosed.
  • methods of assessing the susceptibility or risk for a disease or condition, monitoring disease progression, determination of susceptibility or resistance of a cancer to therapeutic ROSl inhibitor treatment, or determination of suitability of a ROSl inhibitor treatment for a cancer associated with a nucleic acid variation, truncation, or ROSl fusion in a subject comprising detecting the presence or measuring the level or DNA, cDNA, or the expression level of mRNA from a tissue sample from the subject; wherein an increase in the amount of amplification product of ROSl kinase relative to a control absent a corresponding increase in a wild-type ROS l indicates the presence of an ROSl related cancer and therefore a cancer that is susceptible to ROSl inhibitor treatment.
  • Such methods can be accomplished with amplification methods such as PCR, real-time PCR, RT-PCR, or real-time RT-PCR, or by conducting a in situ hybridization methods such as FISH.
  • disclosed herein are methods for determining the susceptibility or resistance to therapeutic treatment of a cancer to a ROSl inhibitor or suitability of a ROSl inhibitor treatment for a cancer in a subject with a cancer comprising detecting the presence of ROSl kinase activity.
  • RT-PCR or real-time PCR reaction comprises the use of a forward and reverse primer pair that specifically hybridizes to a wild-type ROSl sequence (e.g., an extracellular domain sequence of ROSl, such as, SEQ ID NOs: 13, 14, 20 and 21) and/or a forward and reverse primer pair that specifically hybridizes to a wild-type ROSl kinase domain sequence (e.g., SEQ ID NOs: 13, 14, 20 and 21) and/or a forward and reverse primer pair that specifically hybridizes to a wild-type ROSl kinase domain sequence (e.g., SEQ ID NOs: 13, 14, 20 and 21) and/or a forward and reverse primer pair that specifically hybridizes to a wild-type ROSl kinase domain sequence (e.g., SEQ
  • Absence of amplicon or amplicon levels equivalent to normal controls indicates that the cancer is not susceptible to ROSl treatment and would be resistant to such treatment.
  • methods for determining the susceptibility or resistance to therapeutic treatment for a ROSl -related cancer or suitability for a cancer to be treated with a ROS1 inhibitor in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, wherein the nucleic acid from the tissue sample is RNA, wherein the method further comprises synthesizing cDNA from the RNA sample, conducting PCR on the cDNA; and detecting the presence of or measuring the amount of nucleic acid associated with one or a combination of both wild-type ROS1 and ROS1 kinase domain in the tissue sample, wherein an increase in amplicon relative to a normal control or the presence of an amplicon indicates the that the subject has a ROS 1 related cancer.
  • the disclosed methods can utilize a probe that is complementary to a sequence with the product of the real-time RT-PCR (e.g., SEQ DI NOs: 6, 8, 19, and 22) or the method can comprise determining the cycle thresholds (Ct) values for wild-type ROS1 and wild-type ROS1 kinase; wherein a high (Ct) value for wild-type ROS1 relative to ROS1 kinase indicates the presence of a fusion and therefore indicates that the cancer is susceptible to treatment with a ROS1 inhibitor or that a ROS1 inhibitory treatment is suitable for that cancer.
  • the probe can comprise a reporter dye on the end thereof and a quencher dye on the another end thereof.
  • a cancer is determined to be susceptible to or suitable for treatment with ROS 1 inhibitors
  • methods further comprising administering to a subject with a cancer susceptible to ROS1 inhibitor treatment, a ROS1 inhibitor.
  • the method can further comprise treating the subject with the cancer using a form of treatment other than a ROS 1 inhibitor.
  • ROS 1 fusions are detected by qPCR methodology using a two-step detection where the wherein only a forward primer which binds to a ROS 1 fusion partner is used in a first reaction and wherein in a second reaction following the first reaction the amplicon from the first reaction is used as a template for a second amplification, a probe based detection, or a sequencing reaction, wherein the probes or primers used are specific for ROS1 3' to a fusion breakpoint, and wherein detection of ROS1 in the amplicon from the second reaction indicates a ROS1 fusion and therefore a
  • ROS1 related cancer susceptible to ROS1 inhibitor treatment.
  • methods for determining the susceptibility or resistance to therapeutic treatment for a ROS1 -related cancer or suitability for a cancer to be treated with a ROS1 inhibitor in a subject with a cancer comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, conducting a first amplification reaction using a forward primer which binds to a ROSl fusion partner; wherein the amplicon from the first reaction is used as a template for a second amplification reaction; conducting a second amplification reaction following the first reaction, wherein primers specific for a ROS l sequence 3' of the fusion breakpoint are used in the second amplification reaction (e.g., SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18); and detecting the presence of ROSl in the amplicon from the second reaction; wherein detection of ROS l in the amplicon from the second reaction indicates the presence of a ROSl fusion;
  • a cancer is determined to be susceptible to or suitable for treatment with ROSl inhibitors
  • methods further comprising administering to a subject with a cancer susceptible to ROSl inhibitor treatment, a ROSl inhibitor.
  • the method can further comprise treating the subject with the cancer using a form of treatment other than a ROS l inhibitor.
  • ROSl -fusions disclosed herein are targets for cancer treatments.
  • method of screening for an agent that inhibits an ROSl related cancer in a subject comprising
  • the RT-PCR reaction comprises a reverse primer capable of specifically hybridizing to one or more ROSl sequences and at least one forward primer; and wherein a decrease in the amount of amplification product indicative of a ROSl fusion relative to an untreated control indicates an agent that can inhibit an ROS 1 related cancer.
  • one measure of effective treatment by the agent is a decrease in Cycle threshold of an amplicon of ROSl 5' to a fusion breakpoint relative to untreated controls.
  • the process of performing a RT-PCR reaction involves the synthesis of cDNA from the isolated RNA and performing PCR on the cDNA.
  • the disclosed method and compositions make use of various nucleic acids.
  • any nucleic acid can be used in the disclosed method.
  • the disclosed nucleic acids of interest and the disclosed reference nucleic acids can be chosen based on the desired analysis and information that is to be obtained or assessed.
  • the disclosed methods also produce new and altered nucleic acids. The nature and structure of such nucleic acids will be established by the manner in which they are produced and manipulated in the methods.
  • extension products and hybridizing nucleic acids are produced in the disclosed methods.
  • hybridizing nucleic acids are hybrids of extension products and the second nucleic acid.
  • a nucleic acid of interest can be any nucleic acid to which the determination of the presence or absence of nucleotide variation is desired.
  • the nucleic acid of interest can comprise a sequence that corresponds to the wild-type sequence of the reference nucleic acid. It is further disclosed herein that the disclosed methods can be performed where the first nucleic acid is a reference nucleic acid and the second nucleic acid is a nucleic acid of interest or where the first nucleic acid is the nucleic acid of interest and the second nucleic acid is the reference nucleic acid.
  • a reference nucleic acid can be any nucleic acid against which a nucleic acid of interest is to be compared.
  • the reference nucleic acid has a known sequence (and/or is known to have a sequence of interest as a reference).
  • the reference sequence has a known or suspected close relationship to the nucleic acid of interest.
  • the reference sequence can be usefully chosen to be a sequence that is a homolog or close match to the nucleic acid of interest, such as a nucleic acid derived from the same gene or genetic element from the same or a related organism or individual.
  • the reference nucleic acid can comprise a wild-type sequence or alternatively can comprise a known mutation including, for example, a mutation the presence or absence of which is associated with a disease or resistance to therapeutic treatment.
  • the disclosed methods can be used to detect or diagnose the presence of known mutations in a nucleic acid of interest by comparing the nucleic acid of interest to a reference nucleic acid that comprises a wild-type sequence (i.e., is known not to possess the mutation) and examining for the presence or absence of variation in the nucleic acid of interest, where the absence of variation would indicate the absence of a mutation.
  • the reference nucleic acid can possess a known mutation.
  • the disclosed methods can be used to detect susceptibility for a disease or condition by comparing the nucleic acid of interest to a reference nucleic acid comprising a known mutation that indicates susceptibility for a disease and examining for the presence or absence of the mutation, wherein the presence of the mutation indicates a disease.
  • nucleotide variation refers to any change or difference in the nucleotide sequence of a nucleic acid of interest relative to the nucleotide sequence of a reference nucleic acid.
  • a nucleotide variation is said to occur when the sequences between the reference nucleic acid and the nucleic acid of interest (or its complement, as appropriate in context) differ.
  • a substitution of an adenine (A) to a guanine (G) at a particular position in a nucleic acid would be a nucleotide variation provided the reference nucleic acid comprised an A at the corresponding position.
  • nucleic acid determines whether or not a sequence is wild-type.
  • a nucleic acid with a known mutation is used as the reference nucleic acid
  • a nucleic acid not possessing the mutation would be considered to possess a nucleotide variation (relative to the reference nucleic acid).
  • nucleotide for a nucleotide. It is understood and contemplated herein that where reference is made to a type of base, this refers a base that in a nucleotide in a nucleic acid strand is capable of hybridizing (binding) to a defined set of one or more of the canonical bases.
  • nuclease-resistant nucleotides can be, for example, guanine (G), thymine (T), and cytosine (C).
  • G guanine
  • T thymine
  • C cytosine
  • modified or alternative base can be used in the disclosed methods and compositions, generally limited only by the capabilities of the enzymes used to use such bases.
  • Many modified and alternative nucleotides and bases are known, some of which are described below and elsewhere herein.
  • the type of base that such modified and alternative bases represent can be determined by the pattern of base pairing for that base as described herein. Thus for example, if the modified nucleotide was adenine, any analog, derivative, modified, or variant base that based pairs primarily with thymine would be considered the same type of base as adenine. In other words, so long as the analog, derivative, modified, or variant has the same pattern of base pairing as another base, it can be considered the same type of base. Modifications can be made to the sugar or phosphate groups of a nucleotide. Generally such modifications will not change the base pairing pattern of the base.
  • the base pairing pattern of a nucleotide in a nucleic acid strand determines the type of base of the base in the nucleotide.
  • Modified nucleotides to be incorporated into extension products and to be selectively removed by the disclosed agents in the disclosed methods can be any modified nucleotide that functions as needed in the disclosed method as is described elsewhere herein. Modified nucleotides can also be produced in existing nucleic acid strands, such as extension products, by, for example, chemical modification, enzymatic modification, or a combination.
  • nuclease-resistant nucleotides Many types of nuclease-resistant nucleotides are known and can be used in the disclosed methods.
  • nucleotides have modified phosphate groups and/or modified sugar groups can be resistant to one or more nucleases.
  • Nuclease-resistance is defined herein as resistance to removal from a nucleic acid by any one or more nucleases.
  • nuclease resistance of a particular nucleotide can be defined in terms of a relevant nuclease.
  • the nuclease-resistant nucleotides need only be resistant to that particular nuclease.
  • useful nuclease-resistant nucleotides include thio-modified nucleotides and borano-modified nucleotides.
  • nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenine-9-yl (adenine, A), cytosine-l-yl (cytosine, C), guanine-9-yl (guanine, G), uracil- 1- yl (uracil, U), and thymin-l-yl (thymine, T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • a non- limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'- GMP (5'-guanosine monophosphate).
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl ( ⁇ ), hypoxanthin-9-yl (inosine, I), and
  • a modified base includes but is not limited to 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
  • nucleotide analogs such as 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 can increase the stability of duplex formation.
  • time base modifications can be combined with for example a sugar modification, such as 2'-0-methoxyethyl, to achieve unique properties such as increased duplex stability.
  • Nucleotide analogs can also include modifications of the sugar moiety.
  • Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications.
  • Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted CI to CIO, alkyl or C2 to CIO alkenyl and alkynyl.
  • 2' sugar modifications also include but are not limited to -0[(CH2)n 0]m CH3, - 0(CH2)n OCH3, -0(CH2)n NH2, -0(CH2)n CH3, -0(CH2)n -ONH2, and - 0(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10.
  • modifications at the 2' position include but are not limited to: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02 CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar
  • modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Nucleotide analogs can also be modified at the phosphate moiety.
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkage between two nucleotides can be through a 3 '-5' linkage or a 2 '-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3 ' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, 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.
  • 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.
  • PNA aminoethylglycine
  • conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • conjugates 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 l,2-di-0-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 octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
  • a Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute.
  • the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups ( H2 or O) at the C6 position of purine nucleotides.
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps.
  • hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm.
  • Tm the melting temperature at which half of the molecules dissociate from their hybridization partners
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies.
  • Hybridization temperatures are typically higher for DNA-R A and RNA-RNA
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and washing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • selective hybridization conditions would be when at least about, 60, 65,
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their ka, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their ka.
  • selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70,
  • kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagents discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • the kits could include one or more primers disclosed herein to perform the extension, replication and amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended.
  • kits that include at least one reverse primer wherein the reverse primer hybridizes to a portion of wild-type ROS l such as the kinase domain of wild-type ROSl . Additionally, it is understood that the kits disclosed herein can include one or more forward primers that specifically hybridize to a fusion partner of ROSl and/or wild-type ROSl .
  • the forward primer can hybridize to wild-type ROSl, such as a ROSl sequence external to the breakpoint region of ROSl fusions (i.e., 3 ' to the fusion breakpoint), CD74, FIG, SLC34A2, or other fusion partner.
  • wild-type ROSl such as a ROSl sequence external to the breakpoint region of ROSl fusions (i.e., 3 ' to the fusion breakpoint), CD74, FIG, SLC34A2, or other fusion partner.
  • a kit that comprises more than a singular primer or primer pair and could include, for example, a single reverse primer, such as SEQ ID NO: 5, 8, 12, 14, 15, 18, or 21, and multiple forward primers.
  • the kit can include a primer pair for wild-type ROSl external to the fusion breakpoint (i.e., 3 ' to the fusion breakpoint) and a primer pair for ROS l kinase.
  • the kit can further comprise one or more forward primers and/or one or more reverse
  • kits for diagnosing an ROSl related cancer or determining susceptibility of a cancer to a treatment or the suitability of a treatment for a cancer comprising (a) a first primer labeled with a first detection reagent, wherein said first primer is a reverse primer, wherein said reverse primer is one or more polynucleotide(s) that hybridizes, to a first polynucleotide encoding the amino acid sequence of SEQ ID NO 1 or the complement thereof; and (b) at least one second primer, wherein said second primer is a forward primer, wherein said forward primer is one or more polynucleotide(s) that hybridizes to a second polynucleotide encoding wild-type ROSl.
  • kits comprising one or more forward primers that specifically binds to the ECD of ROS l (for example SEQ ID NOs: 13 and 20). Also disclosed are kits comprising one or more forward primers that specifically bind to the ROS1 kinase domain (for example SEQ ID NOS: 4, 7, 15, or 17). It is understood that kits can comprise one or more forward primers specific to the ECD and kinase domains of ROS1. Also disclosed are kits further comprising one or more reverse primers specific for the ECD of ROS1 (e.g., SEQ ID NOS: 14 and 21).
  • kits comprising one or more reverse primers that specifically bind to the ROS1 kinase domain (for example SEQ ID NOS: 5, 8, 12, 16, and 18). Also disclosed are kits comprising a combination of one or more reverse primers specific to the ECD and kinase domains of ROS 1. It is further understood that any one or more forward primers disclosed herein can be combined in a kit with one or more reverse primers. Thus, disclosed herein are kits comprising one or more forward primers, such as, for example the ROS1 specific forward primers SEQ ID NOS: 4, 7, 13, 15, 17, or 20) and/or one or more ROS 1 specific reverse primers SEQ ID NOs: 5, 8, 12, 14, 16, 18, and 21).
  • kits comprising one or more polynucleotide probes, wherein said probe(s) are from about 20 to about 30 nucleotides in length and comprises a reporter dye on one end thereof and a quenching dye on another end thereof, such as, for example SEQ ID NO: 6, 9, 19, or 22.
  • kits can also include controls to insure the methods disclosed herein are properly functioning and to normalize results between assays.
  • positive cDNA controls negative cDNA controls, and control primer pairs.
  • the disclosed kits can include a control primer pairs for the detection of Homo sapiens ATP synthase, H+ transporting, mitochondrial F 1 complex, O subunit (ATP50), nuclear gene encoding mitochondrial protein mRNA;
  • Homo sapiens glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) Homo sapiens H3 histone, family 3A (H3F3A), mRNA;
  • Homo sapiens proteasome Homo sapiens proteasome
  • primers pairs include but are not limited to the primer pairs found in Table 1.
  • NDUFA2 GCCTGAAGACCTGGAATTGG (SEQ ID NO: CTGACATAAGTGGATGCGAATC (SEQ ID NO:
  • GAPDH GGAAGGTGAAGGTCGGAGTC SEQ ID NO: GCTGATGATCTTGAGGCTGTTG (SEQ ID NO:
  • H3F3A CCAGCCGAAGGAGAAGGG (SEQ ID NO: 26) AGGGAAGTTTGCGAATCAGAAG (SEQ ID NO:
  • PSMB4 TACCGCATTCCGTCCACTC (SEQ ID NO: 27) GCTCCTCATCAATCACCATCTG (SEQ ID NO:
  • EIF4A2 CTCTCCTTCGTGGCATCTATG (SEQ ID NO: GGTCTCCTTGAACTCAATCTCC (SEQ ID NO:
  • COX5B ACGCAATGGCTTCAAGGTTAC (SEQ ID NO: CGCTGGTATTGTCCTCTTCAC (SEQ ID NO: 42)
  • kits can include such other reagents and material for performing the disclosed methods such as a enzymes (e.g., polymerases), buffers, sterile water, reaction tubes. Additionally the kits can also include modified nucleotides, nuclease-resistant nucleotides, and or labeled nucleotides. Additionally, the disclosed kits can include instructions for performing the methods disclosed herein and software for enable the calculation of the presence of an ROSl mutation.
  • a enzymes e.g., polymerases
  • buffers e.g., sterile water, reaction tubes.
  • the kits can also include modified nucleotides, nuclease-resistant nucleotides, and or labeled nucleotides. Additionally, the disclosed kits can include instructions for performing the methods disclosed herein and software for enable the calculation of the presence of an ROSl mutation.
  • kits can comprise sufficient material in a single assay run simultaneously or separately to conduct the methods to determine if a sample contains a wild-type ROSl, a known ROSl fusion, or a previously unidentified ROS l fusion.
  • the kits can also include sufficient material to run control reactions.
  • kits comprising a positive cDNA control reaction tube, a negative cDNA control reaction tube, a control primer reaction tube, a reaction tube to detect known ROSl fusions and/or a reaction tube to detect wild-type ROSl, and a reaction tube to detect ROS l kinase activity.
  • the disclosed kit can be used to determine ROS l status - either wild-type expression, kinase domain overexpression, or fusion mutation
  • ROSl status can be determined via an equation of ROSl(numerator)/internal control (denominator) where the resulting quotient is a range of outcomes that indicate tested tissues, cell lines or other samples are either ROSl positive or ROSl negative.
  • the ratio and quotient determined to indicate ROSl positive or negative status will be established separately for each tissue and specimen type.
  • compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
  • the disclosed nucleic acids such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen- Biosearch, Burlington, MA or ABI Model 380B).
  • a Milligen or Beckman System lPlus DNA synthesizer for example, Model 8700 automated synthesizer of Milligen- Biosearch, Burlington, MA or ABI Model 380B.
  • Example 1 Development and validation of a FISH assay to detect ROSl fusions.
  • ROSl partners - FIG, CD74 and SLC34A2 - have been identified thus far as being involved in cancer-associated ROSl fusions. While it is presently unknown, it is unlikely that these three partners of ROSl are the only genes that form fusions with the kinase in cancer based on the experience with other fusion kinases; for example, >15 different genes (some of which are shown in Figure 1) are now known to fuse with the truncated ALK receptor tyrosine kinase (RTK) to form chimeric kinases that drive cancer development.
  • RTK truncated ALK receptor tyrosine kinase
  • ROSl fusion FISH assays can be designed specifically for each of the three known fusion partners involved, such an approach risks the possibility of missing ROSl fusions involving yet to be identified fusion partners; furthermore, the clinical application of three different FISH assays to detect ROS l fusions in tumor specimens is extremely time- and labor-inefficient.
  • the ROSl fusion assay uses large genomic clones (typically BAC and/or PAC clones) that flank the location within the ROS l gene locus at which the breaks occur during cancer-associated chromosomal rearrangements.
  • genomic clones typically BAC and/or PAC clones
  • Such a "breakapart" FISH assay uses flanking genomic clones that are labeled with differently colored fluorochromes. In normal cells, the different colors (e.g., red and green, and sometimes appearing yellow when the colors overlap) are physically paired because the gene locus is intact; by contrast, in cancer cells containing a rearrangement of the gene of interest, the colors are physically separated. As shown in Figure 4, the ROS l breakapart FISH assay works as expected on normal and ROSl fusion-containing cells.
  • Example 2 Development of a PCR-based assay to detect ROSl fusions.
  • ROSl breakapart FISH assay detects the majority of ROS l fusions regardless of the partner gene involved.
  • an additional ROS 1 fusion clinical diagnostic is still significant because 1) some countries and certain situations may prefer diagnostic platforms other than FISH (which can be somewhat technically demanding), and 2) the FIG -ROSl fusion cannot be detected by FISH because it is generated by a micro-deletion within the
  • ROSl locus that does not result in a breakapart of flanking probes (the FIG gene is actually embedded within introns of the ROSl gene, and becomes juxtaposed adjacent to the ROSl exons encoding the portion of ROSl found in FIG -ROSl as a consequence of the micro- deletion within the locus).
  • a PCR-based assay was developed as a complementary platform to FISH for the detection of ROSl fusion mutations.
  • the ROSl PCR diagnostic (to be branded Insight ROSl ScreenTM) is a real-time PCR diagnostic utilizing diverse RT primers to selectively amplify ROSl fusions in lung cancer specimens.
  • the underlying design for Insight ROS l Screen is illustrated in Figures 5 and 6.
  • ROSl is basally expressed in both NSCLC and normal lung tissue. Therefore, an effective ROSl assay must distinguish normal expression from oncogenic expression.
  • Traditional real-time RT-PCR strategies utilize allele-specific primer sets that target each fusion transcript in a one-step reaction. However, this method has drawbacks, such as fusion variability and recurring optimization for newly discovered fusion partners and variants using an ever-expanding mixture of detection primers.
  • the Insight ROSl Fusion Screen avoids these issues by maintaining a universal PCR detection method of the ROSl kinase domain.
  • Oncogenic ROS l -fusions are targeted in a first-strand synthesis step that selects against amplification of basal full-length ROSl expression in tumor lung tissue. In this way, the detection phase of the assay remains constant regardless of fusion partner.
  • the assay utilizes two separate PCR primer sets, one that amplifies a region of the ROSl gene encoding the ROSl extracellular domain found only in the normal RTK (not in ROSl fusions) while the other primer set amplifies a ROSl gene segment encoding the kinase domain, which is found both in normal and fused ROSl.
  • This assay design can not only detect the presence of ROSl fusions but also overexpression of the intact ROS l gene (which most frequently is correlated with DNA amplification of the gene locus).
  • Normal ROSl RTK mRNA expression is correlated with internal control standards; overexpression of normal ROSl is indicated by a threshold Ct value lower than the internal controls whereas the presence of a ROSl fusion is evident by a difference in threshold Ct values between the wild-type and kinase domain real-time PCR reactions (the kinase domain reaction Ct being lower than that of the wild-type reaction).
  • this elegant and simple design identifies all ROSl fusions independent of the fusion partner involved; in addition, it identifies and quantifies overexpression of wild-type ROSl .
  • ROSl amplification/overexpression follows the pattern of amplification/overexpression of other RTKs (e.g., ALK and EGFR) what are known to result in constitutive kinase activation that drives tumor growth when over a certain threshold level; importantly, tumors with such RTK amplification/overexpression are sensitive to pharmacologic inhibition of the driver kinase.
  • RTKs e.g., ALK and EGFR
  • RNA secondary structure reduces RNA secondary structure, but also reduces specificity of the FS-RT primer. If signal is detected in normal lung using the FS-RT primer, the reaction is not specific in discriminating full-length ROS1 from ROS1 fusions (see Thermoscript with 57°C Tm primer). Loss of specificity was addressed by redesigning a series of primers with melt temperatures near the extension temperature (i.e. 63-65°C). A high melt temperature primer eliminated the non-specific cDNA synthesis of the full-length ROS1 transcripts in normal lung samples (Table 2).
  • RNA from a SLC34A2-ROS 1 fusion-expressing cell line (HCC78) or normal lung was subjected to first-strand cDNA synthesis using the Superscript III or Thermoscript reverse transcriptases along with a SLC34A2 FS-RT primer, random hexamer mix, or no primer.
  • the products of these reactions were screened with a ROS1 kinase-specific qPCR assay with mean Ct values reported in the table.
  • RNA from a CD74-ROS1 fusion-expressing Ba/F3 c ell line or norma lung was subjected to first-strand cDNA synthesis using the Thermoscript reverse transcriptase along with a FS-RT CD74 primer, random hexamer mix, or no primer.
  • the products of these reactions were screened with a ROS1 kinase-specific qPCR assay with mean Ct values reported in the table.
  • Routine PCR testing for ROS1 fusions is currently performed using allele-specific primers flanking the fusion breakpoints.
  • the Insight ROSI Fusion Screen in contrast utilizes a universal PCR primer set for detection of the ROS 1 kinase domain following fusion-specific first strand synthesis.
  • cDNA generated using a FS-RT primer and random hexamers was subjected individually to both types of subsequent detection methods.
  • Table 4 shows that a ROS1 kinase-specific assay has similar crossing point (Ct) values when compared to an allele- specific primer set on a specified sample using identical concentrations of starting total RNA.
  • ROS1 kinase-specific assay incorporates the ability to detect differences in fusion transcripts based on amplification/detection of a common genetic region across a variety of fusion inputs.
  • the allele-specific assay in contrast, requires additional template input for each reaction and/or complex multiplexing for the detection phase of the reaction.
  • RNA from an SLC34A2-ROS1 -expressing cell line (HCC78), a CD74-ROS1- expressing cell line (Ba/F3), and normal lung was used as template for first-strand synthesis primed by a fusion-specific primer (FSP) or a random hexamer mix.
  • FSP fusion-specific primer
  • the products of this reaction were screened by qPCR with primers specific for the ROSl kinase domain, the SLC34A2 -ROS l -long fusion, the SLC34A2-ROS1 -short fusion, or the CD74-ROS1 fusion (mean Ct values reported).
  • RNA from the CD74-ROS1- expressing Ba/F3 cell line was diluted into RNA extracted from normal lung. Since normal lung expresses full-length ROSl only and the Ba/F3 cell line solely expresses CD74-ROS 1 fusions, normal lung RNA can be used to dilute Ba/F3 RNA to extinction in order to determine the limit of detection.
  • Each RNA dilution series was subjected to fusion-specific first strand synthesis and amplification with a ROSl kinase qPCR assay (i.e. the Insight
  • Total RNA from Ba/F3 cells expressing the CD74-ROS1 fusion was diluted into total RNA from normal lung and used as template for the Insight ROSl Fusion Screen.
  • the high background levels of constitutively expressed wild-type ROSl in lung tissue may mask any detectable difference in threshold Ct value between the wild-type and kinase domain real-time PCR reactions described in the Insight ROSl Screen PCR based assay.
  • detectable differences are masked, an alternative qPCR approach is used for exclusive detection of the three ROSl fusions reported to date in NSCLC cell lines or patient tumors; SLC34A2-ROS (long form), SLC34A2-ROS (short form) & CD74-ROS.
  • the qPCR assay utilizes two different primers in a multiplexed cDNA synthesis reaction; one cDNA primer can be specific for a region present in both the long and short form of SLC34A2 and another cDNA primer present exclusively in the CD74 gene. Both cDNA primers are strategically placed slightly 5 ' to each fusion break point for ease of cDNA extension through the translocation region of the three different ROSl fusions.
  • the second qPCR Insight ROSl Fusion Screen described herein is referred to as the Insight ROSl Fusion ScreenTM v2 for the duration of this application.
  • the Insight ROS l Fusion ScreenTM v2 consists of a cDNA synthesis phase that generates three potential cDNAs; a truncated wild-type SLC34A2, a truncated wild-type CD74 and potential extension and synthesis of the immediate translocation regions of SLC34A2-ROS1 or CD74-ROS 1 fusions if present in a particular patient specimen.
  • all cDNA primers have a low Tms (to prevent annealing during the higher temperatures of the real time reaction) and the reverse transcriptase is deactivated at 65°C for 20 minutes.
  • the nascent cDNA can then be used in a qPCR detection reaction using primers specific for a ROSl region juxtaposed to the translocation point for both the SLC34A2 or CD74 ROSl fusions ( Figure 7.). Again, the nucleotide distances separating the initiation sites of the cDNA synthesis and the locations of the primers mediating the actual qPCR detection reaction are kept minimal to circumvent problems with degraded FFPE RNA.
  • ROS l specific qPCR kinase reaction then amplifies a cDNA region of ROS l present only in a fused form translocated to either SLC34A2-ROS1 or CD74-ROS1. Wild-type ROS l kinase regions are not be present in each sample due to the lack of any ROS l specific cDNA primers present in the reverse transcription phase of the assay (Figure 7). Specific sequences are listed in Table 7.
  • Example 6 Determination of the sensitivity and specificity of both the Insight ROSl FISH and qPCR based assays.
  • Both the Insight ROS 1 FISH assay and a RT-qPCR assay can be assessed using various cancer cell lines or tissue samples.
  • Table 8 shows cell lines of two types: one cell line expressing the FIG-ROS fusion and one expressing both SLC34A2-ROS fusions (long and short) at varying expression levels. Normal lung total RNA consisting of wild- type levels only of full length ROS 1 can be used as a background control.
  • RNA extraction and one step qPCR procedures follows well-established laboratory protocols.
  • Dugan LC Pattee MS, Williams J, Eklund M, Sorensen K, Bedford JS, Christian AT.
  • Epidermal growth factor receptor (EGFR) gene copy number detection in non-small-cell lung cancer a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization. Histopathology 51 : 631-637, 2007.
  • Moison C Arimondo PB, Guieysse-Peugeot AL. Commerical reverse transcriptase as a source of false-positive strand-specific RNA detection in human cells. Biochimie 93 : 1731- 1737, 201 1.
  • Penault-Llorca F Bilous M, Dowsett M, Hann W, Osamura RY, Ruschoff J, van de Vijver M. Emerging technologies for assessing HER2 amplification. Am J Clin Pathol 132: 539- 548, 2009. Petersen BL, Sorensen MC, Pedersen S, Rasmussen M. Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method. Appl
  • Venkatesh B, Ullrich A Genetic alterations in the tyrosine kinase transcriptome of human cancer cell lines. Cancer Res 67: 11368-1 1376, 2007.

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Abstract

La présente invention concerne des procédés et des compositions permettant de détecter la présence d'un cancer chez un sujet et d'évaluer l'efficacité de traitements de ce cancer. Le procédé de l'invention fait appel à une réaction d'amplification en chaîne par polymérase en temps réel, à une réaction d'amplification en chaîne par polymérase par transcription inverse (RT-PCR), à une hybridation in situ et/ou à une réaction d'amplification en chaîne par polymérase multiplexe pour détecter la présence de mutations ponctuelles, de troncatures ou de fusions de ROS1.
PCT/US2013/025345 2012-02-08 2013-02-08 Procédés et compositions concernant des fusions de ros1 pour diagnostiquer et traiter le cancer WO2013119950A2 (fr)

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CN201380013401.4A CN104364391A (zh) 2012-02-08 2013-02-08 用于诊断和治疗癌症的涉及ros1融合的方法和组合物
AU2013216904A AU2013216904A1 (en) 2012-02-08 2013-02-08 Methods and compositions relating to fusions of ROS1 for diagnosing and treating cancer
JP2014556726A JP2015508644A (ja) 2012-02-08 2013-02-08 癌の診断および治療のためのｒｏｓ１の融合体に関する方法および組成物
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WO2015124697A1 (fr) * 2014-02-20 2015-08-27 Ignyta, Inc. Composés pour traiter des patients présentant des cellules cancéreuses mutantes ros1
US20160313302A1 (en) * 2014-09-10 2016-10-27 Board Of Regents Of The University Of Texas System Targeting emopamil binding protein (ebp) with small molecules that induce an abnormal feedback response by lowering endogenous cholesterol biosynthesis
CN106462669A (zh) * 2014-03-25 2017-02-22 奎斯特诊断投资股份有限公司 通过使用平均循环阈值的基因内差异表达(ide)检测基因融合
US10085979B2 (en) 2014-12-02 2018-10-02 Ignyta, Inc. Combinations for the treatment of neuroblastoma
US10398693B2 (en) 2017-07-19 2019-09-03 Ignyta, Inc. Pharmaceutical compositions and dosage forms
US10577344B2 (en) 2013-09-10 2020-03-03 The Board Of Regents Of The University Of Texas System Therapeutics targeting truncated adenomatous polyposis coli (APC) proteins
US10869864B2 (en) 2015-12-18 2020-12-22 Ignyta, Inc. Combinations for the treatment of cancer
US11007191B2 (en) 2017-10-17 2021-05-18 Ignyta, Inc. Pharmaceutical compositions and dosage forms
WO2024072805A1 (fr) * 2022-09-26 2024-04-04 Lau, Johnson Yiu-Nam Compositions, systèmes et procédés pour la détection du cancer de l'ovaire

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JP2022508651A (ja) * 2018-10-08 2022-01-19 レヴォリューション・メディスンズ，インコーポレイテッド 癌を処置するためのｓｈｐ２阻害剤組成物および方法

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EA033457B1 (ru) * 2014-02-20 2019-10-31 Ignyta Inc Способ лечения рака у пациентов с мутациями в генах ros1, ntrk1, ntrk2 и/или ntrk3
WO2015124697A1 (fr) * 2014-02-20 2015-08-27 Ignyta, Inc. Composés pour traiter des patients présentant des cellules cancéreuses mutantes ros1
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US10682348B2 (en) 2014-02-20 2020-06-16 Ignyta, Inc. Molecules for administration to ROS1 mutant cancer cells
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EP3825421A1 (fr) * 2014-03-25 2021-05-26 Quest Diagnostics Investments Incorporated Détection de fusions de gènes par expression différentielle intragénique (ide) à l'aide de seuils de cycle moyen
US11913079B2 (en) 2014-03-25 2024-02-27 Quest Diagnostics Investments Llc Detection of gene fusions by intragenic differential expression (ide) using average cycle thresholds
US10487367B2 (en) 2014-03-25 2019-11-26 Quest Diagnostics Investments Incorporated Detection of gene fusions by intragenic differential expression (IDE) using average cycle thresholds
US11098373B2 (en) 2014-03-25 2021-08-24 Quest Diagnostics Investments Llc Detection of gene fusions by intragenic differential expression (IDE) using average cycle thresholds
EP3123380A4 (fr) * 2014-03-25 2017-11-22 Quest Diagnostics Investments Incorporated Détection de fusions de gènes par expression différentielle intragénique (ide) à l'aide de seuils de cycle moyen
CN106462669A (zh) * 2014-03-25 2017-02-22 奎斯特诊断投资股份有限公司 通过使用平均循环阈值的基因内差异表达(ide)检测基因融合
US20160313302A1 (en) * 2014-09-10 2016-10-27 Board Of Regents Of The University Of Texas System Targeting emopamil binding protein (ebp) with small molecules that induce an abnormal feedback response by lowering endogenous cholesterol biosynthesis
US10082496B2 (en) * 2014-09-10 2018-09-25 Board Of Regents Of The University Of Texas System Targeting emopamil binding protein (EBP) with small molecules that induce an abnormal feedback response by lowering endogenous cholesterol biosynthesis
US10085979B2 (en) 2014-12-02 2018-10-02 Ignyta, Inc. Combinations for the treatment of neuroblastoma
US10357490B2 (en) 2014-12-02 2019-07-23 Ignyta, Inc. Combinations for the treatment of neuroblastoma
CN104830989A (zh) * 2015-05-25 2015-08-12 上海允英医疗科技有限公司 一种用于ros1与多种基因发生融合突变的检测试剂盒
US10869864B2 (en) 2015-12-18 2020-12-22 Ignyta, Inc. Combinations for the treatment of cancer
US10398693B2 (en) 2017-07-19 2019-09-03 Ignyta, Inc. Pharmaceutical compositions and dosage forms
US11253515B2 (en) 2017-07-19 2022-02-22 Ignyta, Inc. Pharmaceutical compositions and dosage forms
US11007191B2 (en) 2017-10-17 2021-05-18 Ignyta, Inc. Pharmaceutical compositions and dosage forms
WO2024072805A1 (fr) * 2022-09-26 2024-04-04 Lau, Johnson Yiu-Nam Compositions, systèmes et procédés pour la détection du cancer de l'ovaire

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