JP2015508644A - Methods and compositions relating to fusions of ROS1 for the diagnosis and treatment of cancer - Google Patents

Abstract

Disclosed are methods and compositions for assessing the efficacy of detecting and treating the presence of cancer in a subject. The disclosed methods use real-time polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and / or multiple polymerase chain reaction techniques to point mutation, cleavage, or cleavage of ROS1. Detect fusion. [Selection] Figure 1

Description

  This application claims the benefit of US Provisional Application No. 61 / 596,720, filed Feb. 8, 2012, which is incorporated herein by reference in its entirety.

  Oncogenic fusions of ROS1 tyrosine kinases occur in several human cancer subsets, including non-small cell lung cancer (NSCLC), glioblastoma multiforme (GBM) brain tumors, and bile duct cancer (biliary tract tumors) Has recently been reported. Cancer cells expressing ROS1 fusions “use” aberrant signaling associated with constitutively active chimeric forms of kinases for reproduction and survival. In light of the dependence on abnormal ROS1 signaling, preclinical studies have demonstrated that ROS1-induced cancer is extremely sensitive to pharmacological inhibitors of mutant kinases. Although small cell inhibitors are currently under development for the treatment of patients with ROS1-induced cancer, there are no commercially available diagnostic tests available to reliably and efficiently diagnose ROS1 fusion positive cancers. Therefore, what is needed is a ROS1 fusion diagnostic test that meets this requirement.

  The methods and compositions disclosed herein assess susceptibility or risk for a disease or condition associated with nucleic acid mutation, truncation, or gene fusion that detects or diagnoses a disease or condition such as cancer. Relates to the field of monitoring disease progression and determining sensitivity or resistance to therapeutic treatment. In one aspect, the detection and diagnosis methods disclosed herein relate to the detection and / or diagnosis of ROS1 fusion-related cancers.

  In accordance with the purpose of the present invention, as embodied and broadly described herein, the present invention, in one aspect, detects ROS1 by detecting nucleotide mutations, such as fusions, in a nucleic acid of interest. A method for detecting the presence of a related cancer, which is derived from real-time polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), or extracted from a tissue sample mRNA of a subject having cancer, or cancer Performing PCR on cDNA synthesized from RNA extracted from a subject having an presence of an amplification product or an increase in amplification product relative to a control indicates nucleotide mutations, truncations, or overexpression; It relates to a method for detecting the presence of cancer thereby.

  In another aspect, a method of diagnosing a subject having a cancer, such as having a ROS1-related cancer, further comprising determining a cycle threshold (Ct) value of wild-type ROS1 and wild-type ROS1 kinase, Disclosed herein is a method wherein a high (Ct) value for wild-type ROS1 relative to ROS1 kinase indicates the presence of the fusion.

  In another embodiment, a method of diagnosing a subject having a cancer, such as having a ROS1-related cancer, comprising a forward primer that binds to a ROS1 fusion partner 5 ′ of the fusion breakpoint and a fusion breakpoint Comprising a reverse primer that binds 3 'to ROS1, wherein said primer extends throughout the fusion and detection of the presence of an amplicon having both ROS1 and a fusion partner nucleic acid indicates the presence of the fusion; It is disclosed herein.

  In another aspect, a method of diagnosing a subject having a cancer, such as having a ROS1-related cancer, comprising: a) a first amplification reaction using a forward primer that binds to a ROS1 fusion partner, comprising: The amplicon from the first reaction is used as a template for the second amplification reaction, and b) a second amplification reaction after the first reaction, at 3 ′ of the fusion breakpoint A primer specific for the ROS1 sequence is used in the second amplification reaction, and c) detecting the presence of ROS1 in the amplicon from the second reaction, the amplifier from the second reaction Disclosed herein is a method wherein detecting ROS1 in a recon indicates that ROS1 fusion is present.

  In another embodiment, a method for detecting the presence of a ROS1-related cancer by detecting a nucleotide mutation, such as a fusion, in a nucleic acid of interest, comprising a fluorescent in-situ (in situ) hybrid to a tissue sample of a subject having cancer Performing hybridization (FISH), wherein the probe is used for hybridization adjacent to the breakpoint of the ROS1 fusion, the probe is labeled differently, and the separation of the probe indicates the presence of a ROS1-related cancer Are disclosed herein. In the case of a detection method based on FISH, the closely placed hybridized probe shows a wild type RTK such as ROS1, for example.

  In another embodiment, (a) a first primer labeled with a first detection reagent, wherein the first primer is a reverse primer, and the reverse primer is the amino acid sequence of SEQ ID NO: 1 or A first primer that is one or more polynucleotides that hybridize with a first polynucleotide that encodes the complement; and (b) at least one second primer comprising the second And a second primer, wherein the first primer is one or more polynucleotides that hybridize with a second polynucleotide encoding wild-type ROS1. Kits for diagnosing are disclosed herein.

  Additional advantages of the disclosed methods and compositions are set forth in part in the following description and can be learned in part from the description or practice of the disclosed methods and compositions. . The advantages of the disclosed method will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claimed invention.

  The accompanying drawings, which are incorporated in and form a part of this specification, illustrate several embodiments of the disclosed methods and compositions, and together with the description, the disclosed methods and compositions Helps explain the principle of things.

FIG. 2 shows a schematic diagram of a receptor tyrosine kinase (RTK) involved in carcinogenicity resulting from the production of fusion kinase. The figure illustrates a normal transmembrane RTK. Proteins shown in red under each RTK form constitutively active oncogenic fusions with kinases. A representative ROS1 fusion kinase, CD74-ROS1 fusion kinase, is shown. (A) The positions of the human CD74CD74 (5q32) and ROS1 (6q22) genes on the chromosome. In NSCLC, a mutual equilibrium rearrangement involving chromosomes 5 and 6 occurs following a break within the two genes to form a CD74-ROS1 fusion. (B) Schematic representation of normal CD74 and ROS1 protein and CD74-ROS1 fusion protein created by t (5; 6) (q32; q22) chromosome rearrangement. The ROS1 fusion in NSCLC is shown. Figure 2 is a schematic representation of a transcript created by chromosomal translocation by the ROS1 gene. Exon positions have been shown to demonstrate the diversity of fusion configuration. Figure 7 shows a ROS1 breakapartFISH assay. Genomic DNA clones (upper schematics) adjacent to the cleavage site in the ROS1 gene that causes chromosomal rearrangement to produce the ROS1 fusion gene in human cancer are labeled differently with green and red fluorescent dyes, and then normal Hybridized with interphase nuclei and metaphase chromosomes from lower peripheral blood lymphocytes (lower left panel), or SLC34A2-ROS1-positive NSCLC cell line (lower right panel). The Insight ROS1 fusion screen method is shown. The assay uses fusion-specific reverse transcription (FS-RT) at elevated temperatures to prevent confounding conditions where reverse transcriptase primes cDNA synthesis from stem loop structures located within the RNA transcript. By performing the assay at elevated temperatures, these structures are minimized during the extension process, limiting the production of first strand cDNA to ROS1 fusions only. The first strand reaction is then subjected to RNase consumption to remove all RNA, which may allow non-specific amplification downstream of the PCR detection phase. The fusion-specific cDNA is then used as a template for a universal downstream ROS1 kinase quantitative PCR (qPCR) assay. Multiple ROS1 fusions can be targeted by multiplexing multiple FS-RT primers during the reverse transcription phase. This FS-RT primer cocktail is easy to capture newly identified oncogenic fusions without increasing the number of primers used for detection while maintaining specificity and high throughput capability for the reaction. Can be modified. The Insight ROS1 fusion Screen ™ is shown. FIG. 4 is a schematic diagram of ROS1 cDNA, the location of the breakpoint of the cancer-associated ROS1 fusion, and the approximate location of the PCR primer set used for real-time qPCR assays. Ct values indicating normal level expression of wild type ROS1, overexpression of wild type ROS1, or the presence of ROS1 fusions are shown in the lower panel. Shows Insight ROS1Screen ™ v2. FIG. 6 is a schematic diagram of the position of the breakpoint of ROS1 RNA, cancer-associated ROS1 fusion, and the approximate position of primers used for cDNA synthesis. The location of the ROS1 template specific kinase reaction is also shown after the cDNA synthesis qPCR detection phase of the reaction. The blue arrow indicates the relative position of the SLC34A2 cDNA primer, and the red arrow indicates the relative position of the CD74-specific cDNA primer that approximates the actual ROS1 translocation.

  Before the compounds, compositions, articles, devices, and / or methods of the present invention are disclosed and described, they are not specifically specified, unless otherwise specified, or specific synthetic or biotechnological methods. It is to be understood that these are not limited to specific reagents as long as they are of course varied. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

  As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes a mixture of two or more such carriers, and the like.

  Ranges may be expressed herein as “about” from one particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, where a value is expressed as an approximation, it is understood that the use of the antecedent “about” forms that particular value forms another embodiment. It is further understood that the end points of each range are significant whether they are associated with other end points or independent of other end points. It is also understood that there are a number of values disclosed herein, and that each value is disclosed herein as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, “about 10” is also disclosed. Where a value discloses the value “below”, it is also understood that “greater than or equal to” and possible ranges between values are also disclosed, which is well understood by those skilled in the art. For example, when the value “10” is disclosed, “10 or less” and “10 or more” are also disclosed. Throughout the specification, it is also understood that data is provided in a number of different ways, and that this data represents the endpoints and starting points, and ranges, of any combination of data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, then more than 10 and more than, less than, less than or equal to are considered to be disclosed as 10 to 15 It is understood that

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
“Occasionally” or “optionally” includes that the event or situation described below may or may not occur, and that the description includes whether or not the event or situation occurs Means that.

  “Increase” can mean any change that results in a composition or compound such as a large amount of amplification product as compared to a control. Thus, for example, an increase in the amount of amplification product may include an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. However, it is not limited to these. Detecting an increase in the expression or abundance of DNA, mRNA, or protein relative to a control refers to detecting the presence of DNA, mRNA, or protein in a situation where no DNA, mRNA, or protein is present in the control. The need to include is further considered herein.

  “Obtaining a tissue sample” or “obtaining a tissue sample” means collecting a sample of tissue from a subject or measuring tissue in a subject. It is understood and contemplated herein that the tissue sample can be obtained by any method known in the art, including invasive and non-invasive techniques. It is also understood that the method of measurement can be direct or indirect. Examples of methods for obtaining or measuring tissue samples include tissue biopsy, tissue washing, aspiration, tissue wiping, spinal tap, magnetic resonance imaging (MRI), computed tomography (CT) scanning, positron emission tomography ( PET) scanning, and X-gland (with or without contrast agent) may be included, but are not limited to these.

  Sensitive detection of mutations at known sites in DNA has already been performed by existing techniques. Allele-specific primers can be designed to target mutations at known positions so that the signal can be preferentially amplified over wild-type DNA. Unfortunately, this is not possible with unknown mutations that can occur at any position (base) in the target sequence.

  It is understood that the tissue sample can be from any tissue in the body and is contemplated herein. Thus, as used herein, “tissue” refers to blood, nerve tissue (eg, brain tissue or spinal cord tissue), lymphoid tissue, liver tissue, spleen tissue, lung tissue, heart tissue, stomach tissue, intestinal tissue. Mean pancreatic tissue, thyroid tissue, salivary gland tissue, joint tissue, and skin tissue. It will be appreciated that the tissue sample may contain at least a single cell or fraction from the target sample. For example, the tissue cells can be peripheral blood mononuclear cells, B cells, T cells, macrophages, erythrocytes, platelets, or other blood cells. Similarly, the cells can be epithelial cells, hepatocytes, nerve cells, or other cells.

Methods for detecting ROS1-related cancers In a subject previously diagnosed with cancer, the presence of a disease or condition such as ROS1-related cancer (eg, small cell lung cancer (NSCLC), glioblastoma, or cholangiocarcinoma) ROS1-related cancers (e.g., NSCLC, glioblastoma, glioblastoma, ROS1-related cancers, e.g., NSCLC, glioblastoma, or cholangiocarcinoma) that assess or detect the susceptibility or risk of a disease or condition Or progression of a disease or condition such as (bile duct cancer) and determining susceptibility or resistance to therapeutic treatment of a disease or condition such as ROS1-related cancer (eg, NSCLC, glioblastoma, or cholangiocarcinoma) Obtaining a tissue sample, detecting the presence of ROS1 mRNA in a tissue sample of a subject, or expressing an expression level. An increase in the amount of amplification product relative to a control, or a simple detection of an amplicon indicates the presence of cancer, which is associated with a nucleic acid mutation, cleavage, or gene fusion of ROS1 A method disclosed in one aspect relating to a (ie ROS1-related cancer) method. In another embodiment, detecting or diagnosing the presence of a disease or condition such as a ROS1-related cancer can be achieved by use of in situ hybridization, where it is a fusion site to another gene within the cancer. Separation of the probe adjacent to the gene breakpoint indicates the presence of ROS1-related cancer. In another embodiment,

Oncogene tyrosine protein kinase ROS precursor (ROS1)
Receptor tyrosine kinases (RTKs) are cell signaling pathways that play an important role in normal development by regulating cell proliferation, differentiation, migration, and death. Perturbations in RTK signaling through diverse genetic alterations can lead to deregulated kinase activity followed by malignant transformation (FIG. 1).

  Phylogenetic analysis of all 58 known human RTKs shows that ROS1 is a distinct receptor that is distantly related to the anaplastic lymphoma kinase / leukocyte tyrosine kinase (ALK / LTK) and insulin receptor (INSR) families It is proved that. ROS1 is the vertebrate counterpart of the Drosophila Sevenless Receptor Tyrosine Kinase (Sevenless Receptor, also known as ROS), that is, the ligand has not yet been determined, by the ligand BOSS (Sevenless Bride) When activated, it is involved in the differentiation of R7 photoreceptors in the development of fly compound eyes. Mammalian ROS function considers that only the abnormalities observed in Ros1 null mice are involved in male infertility due to defective sperm function associated with epithelial incompetence (ROS expressing) Then, it seems almost unnecessary to support differentiation of spermatocytes. In addition to leukocyte tyrosine kinase (LTK), ROS is the most highly related receptor tyrosine kinase for ALK.

  Recently, three types of ROS1 that have occurred spontaneously have been described. In 2003, an intermediate microdeletion in chromosome 6q21 resulting in a fusion event of a novel gene called FIG (fused to glioblastoma) and a sequence encoding the intracellular portion of ROS1 was Found in blastoma cell lines. Experimentally forced expression of FIG-ROS1 in the genetically engineered adult mouse CNS results in the formation of glioblastoma multiforme tumors, confirming the oncogenic activity of this fusion kinase. No clinical studies have yet been reported that describe the precise and detailed development of FIG-ROS1 fusions in glioblastoma multiforme brain tumors. The FIG-ROS1 fusion is not limited only to glioblastoma. In 2011, it was shown that this chimeric kinase is also expressed in cholangiocarcinoma (biliary tract cancer). In 8.7% (2 out of 23) in primary tumor specimens. Similar to glioblastoma and NSCLC, the prognosis for patients with bile duct cancer, the second most common primary liver cancer, is very poor, with a median survival of less than 2 years.

  In 2007, two new ROS1 fusions were first reported in NSCLC, and interchromosomal translocations were found in both the NSCLC cell line and patient tumors, and in the vicinity of the outer membrane of ROS1 and the SLC34A2 and CD74 genes. It has been shown to result in a fusion with the moiety (for example, the CD74-ROS1 fusion is shown in FIG. 2). Fusion of the N-terminal portion of FIG, CD74, SLC34A2, TPM3, SDC4, EZR, and LRIG3 with the ROS1 kinase domain in glioblastoma, cholangiocarcinoma, and NSCLC results in constitutive kinase activity that induces tumor growth (Figure 3). Consistent with the role of these ROS1 fusions as carcinogenic mutants, siRNA-mediated downregulation of SLC34A2-ROS1 induced apoptotic death of human NSCLC cells. Similarly, treatment of cells experimentally made tumorigenic by FIG-ROS1 expression with a ROS1 small molecule inhibitor is associated with potent tumor cell killing.

  Although not as common as the EGFR or ALK RTK mutations in NSCLC, ROS fusions are clearly repetitive, with 2.6% (17/656) of NSCLS specimens being immune tissue in recent studies. Chemistry and FISH showed positive for ROS1 fusion, and no fusion of ALK and ROS1 was observed in the same tumor. In addition, several independent analyzes of tumor specimens revealed significantly elevated expression of ROS1 in 20-30% of NSCLC, supporting a broader role for kinases in lung cancer pathogenesis. Still additional malignant tumors can be induced by abnormal ROS1 signaling, for example, high levels of ROS1 expression have been found in 30-40% of glioblastoma surgical tumors, and ROS1 mutants Have been identified in colorectal and renal cancer cell lines. Commercial efficient and reliable diagnostic tests that detect the presence of ROS1 fusions in tumors also greatly facilitate bridging studies to profile other cancers with these mutants, while at the same time being more effective for certain cancers Provides a more efficient and less costly diagnosis and also allows for more effective and less costly personalized therapy for patients with ROS1 fusion positive tumors through the use of inhibitors of this mutant kinase.

  Thus, known ROS1 fusions do not represent the most common mutants of this tyrosine kinase, but still account for a significant proportion of tyrosine kinase fusion-related cancers, the significance of which is additional Increased by identifying fusion partners. Such fusions include (FIG) -ROS1, SLC34A2-ROS1, TPM3-ROS1, SDC4-ROS1, EZR-ROS1, LRIG3-ROS1, and CD74-ROS1 fused to glioblastoma, It is not limited to these. All three fusions have been found in cholangiocarcinoma, non-small cell lung cancer (NSCLC), and glioblastoma. Thus, in one aspect, ROS1 assesses susceptibility or risk of developing a disease or condition, such as ROS1-related cancer, detecting or diagnosing the presence of a disease or condition, such as ROS1-related cancer, in a subject having cancer. A method of monitoring the progression of a disease, such as a related cancer, and determining the sensitivity or resistance to a therapeutic treatment of a ROS1-related cancer, comprising detecting the presence or amount of a ROS1 amplicon in a tissue sample of a subject. Disclosed herein is a method wherein the presence of a nucleic acid or an increase in nucleic acid relative to a control indicates the presence of a ROS1-related cancer, such as a cancer with a RO1 fusion. . As used herein, “ROS-1-related cancer” means any cancer in which ROS1 is dysregulated through the presence, overexpression, mutation, or other mechanism of a ROS1 fusion.

  In one aspect, the disclosed methods can be accomplished by quantitative PCR (qPCR) assays. Accordingly, in one aspect, a method of detecting the presence of a ROS1-related fusion in a subject having cancer (ie, diagnosing the presence), obtaining a tissue sample, isolating nucleic acid from the sample, Performing PCR on nucleic acid isolated from a tissue sample of interest, wherein a primer for the qPCR assay is specific for a primer pair specific for ROS1 kinase and wt ROS1 at 5 ′ of the fusion breakpoint Cycle threshold (Ct) values are determined, and the high (Ct) value of wild-type ROS1 (eg, the extracellular domain (ECD) of ROS1) compared to ROS1 kinase is higher in the fusion and thus ROS1. Methods for indicating the presence of a related cancer are disclosed herein. Alternatively, a Ct value is not measured, but includes a forward primer that binds to the ROS1 fusion partner at 5 ′ of the fusion breakpoint, and a reverse primer that binds to ROS1 at the fusion breakpoint 3 ′, Disclosed herein is a method involving qPCR, wherein the detection of the presence of an amplicon extending throughout the fusion and having both ROS1 and a fusion partner nucleic acid indicates the presence of the fusion and thus a ROS1-related cancer. Has been. Thus, the disclosed methods can be used for diagnosis of ROS1-related cancers.

  It is understood and contemplated herein that nucleic acid (eg, RNA, such as DNA or mRNA) can be isolated from tissue cells after the tissue sample has been removed from the subject. Accordingly, in a further aspect, the disclosed method comprises obtaining a tissue sample and isolating nucleic acid from the tissue sample. For example, the method can include taking a lung tissue biopsy or sputum sample and isolating mRNA from the sample. It is further understood that if mRNA is isolated from a tissue sample, cDNA can be synthesized from mRNA and PCR can be performed on the cDNA (eg, as part of an RT-PCR reaction). Thus, in one aspect, a method of diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid (eg, mRNA) from the tissue sample, and RT Performing PCR, real-time PCR, or real-time RT-PCR and detecting the presence of nucleic acid associated with one or a combination of wild-type ROS1 (eg, ECD of ROS1) and ROS1 kinase domain in a tissue sample A forward and reverse primer pair that specifically hybridizes to a wild-type ROS1 sequence (eg, a PCR, RT-PCR, real-time PCR, or real-time RT-PCR reaction (eg, ROS1 cells such as SEQ ID NOs: 13, 14, 20, and 21 Primer that hybridizes to the domain (ECD)) and / or the sequence that specifically hybridizes to the wild-type ROS1 kinase domain sequence (eg, SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18) Including the use of directional and reverse primer pairs and detecting the presence or measuring the amount of nucleic acid associated with one or a combination of both wild type ROS1 and ROS1 kinase domains in a tissue sample Disclosed herein are methods, comprising: an increase in amplicon relative to a normal control, or the presence of an amplicon indicating that the subject has a ROS1-related cancer. A method for diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, wherein the nucleic acid in the tissue sample is RNA Wherein the method synthesizes cDNA from the RNA sample, performs PCR on the cDNA, and one of the wild-type ROS1 (eg, ECD of ROS1) and ROS1 kinase domain in the tissue sample, or Detecting the presence or measuring the amount of nucleic acid associated with both combinations, wherein an increase in amplicon relative to a normal control, or the presence of an amplicon, indicates that the subject has a ROS1-related cancer. Showing further. The disclosed method is to determine a cycle threshold (Ct) value, where a high (Ct) value of wild type ROS1 compared to ROS1 kinase indicates the presence of a fusion and thus a ROS1-related cancer; It is also disclosed that the amplicon can further comprise contacting the amplicon with a labeled probe complementary to the sequence of the amplicon to detect and measure the amount of amplicon.

  In a further alternative method, the ROS1 fusion uses only the forward primer that binds to the ROS1 fusion partner in the first reaction, and in the second reaction following the first reaction, the first reaction amplifier. The recon is used as a template in a second amplification, probe-based detection, or sequencing reaction, and the probe or primer used is specific for ROS1 at the fusion breakpoint 3 ', Detection of ROS1 in the amplicon of the reaction is detected by the qPCR method using two-step detection, indicating the fusion. Also, a method of diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, and using a forward primer that binds to the ROS1 fusion partner Performing the first amplification reaction, wherein the amplicon of the first reaction is used as a template for the second amplification reaction, and performing the second amplification reaction after the first reaction. A primer specific for the ROS1 sequence at 3 ′ of the fusion breakpoint is used in the second amplification reaction, and c) detecting the presence of ROS1 in the amplicon of the second reaction The detection of ROS1 in the amplicon of the second reaction indicates the presence of the ROS1 fusion and the nucleic acid associated with the ROS1 kinase domain in the tissue sample. And detecting a standing, presence of amplicon, the method comprising to show that it has a target ROS1 related cancer, are disclosed. As with the methods described above, this two-step detection can further include determining a C (t) value, or contacting the amplicon with a labeled probe that is complementary to the amplicon.

  It is understood and considered herein that additional qPCR assays can be used to reach the same information. For example, in one embodiment, an allele-specific method for detecting the presence of a ROS1-related fusion, wherein qPCR is performed on a tissue sample of interest, wherein the qPCR assay primer is a reverse that is specific for ROS1 kinase. A forward primer that binds 5 'to the fusion breakpoint of the ROS1 fusion partner, and the presence of an amplicon that reads across the fusion breakpoint indicates the presence of the fusion, thus detecting ROS1-related cancer A method including indicating is disclosed herein. In such methods, there are amplicons derived from reverse or forward primers, but such amplicons are derived only from unidirectional primers and the signal is more than the signal from the fusion event. It is understood that it is significantly less. Furthermore, the size of such amplicons is such that the forward primer amplifies only the remaining portion of the fusion partner and the reverse primer amplifies only the portion of ROS1 at 5 'of the reverse ROS1 kinase primer used. So it is different from the size of the fusion amplicon.

  In yet another aspect, a method for detecting the presence of a ROS1-related cancer by detecting a nucleotide mutation such as a fusion in a nucleic acid of interest, comprising fluorescence in situ hybridization to a tissue sample of a subject having cancer A method wherein the probe is used for hybridization adjacent to the breakpoint of the ROS1 fusion, the probe is labeled differently, and the separation of the probe indicates the presence of a ROS1-related cancer. It is disclosed in the document. In the case of a detection method based on FISH, the closely placed hybridized probe shows a wild type RTK such as ROS1, for example.

  ROS1 fusions are associated with several known types of cancer. It will be understood that one or more ROS1 fusions may be associated with a particular cancer. Anaplastic large cell lymphoma (ALCL), neuroblastoma, breast cancer, ovarian cancer, colorectal cancer, renal cancer, liver cancer, bile duct cancer, non-small cell lung cancer (NSCLC), diffuse large B cell lymphoma, esophageal flatness Some associated with ROS1 fusions, including but not limited to epithelial cancer, anaplastic large cell lymphoma, neuroblastoma, inflammatory myofibroblastic tumor, malignant histiocytosis, and glioblastoma It is further understood that there are several types of cancer. Accordingly, in one aspect, disclosed herein is a method of diagnosing a ROS-1-related cancer, wherein the cancer comprises NSCLC, glioblastoma, or cholangiocarcinoma. Also disclosed is a method of determining the sensitivity or resistance of a ROS1-related cancer to therapeutic treatment with a ROS1 inhibitor, wherein the cancer is NSCLC, glioblastoma, or cholangiocarcinoma. . In one aspect, the subject is understood to have previously been diagnosed with a cancer, such as, for example, NSCLC, glioblastoma, or cholangiocarcinoma.

  Recent studies have shown that inhibition of mutant forms of receptor tyrosine kinases (RTKs) by small molecule drug candidates abrogated this abnormal cell proliferation and apoptosis in neuroblastoma and other RTK-induced tumor cell lines. Has been demonstrated to promote These findings highlight the need for a personalized diagnostic test for RTK mutations, a test with multiple clinical uses. For example, such an assay can be used to screen family children suffering from hereditary neuroblastoma to help facilitate the detection of tumors in the early stages where treatment is more acceptable. Early detection and diagnosis of RTK-mediated cancer significantly increases survival in the patient population. In one aspect, a method of detecting or diagnosing the presence of a disease or condition, such as a cancer, eg, a ROS1-related cancer, associated with a wild-type ROS1 nucleic acid, or a mutation, truncation, or fusion thereof in a tissue sample of interest. Detecting in or measuring the presence of a level or expression level of DNA, cDNA, or mRNA, the corresponding increase in the amount of ROS1 kinase amplification product compared to a control that is absent in wild-type ROS1 However, methods for indicating the presence of a ROS1-related cancer are disclosed herein.

  Thus, in one aspect, detection of abundance relative to a ROS1 kinase sequence that does not have a corresponding ROS1 wild type sequence, or ROS1 wild type sequence, indicates the presence of a ROS1 fusion sequence and thus the presence of cancer. Thus, a method of diagnosing ROS1-related cancer in a subject comprising detecting the presence of mRNA from a tissue sample of the subject or measuring the expression level, wherein the mRNA is specific for a ROS1 fusion, Disclosed herein is a method wherein an increase in the amount of mRNA relative to a control indicates the presence of a ROS1-related cancer.

  The disclosed methods for diagnosing and determining susceptibility or resistance to ROS1 inhibitor treatment are not only for confirming that a subject has cancer, but also for subjects not previously diagnosed as having cancer, It can also be used on subjects previously diagnosed with cancer, and the method diagnoses whether the cancer is particularly ROS1-related or sensitive to treatment, and thus diagnoses cancer Instead, it is understood and considered herein that it can be used to determine whether a known cancer in a subject is ROS1-related or susceptible to treatment with a ROS1 inhibitor.

  It is also understood and considered herein that the cause of ROS1-related cancers can be attributed not only to dysregulation of wild-type ROS1 or known ROS1 fusions, but also to one or more unidentified ROS1 fusions. Methods that can detect only known fusions cannot detect previously unknown fusions or mutations in ROS1. By detecting ROS1, ROS1 fusion, and / or wild-type ROS1 cleavage, the presence of nucleic acid mutations, as well as detecting the presence of ROS1 kinase activity or ROS1 kinase amplicons, one of skill in the art can control cancer It can be determined whether due to defective wild type ROS1, a known ROS1 fusion, or a previously unidentified ROS1 fusion or mutation of ROS1. Therefore, diagnosing ROS1-related cancer, assessing cancer susceptibility or risk, or detecting the presence of dysregulation of ROS1 and / or the presence of wild-type ROS1, and detecting the presence of ROS1 kinase activity Methods involving are disclosed herein. Thus, for example, a method of diagnosing a ROS1-related cancer in a subject having cancer, comprising detecting the presence of a nucleic acid associated with a ROS1 fusion, wild-type ROS1, and / or ROS1 kinase domain in a tissue sample of the subject Methods involving are disclosed herein. In one aspect of the methods disclosed herein, it is understood and considered herein that the presence or increase of wild type ROS1 and ROS1 kinase alone can be examined. In such embodiments, the presence of wild-type ROS1 and ROS1 kinase, or an increase in its amplification compared to a control, may indicate a dysregulation of ROS1 that may be involved in a ROS-1-related cancer that is not due to a fusion event. The absence of change compared to the control indicates that ROS1 is not involved in cancer. In contrast, the presence or amplification compared to the ROS1 kinase only control indicates a ROS1 fusion. For example, in assays that measure cycle threshold (Ct) values, it is understood that the cycle threshold is a relative value based on an internal control, with a high Ct indicating a low expression level. Thus, it is understood that normal ROS1 expression is observed when the Ct values of both the wild type ROS1 primer pair and the ROS1 kinase primer pair are high compared to the internal control and there is no statistically significant difference from each other. ROS1 is overexpressed if the Ct value for both wild type and ROS1 kinase is low (ie, the expression level is high) but there is no statistically significant difference between the kinase and wild type primer pair. A ROS1 fusion is present when the Ct value of ROS1 is high (ie, low expression) compared to the Ct value of ROS1 kinase.

  In an alternative method of diagnosing a ROS1-related cancer, the method can include a first amplification reaction, wherein only the forward primer that binds to the ROS1 fusion at 5 ′ of the fusion breakpoint is responsible for the generation of the amplicon. used. The amplicon is used as a template for sequencing, amplification, or probe-based detection using a primer or probe specific for ROS1 at 3 ′ of the fusion breakpoint. The presence of ROS1 in the amplicon indicates the presence of a ROS1 fusion and thus a ROS1-related cancer.

  Accordingly, as disclosed above, a method for diagnosing ROS1-related cancer in a patient having cancer, comprising performing a first PCR-based reaction on a subject nucleic acid, Performing a second PCR-based reaction, sequencing reaction, or probe-based detection on the amplicon, wherein the primer for the first PCR reaction is ROS1 at 5 ′ of the fusion breakpoint Any primer or probe that is a forward primer that binds to the fusion partner and is used for the second PCR reaction or detection is specific for ROS1 at 3 ′ of the fusion breakpoint (eg, SEQ ID NO: 4, 5, 7, 8, 15, 16, 17, and 18), the presence of ROS1 in the amplicon of the first reaction indicates the presence of the ROS1 fusion and thus the ROS1-related cancer. Way to indicate is disclosed herein.

  In another aspect, a method of diagnosing a ROS1-related cancer in a subject having cancer, comprising performing a PCR-based reaction on the subject nucleic acid, wherein the forward primer is specific for the ROS1 fusion partner Detection of an amplicon bound to 5 'of the fusion breakpoint and a reverse primer specific for ROS1, bound to 3' of the fusion breakpoint and containing ROS1 and ROS1 fusion partner Methods for indicating the presence of a related cancer are disclosed herein.

  Alternatively, when FISH is used as a detection assay, the presence of spaced apart probes that hybridize to sequences adjacent to the breakpoint of ROS1 indicates the presence of a fusion event and thus cancer.

  Once ROS1-related cancer is detected by use of any of the aforementioned diagnostic methods, or its presence is diagnosed, the method then administers a ROS1 inhibitor to a subject with ROS1-related cancer. Further, it can be included.

  In one aspect, disclosed herein is a method of diagnosing ROS1-related cancer in a subject having cancer, comprising detecting the presence of ROS1 kinase activity. Thus, for example, a method of diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, RT-PCR, real-time PCR, or Performing real-time RT-PCR and detecting the presence or measuring the amount of nucleic acid associated with wild-type ROS1 and ROS1 kinase domains in a tissue sample, wherein RT-PCR or real-time PCR reaction comprises: Forward and reverse primer pairs that specifically hybridize to wild type ROS1 sequences (eg, SEQ ID NOs: 13, 14, 20, and 21), and wild type ROS1 kinase domain sequences (eg, SEQ ID NOs: 4, 5, 7 , 8, 15, 16, 17, and 18) Include the use of one or a combination of forward and reverse primer pairs and detect the presence of nucleic acids associated with one or both of wild-type ROS1 and ROS1 kinase domains in a tissue sample Or measuring the amount, wherein the increase in amplicon relative to a normal control or the presence of the amplicon indicates that the subject has a ROS1-related cancer. It is disclosed in the specification. Absence of amplicon, or amplicon levels equal to normal controls, indicate that the cancer is a ROS1-related cancer. A method for diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, wherein the nucleic acid in the tissue sample is RNA A method comprising: synthesizing cDNA from an RNA sample; performing PCR on the cDNA; and one of wild-type ROS1 and ROS1 kinase domains in a tissue sample, Or detecting the presence of a nucleic acid associated with a combination of both or measuring the amount, wherein an increase in amplicon relative to a normal control, or the presence of an amplicon, the subject has a ROS1-related cancer Indicates. In further embodiments, the disclosed methods can utilize probes complementary to sequences of real-time RT-PCR products (eg, SEQ ID NOs: 6, 8, 19, and 22) or Determining the cycle threshold (Ct) value of wild type ROS1 and wild type ROS1 kinase, a high (Ct) value of wild type ROS1 relative to ROS1 kinase indicates the presence of the fusion. Also disclosed is a method further comprising administering a ROS1 inhibitor to a subject having a cancer susceptible to ROS1 inhibitor treatment when the cancer is determined to be a ROS1-related cancer. Conversely, if the cancer is not an rOS1-related cancer, the method can further comprise treating the patient with the cancer using a form of treatment other than a ROS1 inhibitor.

  In a further alternative method of diagnosing a ROS1-related cancer in a subject with cancer, the ROS1 fusion uses only a forward primer that binds to the ROS1 fusion partner in the first reaction, followed by the first reaction. In the second reaction, the amplicon of the first reaction is used as a template in a second amplification, probe-based detection, or sequencing reaction, and the probe or primer used is 3 ′ of the fusion breakpoint. Detection of ROS1 in the amplicon of the second reaction is detected by qPCR using two-step detection, indicating fusion and thus ROS1-related cancer. Also, a method of diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, and using a forward primer that binds to the ROS1 fusion partner Performing the first amplification reaction, wherein the amplicon of the first reaction is used as a template for the second amplification reaction, and performing the second amplification reaction after the first reaction. A primer specific for the ROS1 sequence (eg, SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18) at 3 ′ of the fusion breakpoint in the second amplification reaction. And detecting the presence of ROS1 in the amplicon of the second reaction, wherein the detection of ROS1 in the amplicon of the second reaction is a ROS1 fusion, and thus RO The method comprising indicate that it has one associated cancer, it is disclosed. Also disclosed is a method further comprising administering a ROS1 inhibitor to a subject having a cancer susceptible to ROS1 inhibitor treatment when the cancer is determined to be a ROS1-related cancer. Conversely, if the cancer is not an rOS1-related cancer, the method can further comprise treating the patient with the cancer using a form of treatment other than a ROS1 inhibitor.

  Also, a method for diagnosing ROS1-related cancer in a subject, wherein a nucleic acid in a cell hybridizes with a first probe that hybridizes with ROS1 kinase and a ROS1 sequence 3 ′ of the fusion breakpoint of ROS1. Detection of a split locus indicated by a probe that is differentially labeled, isolated, including contact with two probes, indicates the presence of a ROS1 fusion, which indicates the presence of a ROS1-related cancer. The method shown is disclosed. In one aspect, it is disclosed that the probe comprises a sequence that is complementary to the sequence of the real-time RT-PCR product, and the probe has a reporter dye at its end and a quencher dye at its other end. It is further understood that the probes can be selected from any of the probes in Table 7, including but not limited to SEQ ID NOs: 6, 8, 19, and 22.

mRNA detection and quantification The methods disclosed herein include, for example, detection of nucleic acid mutations in the form of point mutations and truncations, or expression of ROS1 fusions, abnormal wild-type ROS1 expression, wild-type ROS1 It relates to the detection of expression or expression of a ROS1 truncation mutant. In detecting these latter expression levels, the method involves detecting either the abundance or presence of mRNA, or both. Accordingly, methods and compositions for diagnosing ROS1-related cancer in a subject comprising measuring the presence or level of mRNA in a subject tissue sample, wherein an increase in the amount of mRNA relative to the subject is associated with ROS1-related cancer. Methods and compositions that indicate presence are disclosed herein.

  A number of widely used procedures exist for detecting and determining the abundance of specific mRNAs in total or poly (A) RNA samples. For example, specific mRNAs can be used for Northern blot analysis, nuclease protection assay (NPA), in situ hybridization (eg, fluorescence in situ hybridization), real-time PCR reaction, or reverse transcription polymerase chain reaction (RT-PCR), and use of microarrays Can be detected.

  Each of these techniques can be used to detect specific RNAs and to accurately determine their expression levels. In general, Northern analysis is the only way to provide information about transcript size, while NPA is the easiest way to examine multiple messages simultaneously. In situ hybridization is used to localize the expression of specific genes within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantifying gene expression.

  Real-time PCR, RT-PCR, and real-time RT-PCR allow detection of RNA transcripts of any gene, regardless of lack of starting material or relative abundance of specific mRNAs. In RT-PCR, the RNA template is cloned into complementary DNA (cDNA) using retroviral reverse transcriptase. The cDNA is then exponentially amplified by PCR using DNA polymerase. Reverse transcription and PCR reactions can occur in the same or different tubes. RT-PCR is somewhat tolerated by degraded RNA. As long as the RNA is intact in the region spanned by the primer, the target is amplified.

  Relative quantitative RT-PCR involves amplifying an internal control simultaneously with the gene of interest. An internal control is used to normalize the sample. Once normalized, a direct comparison of the relative abundance of a particular mRNA can be made across the sample. It is important to select an internal control that has a constant level of expression across all experimental samples (ie not affected by the experimental treatment). Commonly used internal controls (eg, GAPDH, β-actin, cyclophilin) often vary in expression and therefore cannot be suitable internal controls. In addition, the most common internal controls are expressed at much higher levels than the mRNA studied. All products of the PRC reaction must be analyzed in the linear range of amplification because the relative RT-PCR results are meaningful. This is difficult with transcripts whose abundance levels vary widely.

  Competitive RT-PCR is used for absolute quantification. This technique involves designing, synthesizing, and accurately quantifying competitor RNA that can be distinguished from endogenous targets by small differences in size or sequence. A known amount of competitor RNA is added to the experimental sample and RT-PCR is performed. The signal from the endogenous target is compared to the competitor's signal to determine the amount of target present in the sample.

  In one aspect, a method of diagnosing ROS1-related cancer in a subject comprising performing real-time PCR, RT-PCR, or other PCR reaction on a nucleic acid, such as mRNA or DNA, in a test sample of the subject. The polymerase chain reaction comprises a reverse primer capable of specifically hybridizing with one or more ROS1 sequences, and at least one forward primer, wherein an increase in the amount of amplified product relative to the control is ROS1 Methods for indicating the presence of related cancers are disclosed herein. Also, a method of diagnosing ROS1-related cancer in a subject comprising performing FISH on a tissue sample of the subject, wherein the polymerase chain reaction comprises one or more ROS1 sequences on separate sides of the ROS1 fusion breakpoint and Disclosed herein are methods in which the mitotic locus that includes a probe that can specifically hybridize and is indicated by an isolated probe indicates the presence of a ROS1-related cancer. Examples of probes used in this assay include those found in Table 7.

  Since the disclosed method can be used to detect wild-type ROS1, ROS1 fusion, and ROS1 kinase domain activity, it is also a method of diagnosing ROS1-related in a subject or detecting dysregulation of ROS1 kinase. Performing a first RT-PCR on mRNA from a tissue sample of interest, wherein a reverse transcription polymerase chain reaction (RT-PCR) is performed with a ROS1 kinase sequence (eg, SEQ ID NOs: 4, 5, 7, 8). , 15, 16, 17 and 18) and one primer pair that can specifically hybridize, and any fusion breakpoints (ie external such as eg SEQ ID NOs: 13, 14, 20, and 21). At least one protein capable of specifically hybridizing with ROS1 5 'of the wild-type ROS1 site) And determining the amplicon cycle threshold for each primer pair, wherein the amplicon cycle threshold for the wild-type primer pair is greater than the cycle threshold for ROS1 kinase in a statistically significant amount. Disclosed herein is a method comprising high indicating the presence of a fusion or a sudden displacement ROS1. A method of diagnosing ROS1-related cancer in a subject or detecting dysregulation of ROS1 kinase, comprising performing a first RT-PCR reaction on mRNA from a tissue sample of the subject, comprising reverse transcription The polymerase chain reaction (RT-PCR) contains one primer pair that can specifically hybridize with the ROS1 fusion partner 5 'at any fusion breakpoint and amplifies only in the forward direction , Wherein the method comprises using one or more primers that specifically hybridize with the ROS1 sequence at 3 ′ of the fusion breakpoint to amplicons in the first reaction. Detecting the presence or further amplifying, wherein the presence of the ROS1 sequence in the amplicon of the first reaction indicates the presence of the ROS1 fusion. It is.

  Northern analysis is the easiest way to determine the size of transcripts and to identify alternatively spliced transcripts and multigene family members. This can also be used to directly compare the relative abundance of a given message between all samples in the blot. Northern blot procedures are straightforward and provide an opportunity to assess progression at various time points (eg, the integrity of the RNA sample and the efficiency with which the RNA sample migrates to the membrane). RNA samples are first separated by size via agarose gel electrophoresis under denaturing conditions. The RNA is then transferred to the membrane, crosslinked and hybridized with the labeled probe. Non-isotopic or high specific activity radiolabeled probes can be used, including random stimulation, nick translation, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. In addition, sequences with only partial homology (eg, cDNA from different species, or genomic DNA fragments that may contain exons) can be used as probes.

  Nuclease protection assays (NPAs) (including both ribonuclease protection assays and S1 nuclease assays) are sensitive methods for detecting and quantifying specific mRNAs. The basis of NPA is solution hybridization between an antisense probe (radiolabeled or non-isotopic) and an RNA sample. After hybridization, the single stranded non-hybridizing 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 20-30 μg can accommodate up to 100 μg of sample RNA compared to maximum blot hybridization. NPA is also less sensitive to RNA sample degradation than Northern analysis because it is detected only in the region where cleavage overlaps with the probe (probes are typically about 100-400 bases in length). ).

  NPA is a selection method in the simultaneous detection of several RNA species. During solution hybridization and subsequent analysis, the individual probe / target interactions are completely independent of each other. Thus, several RNA targets and appropriate controls can be assayed simultaneously (up to 12 used in the same reaction) as long as the individual probes are of different lengths. NPA is also commonly used to accurately map mRNA ends and intron / exon junctions.

  In situ hybridization (ISH) is a powerful and versatile tool for localizing specific mRNAs in cells or tissues. Unlike Northern analysis and nuclease protection assays, ISH does not require RNA isolation or electrophoretic separation. Probe hybridization occurs in cells or tissues. As cell structure is maintained throughout the procedure, ISH provides information about the location of mRNA within the tissue sample. ISH can be combined with fluorescent markers that reach fluorescence in situ hybridization (FISH).

  The procedure begins by fixing the sample in neutral buffered formalin and embedding the tissue in paraffin. The sample is then cut into thin sections and placed on a microscope slide. (Alternatively, the tissue can be frozen and sliced and post-fixed in paraformaldehyde.) After a series of washes to dewax and rehydrate the sections, proteinase K digestion was performed to Increased availability and then the labeled probe is hybridized with the sample section. Radiolabeled probes are visualized by a dried liquid film on the slide, while nonisotopically labeled probes are conveniently detected by colorimetric or fluorescent reagents.

DNA detection and quantification The methods disclosed herein include, for example, detection of nucleic acid mutations in the form of point mutations and truncations, or expression of ROS1 fusions, abnormal wild-type ROS1 expression, or ROS1 truncation suddenly. It relates to the detection of mutant expression. In detecting these latter expression levels, the method involves detecting either the abundance or presence of mRNA, or both. Alternatively, detection can be directed to the abundance or presence of DNA, such as cDNA. Accordingly, methods and compositions for diagnosing ROS1-related cancer in a subject comprising measuring the presence or level of DNA in a tissue sample of the subject, wherein an increase in the amount of DNA relative to a control is associated with ROS1-related cancer. Methods and compositions that indicate presence are disclosed herein.

  A number of widely used procedures exist for detecting and determining the abundance of specific DNA in a sample. For example, PCR techniques allow for the amplification of trace amounts of target nucleic acid followed by detection. Details of PCR are well described in the art, eg, US Pat. No. 4,683,195, Mullis et al, 4,683,202, Mullis, and 4,965,188. , Mullis et al. In general, the oligonucleotide primer is annealed to the denatured strand of the target nucleic acid, and the primer extension product is formed by polymerization of deoxynucleoside triphosphates by polymerase. A typical PCR method involves repeated cycles of template nucleic acid denaturation, primer annealing, and extension of the annealed primer by the action of a thermostable polymerase. The process results in exponential amplification of the target nucleic acid, thus allowing detection of targets that are present at very low concentrations in the sample. It is understood that there are variations of the PCR methods known in the art that can also be utilized in the disclosed methods, and are considered herein, eg, quantitative PCR (QPCR), microarray, real-time PCT, hot Start PCR, nested PCR, allele specific PCR, and touchdown PCR.

A microarray array is an ordered array of samples that provides a medium that automates the process of aligning known and unknown DNA samples according to base-pairing rules and identifying unknowns. Array experiments can use common assay systems such as microplates or standard blot membranes, can be created manually, or samples can be deposited using robotics. In general, an array is described as a macroarray or a microarray, the difference being the size of the sample spot. Macroarrays contain sample spot sizes greater than about 300 microns and can be easily imaged by existing gel and blot scanners. Microarray sample spot sizes are 300 microns or less, but typically are less than 200 microns in diameter, and these arrays usually contain thousands of spots. Microarrays require specialized robotics and / or imagers that are not generally commercially available as complete systems. The terms used in the literature to describe this technique include biochip, DNA chip, DNA microarray, GENECHIP® (Affymetrix, Inc., high density, oligonucleotide-based DNA array And) and gene arrays.

  DNA microarrays or DNA chips are made by high-speed robotics, generally on glass or nylon substrates, to determine complementary binding using probes with known identities, and thus large amounts of parallel gene expression and Enables research on gene discovery. Experiments with a single DNA chip can provide information on thousands of genes simultaneously. It is contemplated herein that the disclosed microarrays can be used to monitor gene expression, disease diagnosis, gene discovery, drug discovery (pharmacogenomics), and toxicology research or toxicogenomics.

  There are two variations in DNA microarray technology with respect to the properties of aligned DNA sequences with known identity. Type I microarrays are immobilized on a solid surface such as glass using robotic spot formation and probe cDNA (500-5,000 bases long) exposed to a series of targets separately or as a mixture. )including. This method is traditionally called a DNA microarray. A type I microarray immobilizes localized copies of one or more polynucleotide sequences, preferably copies of a single polynucleotide sequence, into a plurality of defined regions of the substrate surface. Polynucleotide means a chain of nucleotides ranging from 5 to 10,000 nucleotides. Immobilized copies of these polynucleotide sequences are suitable for use as probes in hybridization experiments.

  To prepare beads coated with immobilized probes, the beads are immersed in a solution containing the desired probe sequence and then immobilized on the beads by a covalent or non-covalent method. Alternatively, if a probe is immobilized on a rod, a given probe can be spotted on a defined area of the rod. A typical dispenser includes a micropipette that delivers a solution to a substrate by a robotic system that controls the position of the micropipette relative to the substrate. The distributor can be multiple so that the reagents can be delivered to the reaction area simultaneously. In one embodiment, the microarray is formed by the use of inkjet technology based on piezoelectric effects, in which a capillary containing the liquid of interest, such as an oligonucleotide synthesis reagent, is surrounded by an adapter. The charge delivered across the adapter causes the adapter to expand at a different rate than the tube and pushes a droplet of liquid toward the substrate.

  The sample can be any sample containing the polynucleotide of interest (polynucleotide target) and can be any bodily fluid (blood, urine, saliva, mucus, gastric fluid, etc.), cultured cells, biopsy, or other tissue preparation. Can be obtained from DNA or RNA can be isolated from the sample according to any of a number of methods well known to those skilled in the art. In one embodiment, total RNA is isolated using TRIzol total RNA isolation reagent (Life Technologies, Inc., Rockville, Md.), And RNA is subjected to oligo d (T) column chromatography or glass beads. Isolated using. After hybridization and processing, the resulting hybridization signal should accurately reflect the amount of control target polynucleotide sequence added to the sample.

  The plurality of defined regions on the substrate can be arranged in a variety of ways. For example, the regions can be arranged perpendicular or parallel to the length of the casing. Furthermore, the target need not be directly attached to the substrate, but rather can be attached to the substrate via a linker group. The linker group typically can vary from 6 to 50 atoms in length. Linker groups include ethylene glycol oligomers, diamines, diacids, and the like. The reactive group on the substrate surface reacts with one end portion of the linker to attach the linker to the substrate. The other end portion of the linker is then functionalized to attach to the probe.

The sample polynucleotide can be labeled with one or more label moieties to allow detection of the hybridized probe / target polynucleotide complex. The label moiety can include a composition that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunological, electrical, or chemical methods. Labeling moieties include radioisotopes such as ³² P, ³³ P, or ³⁵ S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, magnetic labels, binding enzymes, mass spectrometry Tags, spin labels, electron transfer donors and acceptors, biotin and the like are included.

  Labeling can be performed during polymerase chain reactions and amplification reactions such as in vitro or in vivo transcription reactions. Alternatively, the label moiety can be incorporated after the probe target complex is formed. In one embodiment, biotin is first incorporated during the amplification step described above. After the hybridization reaction, unbound nucleic acid is rinsed away, so that only biotin bound to the target polynucleotide hybridized with the polynucleotide probe remains bound to the substrate. Next, an avidin-binding fluorophore such as avidin-phycoerythrin that binds with high affinity to biotin is added.

  Hybridization causes the polynucleotide probe and complementary target to form a stable duplex via base pairing. Hybridization methods are well known to those skilled in the art. The exact conditions for hybridization can be determined by salt concentration, temperature, and other chemicals and conditions. A variety of additional parameters such as hybridization time, detergent (sodium dodecyl sulfate, SDS) or solvent (formamide) concentration, and inclusion or exclusion of carrier DNA are also well known to those skilled in the art. Additional variations on these conditions will be readily apparent to those skilled in the art.

  Methods for detecting complex formation are well known to those skilled in the art. In one embodiment, the polynucleotide probe is labeled with a fluorescent label, and the level and pattern of complex formation is achieved by fluorescence microscopy, preferably confocal fluorescence microscopy. An argon ion laser excites a fluorescent label and radiation is directed to a photomultiplier tube to detect and quantify the amount of luminescence. The detected signal should be proportional to the amount of probe / target polynucleotide complex at each location of the microarray. A fluorescence microscope can be associated with a computer-operated scanner device to generate a quantitative two-dimensional image of hybridization intensity. The scanned image is examined to determine the abundance / expression level of each hybridization target polynucleotide.

  In different hybridization experiments, polynucleotide targets from two or more different biological samples are labeled with two or more different fluorescent labels having different emission wavelengths. Fluorescent signals are detected separately by different photomultiplier tube sets to detect specific wavelengths. A relative abundance / expression level of the target polynucleotide in more than one sample is obtained. Typically, microarray fluorescence intensity can be normalized to account for variations in hybridization intensity when more than one microarray is used under similar test conditions. In one embodiment, the hybridization intensity of individual polynucleotide probes / target complexes is normalized using an intensity derived from an internal normalization standard contained in each microarray.

  Type II microarrays are arrays of oligonucleotides (20-80 bases long) or peptide nucleic acid (PNA) probes synthesized either in situ (on chip) or by conventional synthesis followed by immobilization on the chip. Including. The array is exposed to labeled sample DNA and hybridized to determine the identity / abundance of complementary sequences. This method, referred to “historically” as a DNA chip, is described in Affymetrix, Inc. Products that are developed by and photolithographically produced under the GENECHIP® trademark are sold.

  The basic concept of using type II arrays for gene expression is simple. Labeled cDNA or cRNA targets derived from experimental sample mRNA are hybridized to nucleic acid probes bound to a solid support. By monitoring the amount of label associated with each DNA position, it is possible to infer the abundance represented by each mRNA species. Hybridization has been used for decades to detect and quantify nucleic acids, but the combination of miniaturization techniques and a large amount of increasing sequence information has made it extremely large to study gene expression. It is spreading.

  Microarray fabrication can begin with a 5 square inch quartz wafer. First, the quartz is cleaned to ensure uniform hydroxylation across the surface. Quartz is naturally hydroxylated, thus providing a substrate that is excellent for binding chemicals such as linker molecules that are later used to locate probes in the array.

  The wafer is placed in a bath of silane that reacts with the hydroxyl groups of quartz and forms a matrix of covalent molecules. The distance between these silane molecules determines the probe packing density and allows the array to hold more than 500,000 probe positions or features within a range of only 1.28 square centimeters. Each of these features has millions of identical DNA molecules. The silane film provides a uniform hydroxyl density that initiates probe assembly. Linker molecules attached to the silane matrix provide a surface that can be spatially activated by light.

  Probe synthesis occurs in parallel, resulting in the addition of A, C, T, or G nucleotides to multiple growing strands. In order to determine which oligonucleotide strand receives the nucleotide in each step, a photolithographic mask with an 18-20 square micron window corresponding to the size of the individual features is placed over the coated wafer. The windows are distributed throughout the mask based on the desired arrangement of each probe. When UV light is illuminated from above the mask in the first step of the synthesis, the exposed linker is deprotected and made available for nucleotide coupling.

  Once the desired feature is activated, a solution containing a single type of deoxynucleotide having a removable protecting group is fused to the surface of the wafer. Nucleic acids bind to the activated linker and initiate the synthesis process.

  Although each position in the oligonucleotide sequence is occupied by 1 to 4 nucleotides, this can lead to the appearance of 25 x 4 or 100 different masks per wafer, but the synthesis process must meet this requirement. Can be designed to significantly reduce. Algorithms that help minimize the use of masks show how to optimally coordinate probe growth by adjusting the synthesis speed of individual probes and seeing when the same mask can be used multiple times. calculate.

  Several key factors in selection and design are common to all microarray manufacturing, regardless of the intended application. Strategies for optimizing probe hybridization are always included, for example, in the process of probe selection. Hybridization under specific pH, salt, and temperature conditions can be optimized using empirical rules that take into account the melting temperature and correlate with the desired hybridization behavior.

  In order to obtain a complete picture of the activity of the gene, some probes are selected from regions shared by multiple splices or polyadenylation variants. In other cases, unique probes that distinguish between variants are preferred. The distance between probes is also included as an element in the selection process.

  A series of different strategies are used to select probes for genotyping arrays that rely on multiple probes to probe individual nucleotides in the sequence. Target base identity can be estimated using four identical probes, each containing one of the four possible bases, differing only in the target position.

  Alternatively, the presence of a consensus sequence can be examined by the use of one or two probes representing a particular allele. To genotype heterozygous or gene-mixed samples, an array with many probes can be created to provide redundancy information and provide a clear genetic determination. In addition, gene probes can be used for some applications to maximize flexibility. For example, some probe arrays allow separation and analysis of individual reaction products from complex mixtures, such as those used in some protocols to identify single nucleotide polymorphisms (SNPs) To do.

Real-time PCR
In one aspect, the disclosed diagnostic methods can be performed by real-time PCR reaction methods. For example, in one aspect, a method of detecting the presence of a ROS1-related fusion, or a method of diagnosing ROS1-related cancer in a subject or detecting dysregulation of ROS1 kinase, wherein mRNA is expressed in a subject tissue sample. One primer pair that allows the reverse transcription polymerase chain reaction (RT-PCR) to specifically hybridize with the ROS1 kinase sequence, and optional fusion cleavage. Including at least one primer pair capable of specifically hybridizing to ROS1 at 5 'of the point (ie, external wild type ROS1 site) and determining the amplicon cycle threshold of each primer pair Where the amplicon cycle threshold of the wild-type primer pair is statistically significant In higher than the cycle threshold ROS1 kinases, and indicate the presence of a fusion or sudden displacements ROS1, the method comprising is disclosed herein.

Real-time PCR, in contrast to endpoint detection, monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (ie, in real time). Real time progression of the reaction can be seen in several systems. Real-time PCR does not detect the size of the amplicon, so differentiation of DNA and cDNA amplification is not possible, but is not affected by non-specific amplification unless SYBR green is used. Real-time PCR quantification eliminates post-PCR processing of PCR products. This helps to increase throughput and reduce the chance of carrying over contamination. Real-time PCR also provides a wide dynamic range up to 10 ⁷ times. The dynamic range of any assay determines how the target concentration changes and can still be quantified. A wide dynamic range means that a wide range of ratios between target and normalization group can be assayed with equal sensitivity and specificity. Therefore, the wider the dynamic range, the more accurate the quantification. When combined with RT-PCR, the real-time RT-PCR reaction reduces the time required to measure the amount of amplicon by providing amplicon visualization as the amplification process proceeds.

  Real-time PCR systems are based on the detection and quantification of fluorescent reporters. This signal increases in direct proportion to the amount of PCR product in the reaction. By recording the amount of fluorescent emission in each cycle, it is possible to monitor the PCR reaction in logarithmic phase where the first significant increase in the amount of PCR product correlates with the initial amount of target template. The higher the starting copy number of the nucleic acid target, the earlier a significant increase in fluorescence is observed. A significant increase in fluorescence over the baseline value measured in 3-15 cycles can indicate the detection of accumulated PCR products.

The fixed fluorescence threshold is set significantly above the baseline and can be changed by the operator. The parameter C _T (threshold cycle) is defined as the number of cycles where the fluorescence emission exceeds a fixed threshold.

  There are three main fluorescence monitoring systems for DNA amplification: (1) hydrolysis probes, (2) hybridization probes, and (3) DNA binders. Hydrolysis probes include TaqMan probes, molecular beacons, and scorpions. These use the fluorogenic 5 'exonuclease activity of Taq polymerase to measure the amount of target sequence in a cDNA sample.

  TaqMan probes are usually more than primers (20-30 base lengths with Tm values above 10 ° C.) containing fluorescent dyes at the 5 ′ base and typically quenching dyes (usually TAMRA) at the 3 ′ base. Long oligonucleotide. When illuminated, the excited fluorescent dye transfers energy to nearby quenching dye molecules rather than fluorescence (this is called FRET = Forster or fluorescence resonance energy transfer). Thus, the proximity of the reporter and quencher prevents any fluorescence emission, while the probe is intact. The TaqMan probe is designed to anneal to the internal region of the PCR product. When the polymerase replicates the template to which the TaqMan probe is bound, its 5 'exonuclease activity cleaves the probe. This terminates the activity of the quencher (no FRET) and the reporter dye begins to emit fluorescence, which increases in proportion to the rate of probe cleavage in each cycle. PCR product accumulation is detected by monitoring the increase in fluorescence of the reporter dye (note that the primer is not labeled). The TaqMan assay uses global thermal cycling parameters and PCR reaction conditions. Since cleavage occurs only when the probe hybridizes with the target, the detected source of fluorescence is specific amplification. The process of hybridization and cleavage does not interfere with the exponential accumulation of product. One particular requirement of the fluorogenic probe is the absence of G at the 5 'end. The “G” adjacent to the reporter dye can quench the reporter fluorescence even after cleavage.

  Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL) at either end, but they are designed to fit the hairpin structure, while in solution It is designed to bring the fluorescent dye and the quencher in close proximity so that they are released in and FRET occurs. They have two arms with complementary sequences that form a very stable hybrid or stem. The proximity of the reporter and quencher in this hairpin configuration suppresses reporter fluorescence. If the beacon hybridizes with the target during the annealing step, the reporter dye is separated from the quencher and the reporter fluoresces (FRET does not occur). Molecular beacons must remain intact during PCR and must bind to the target in every cycle of fluorescence emission. This correlates with the amount of available PCR product. All real-time PCR chemicals can be generated by designing each probe / beacon with a spectrally unique fluorescence / quenching pair as long as the platform is suitable for melting curve analysis when SYBR Green is used. Detection of multiple DNA species (multiplexed). Multiplexing allows the target (s) and endogenous control to be amplified in a single tube.

  With a scorpion probe, sequence specific stimulation and PCR product detection is achieved through the use of a single oligonucleotide. The scorpion probe maintains a stem-loop configuration in a non-hybridized state. The phosphor is bound to the 5 'end and quenched by the moiety bound to the 3' end. The 3 'portion of the stem also contains a sequence that is complementary to the primer extension product. The sequence is attached to the 5 'end of a specific primer via a non-amplifiable monomer. After the scorpion primer is extended, specific probe sequences can bind to complement within the extended amplicon, thereby opening the hairpin loop. This prevents the fluorescence from quenching and a signal is observed.

  Another alternative is double-stranded DNA binding dye chemistry, which can be achieved by using non-sequence specific fluorescent intercalating agents (SYBR-Green I or ethidium bromide) (non-specific amplification and primer-dimer Quantify amplicon products (including body complexes). This does not bind to ssDNA. SYBR Green is a fluorescence-generating minor groove binding dye that exhibits little fluorescence in solution but emits a strong fluorescent signal when bound to double-stranded DNA. The drawbacks of real-time PCR based on SYBR Green include the need for extensive optimization. Furthermore, non-specific amplification requires a follow-up assay (melting curve or dissociation analysis) for amplicon identification. The method has been used for HFE-C282Y genotyping. Another controllable problem is that the longer the amplicon produces a stronger signal (when combined with other factors, this causes saturation in the CDC camera, see below). Usually, SYBR green is used for a single reaction, but when combined with a melting point analysis, it can be used for multiple reactions.

The threshold cycle or C _T value is the cycle at which a significant increase in ΔRn is first detected (definition of ΔRn, etc., see below). The threshold cycle is when the system begins to detect an increase in signal associated with exponential growth of the PCR product in the log-linear phase. This phase provides the most useful information about the reaction (which is of course more important than the endpoint). The slope of the log linear phase is a reflection of amplification efficiency. The efficiency of the reaction (Eff) can be calculated by the formula: Eff = 10 ^{(−1 / gradient)} −1. The efficiency of PCR should be 90-100% (3.6>slope> 3.1). A number of variables can affect the efficiency of PCR. These factors include amplicon length, secondary structure, and primer quality. Although valid data outside the efficiency range can be obtained, qRT-PCR should be further optimized or alternative amplicons should be designed. In order for the slope to be a measure of actual amplification (not signal drift), the presence of an inflection point is necessary. This is the point on the growth curve when the log-linear phase begins. This also represents the maximum rate of change along the growth curve. (Drift signal is characterized as a gradual increase or decrease in fluorescence without amplification product.) An important parameter to quantify a C _T. The greater the initial amount of genomic DNA, the earlier the accumulated product is detected by the PCR process and the lower the _CT value. The threshold is placed within an exponential increasing phase (which appears linear by logarithmic transformation) beyond any baseline activity. Some software allows the cycle threshold (C _T ) to be determined by mathematical analysis of the growth curve. This provides good run-to-run reproducibility. A _CT value of 40 means no amplification, and this value cannot be included in the calculation. In addition to being used to quantify, C _T value can be used to quantify analyzed as acceptance means.

  Multiplex TaqMan assays can be performed using multiple dyes with different emission wavelengths. Dyes that can be used for this purpose are FAM, TET, VIC and JOE (the most expensive). TAMRA is secured as a probe quencher and ROX as a passive reference. For optimal results, a combination of FAM (target) and VIC (intrinsic control) is recommended (these have the greatest difference in maximum emission), while JOE should not be combined with VIC. If the dye phase is not selected correctly, it is important that the machine still reads the spectra of other dyes. For example, when both VIC and FAM emit fluorescence in a similar range to each other, and a single dye is used, the well should be correctly labeled. In the case of multiplexing, spectral correction for post-run analysis should be turned on (ABI 7700: Instrument / Diagnostics / Advanced Options / Micellaneous). Activating spectral correction improves dye spectral resolution.

Nested PCR
The disclosed method can further utilize nested PCR. Nested PCR increases the specificity of DNA amplification by reducing the background due to non-specific amplification of DNA. Two sets of primers are used for two consecutive PCRs. In the first reaction, a pair of primers is used to generate a DNA product, which, in addition to the intended target, is still composed of non-specifically amplified DNA fragments. The product (s) is then used in a second PCR using a set of primers whose binding sites are completely or partially different from the respective primers used in the first reaction and located 3 '. Is done. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but requires more detailed knowledge of the target sequence.

  Accordingly, in one aspect, a method of diagnosing ROS1-related cancer in a subject comprising performing a PCR reaction on DNA in a test sample of the subject, wherein the PCR reaction is specifically hybridized with one or more ROS1 sequences. Disclosed herein are reverse primers that can be formed, and methods that include at least one forward primer, wherein an increase in the amount of amplification product relative to a control indicates the presence of a ROS1-related cancer.

Primers and probes As used herein, a “primer” is a subset of probes that can support a type of enzymatic manipulation and can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. is there. Primers can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with enzymatic manipulation.

  As used herein, a “probe” is a molecule that can interact with a target nucleic acid, typically in a sequence specific manner, for example via hybridization. Nucleic acid hybridization is well understood in the art and discussed herein. Typically, probes can be made from any combination of nucleotides, or nucleotide derivatives or analogs available in the art. As used herein, a probe can include a reporter dye at its end and a quencher dye at its other end.

  Primers and probes capable of interacting with the disclosed nucleic acids, such as SEQ ID NO: 1, or their complements such as those listed in Table 7 (eg, sequences that are primers that interact with SEQ ID NO: 1) Nos. 4, 5, 7, 8, 12, 13, 14, 15, 16, 17, 18, 20, and 21, and SEQ ID NO: 6, 8, 19, and 22 that are probes that interact with SEQ ID NO: 1) A composition comprising is disclosed. The disclosed plumers and probes of the disclosed methods for diagnosing ROS1-related cancers disclosed herein and methods for determining whether a cancer is sensitive or resistant to treatment with a ROS1 inhibitor Can be used for either. In certain embodiments, primers are used to support nucleic acid extension reactions, nucleic acid replication reactions, and / or nucleic acid amplification reactions. Typically, primers can be extended in a sequence specific manner. For extension of a primer by a sequence specific method, the sequence and / or composition of the nucleic acid molecule to which the primer hybridizes or otherwise is related to the composition or sequence of the product produced by the extension of the primer. Any method of indicating or influencing is included. Thus, primer extension by sequence-specific methods includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions for amplifying primers in a sequence specific manner have been disclosed. In certain embodiments, primers are used for DNA amplification reactions such as PCR or direct sequencing. In certain embodiments, it is understood that the primer can also be extended by the use of non-enzymatic techniques, for example, the nucleotide or oligonucleotide used to extend the primer chemically reacts to make the primer sequence specific. It is modified to extend in a simple manner. Typically, a disclosed primer hybridizes to a disclosed nucleic acid or region of a nucleic acid, or hybridizes to the complement of a nucleic acid or region of a nucleic acid. As an example of the use of a primer, one or more primers can be used to create an extension product templated from or by the first nucleic acid.

  The size of the primer or probe that interacts with the nucleic acid can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification, or simple hybridization of the probe or primer. Typical primers or probes are 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, 99, 100, 125 , 150, 175, 200, 225, 250, 275, 00, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, It is 2750, 3000, 3500, or 4000 nucleotides long.

  In other embodiments, the primer or probe is 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, 27 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500 2750, 3000, 3500, or 4000 nucleotides in length.

  Primers of the nucleic acid of interest are typically used to produce extension products and / or other replication or amplification products that contain regions of the nucleic acid of interest. The size of the product can be such that the size can be accurately determined within 3, or 2 or 1 nucleotide.

  In certain embodiments, the product is, 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, 50,600,650,700,750,800,850,900,950,1000,1250,1500,1750,2000,2250,2500,2750,3000,3500 or may be 4000 nucleotides in length.

  In other embodiments, the product is, for example, 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, 60 It may be 650,700,750,800,850,900,950,1000,1250,1500,1750,2000,2250,2500,2750,3000,3500 or 4000 nucleotides or less in length.

  Accordingly, it is understood that the disclosed RT-PCR and PCR reactions require at least one forward primer and / or at least one reverse primer to amplify the target nucleic acid and are considered herein. Is done. Disclosed herein is that the forward primer is, for example, an intracellular (ie, 3 ′ of ROS1 breakpoint) ROS1 primer, or a ROS1 kinase such as SEQ ID NO: 4, 7, 15, 17, or 20. It can be a forward primer. The reverse primer can be, for example, a ROS1 kinase reverse primer, eg, SEQ ID NO: 5, 8, 12, 16, 18, or 21. It is understood that the method can include at least one forward primer and a reverse primer. Accordingly, a method of diagnosing a ROS1-related cancer in a subject comprising performing real-time PCR, RT-PCR, or other PCR reactions on nucleic acids such as mRNA or DNA in a subject tissue sample, comprising polymerase chain The reaction (RT-PCR) comprises one or more reverse primers and at least one forward primer capable of specifically hybridizing with one or more ROS1 sequences, wherein the forward primer is for example CD74-ROS1 Primer (eg, SEQ ID NO: 2 and 10), FIG-ROS1 primer, SLC34A2-ROS (short) primer, SLC34A2-ROS (short and long) primer (eg, SEQ ID NO: 3 and 11), SLC34A2-ROS (long) primer , ROS1 kinase A wild-type ROS1 primer (eg, SEQ ID NO: 13 or 20) comprising an immer (eg, SEQ ID NO: 4, 7, 15, or 17), and / or a wild-type ROS1 primer that binds 5 ′ of the fusion junction Thus, disclosed herein is a method wherein an increase in the amount of amplification product of ROS1 kinase relative to a control that does not have a corresponding increase in wild-type ROS1 indicates the presence of a ROS1-related cancer. When primers that bind to the ROS1 fusion partner are used, the presence of a known ROS1 fusion-associated cancer is indicated by an increase in the amount of amplification product of ROS1 kinase and an increase in amplification of the fusion partner relative to the control. Amplification of the fusion partner includes a reverse primer that hybridizes with ROS1 at the 3 ′ breakpoint and a forward primer that hybridizes with the fusion partner at the 5 ′ breakpoint or only with the fusion partner. It is understood that this can occur through the use of primer pairs. In a method using a primer pair consisting of a forward primer that binds to a fusion partner and a reverse primer that binds to ROS1 at 3 'of the fusion breakpoint, detection of both ROS1 and the fusion partner in the amplicon, between the primers Detection of an amplicon of the appropriate size for the amplicon containing the fusion, or detection of a larger amount of amplicon than the control, using only forward or reverse primers, indicates a ROS1 fusion. When a primer pair is used that has a forward primer that binds to the fusion partner, the primer pair that hybridizes with ROS1 at the 5 ′ breakpoint serves as a control for misidentification of fusion events and determination of unknown fusions. Alternatively, in one aspect, the methods disclosed herein use only a forward primer associated with a fusion partner in a first reaction to amplify nucleic acid from a subject, and fusion cleavage. A primer pair bound to ROS1 at point 3 'can be used to amplify the amplicon of the first reaction, wherein the amplicon containing the ROS1 sequence indicates the presence of a ROS1-related cancer.

  In yet another aspect, a method of diagnosing ROS1-related cancer in a subject comprising performing real-time PCR, RT-PCR, or other PCR reaction on a nucleic acid such as mRNA or DNA in a subject test sample. A polymerase chain reaction comprising one or more reverse primers capable of specifically hybridizing with one or more ROS1 sequences, and at least one forward primer, wherein the forward primer is an external wild type ROS1 Disclosed herein is a method that is a primer (ie, 5 'of the fusion breakpoint) and an increase in the amount of amplification product relative to the control indicates the presence of a ROS1-related cancer.

  It is understood and contemplated herein that there are situations where it may be advantageous to utilize more than one primer pair to detect the presence of a fusion, truncation, or overexpression mutation. Such real-time PCR, RT-PCR, or other PCR reactions can be performed separately or as a single reaction. If multiple primer pairs are arranged for a single reaction, this is called “multiplex PCR”. For example, the reaction can include a wild type ROS1 primer pair with a reverse primer as well as a ROS1 kinase primer pair with the same reverse primer. Accordingly, a method of diagnosing ROS1-related cancer in a subject comprising performing a real-time or RT-PCR reaction on mRNA in a test sample of the subject, wherein the polymerase chain reaction is specifically associated with one or more ROS1 sequences. A method comprising a reverse primer capable of hybridizing, and at least two forward primers, wherein an increase in the amount of amplification product relative to a control in ROS1 kinase but not both primers, indicates the presence of a ROS1-related cancer. , As disclosed herein. A method involving at least three forward primers is also disclosed. It is understood and contemplated herein that any combination of two or more or three or more forward primers disclosed herein can be used in a multiplex reaction. Thus, for example, disclosed herein are diagnostic methods in which the forward primer is a ROS1 fusion primer (eg, CD74-ROS1, FIG-ROS1, or SLC34-ROS1), a ROS1 kinase primer, and a wild type ROS1 primer. Has been.

  A method for diagnosing ROS1-related cancer in a subject having cancer, comprising obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, performing a nucleic acid amplification process on the nucleic acid, tissue Detecting the presence or measuring the amount of nucleic acid associated with one or a combination of one or both of wild-type ROS1 and ROS1 kinase domains in a sample, wherein the amplification process comprises PCR to cDNA or mRNA Real-time PCR, RT-PCR, or real-time RT-PCR against which forward, reverse primers in which the PCR, RT-PCR, real-time PCR, or real-time RT-PCR reaction specifically hybridizes with the wild-type ROS1 sequence Pair (eg, the order of SEQ ID NOs: 13 and 20 Primers and primers that bind to the extracellular domain of ROS1, such as the reverse primers of SEQ ID NOS: 14 and 21, and forward and reverse primer pairs that specifically hybridize to the wild type ROS1 kinase domain sequence (eg, A method comprising the use of a forward primer of SEQ ID NO: 4, 7, 13, 15, 17, or 20 and (a reverse primer of SEQ ID NO: 5, 8, 12, 14, 18, or 21) is described herein. It is understood that any combination of forward and reverse primers can be used for a particular wild type ROS1 or ROS1 kinase domain, eg, the forward direction of wild type ROS1 (eg, ECD of ROS1) and The reverse primer pair consists of SEQ ID NO: 13 and 14, SEQ ID NO: 13 and 21, No. 20 and 21, or SEQ ID NOs: 20 and 14. Similarly, primers specific for the kinase domain of ROS1 are, for example, SEQ ID NO: 4 and 5, SEQ ID NO: 4 and 8, SEQ ID NO: 4 and 14, sequence SEQ ID NO: 4 and 16, SEQ ID NO: 4 and 18, SEQ ID NO: 4 and 21, SEQ ID NO: 7 and 5, SEQ ID NO: 7 and 8, SEQ ID NO: 7 and 14, SEQ ID NO: 7 and 16, SEQ ID NO: 7 and 18, SEQ ID NO: 7 And 21, SEQ ID NO: 13 and 5, SEQ ID NO: 13 and 8, SEQ ID NO: 13 and 14, SEQ ID NO: 13 and 16, SEQ ID NO: 13 and 18, SEQ ID NO: 13 and 21, SEQ ID NO: 15 and 5, SEQ ID NO: 15 and 8 , SEQ ID NO: 15 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 15 and 18, SEQ ID NO: 15 and 21, sequence SEQ ID NO: 17 and 5, SEQ ID NO: 17 and 8, SEQ ID NO: 17 and 14, SEQ ID NO: 17 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 17 and 21, SEQ ID NO: 20 and 5, SEQ ID NO: 20 and 8, SEQ ID NO: 20 And 14, 14, SEQ ID NOs: 20 and 16, SEQ ID NOs: 20 and 18, and SEQ ID NOs: 20 and 21, can be any combination of forward and reverse primers for the kinase domain.

Fluorescent Change Probes and Primers Fluorescent Change Probes and Fluorescent Change Primers are probes, primers, or primers that are detected, assayed, or replicated, and all probes with changes in fluorescence intensity or wavelength based on changes in nucleic acid morphology or structure. Means primer. Examples of fluorescent change probes and primers include, but are not limited to, molecular beacons, Amplifluor, FRET probes, cleavable FRET probes, TaqMan probes, scorpion primers, triple molecular beacons or triple FRET probes, Fluorescent water soluble conjugated polymers, PNA probes, and QPNA probes are included.

  Fluorescent change probes and primers can be classified according to their structure and / or function. Fluorescence change probes include hairpin quenching probes, cleavage quenching probes, cleavage activation probes, and fluorescence activation probes. Fluorescent change primers include stem quenching primers and hairpin quenching primers.

  A hairpin quenching probe is a probe that, when not bound to a target sequence, forms a hairpin structure (typically a loop) that brings the fluorescent label and the quenching moiety into close proximity to quench the fluorescence from the label. When the probe binds to the target sequence, the stem breaks and the quenching moiety is no longer in close proximity to the fluorescent label and fluorescence increases. Examples of hairpin quenching probes are molecular beacons, fluorescent triple oligos, triple molecular beacons, triple FRET probes, and QPNA probes.

  A cleavage activated probe is a probe whose fluorescence increases upon cleavage of the probe. The cleavage activation probe can include a fluorescent label and a quenching moiety in close proximity so that the fluorescence from the label is quenched. When the probe is sheared or digested (typically due to the 5'-3 'exonuclease activity of the polymerase during amplification), the quenching moiety is no longer in close proximity to the fluorescent label and fluorescence increases. . The TaqMan probe is an example of a cleavage activation probe.

  A cleavage-quenched probe is a probe whose fluorescence decreases or changes upon probe cleavage. The cleavage quencher probe can include an acceptor fluorescent label and a donor moiety such that fluorescence acceptor energy transfer from donor to acceptor causes the acceptor to fluoresce when the acceptor and donor are in close proximity. . Thus, the probe fluoresces when hybridized with the target sequence. If the probe is sheared or digested (typically due to the 5'-3 'exonuclease activity of the polymerase during amplification), the donor moiety is no longer in close proximity to the acceptor fluorescent label and accepts Fluorescence from the body decreases. If the donor moiety is itself a fluorescent label, energy can be emitted as fluorescence (typically at a different wavelength than the acceptor fluorescence) if not in proximity to the acceptor. The overall effect is a reduction in acceptor fluorescence and an increase in donor fluorescence. In the case of a cleavage quencher probe, the donor fluorescence is equivalent to the fluorescence generated by the cleavage activation probe, the acceptor is the quenching moiety and the donor is the fluorescent label. A cleavable FRET (fluorescence resonance energy transfer) probe is an example of a cleaved quenching probe.

  A fluorescence activated probe is a probe or probe pair in which fluorescence increases or changes upon hybridization of the probe and target sequence. Fluorescently activated probes have an acceptor fluorescent label and a donor moiety that, when the acceptor and donor are in close proximity (when the probe is hybridized to the target sequence), donor to acceptor fluorescence resonance. Energy transfer can be included to cause the receptor to fluoresce. A fluorescence activated probe is typically a probe pair designed to hybridize to adjacent sequences so that the acceptor and donor are in close proximity. A fluorescently activated probe is not in close proximity to the donor and acceptor when the probe is not hybridized to the target sequence, but is in close proximity to the donor and acceptor when the probe is hybridized to the target sequence. As well as a single probe containing both a donor and an acceptor. This can be achieved, for example, by placing the donor and acceptor at opposite ends of the probe and placing a target complementary sequence at each end of the probe, where the target complementary sequence is complementary to the adjacent sequence of the target sequence. Is. When the donor portion of a fluorescently activated probe is itself a fluorescent label, when not in close proximity to the acceptor (ie, when the probe is not hybridized to the target sequence) as fluorescence (typically acceptor Energy can be emitted (at a wavelength different from that of the fluorescence). When the probe hybridizes to the target sequence, the overall effect is a reduction in donor fluorescence and an increase in acceptor fluorescence. A FRET probe is an example of a fluorescence activated probe.

  When the stem quencher primer does not hybridize with a complementary sequence, it forms a stem structure (either an intramolecular stem structure or an intermolecular stem structure) that brings the fluorescent label and the quenching moiety into close proximity so that the fluorescence from the label is quenched. It is a primer. When the primer binds to the complementary sequence, the stem is split and the quenching moiety is no longer in close proximity to the fluorescent label and fluorescence increases. In the disclosed method, stem-quenched primers are used as primers for nucleic acid synthesis and are therefore incorporated into the synthesized or amplified nucleic acid. Examples of stem quenching primers are peptide nucleic acid quenching primers and hairpin quenching primers.

  A peptide nucleic acid quenching primer is a primer that forms a stem structure in association with a peptide nucleic acid quencher or a peptide nucleic acid fluorophore. The primer includes a fluorescent label or quenching moiety and is associated with either a peptide nucleic acid quencher or a peptide nucleic acid fluorophore, respectively. This places the fluorescent label close to the quenching portion. When the primer is replicated, the peptide nucleic acid is replaced, thus allowing the fluorescent label to produce a fluorescent signal.

  A hairpin quenching primer is a primer that forms a hairpin structure (typically a loop) that, when not hybridized to a complementary sequence, brings the fluorescent label and the quenching moiety into close proximity to quench the fluorescence from the label. When the primer binds to the complementary sequence, the stem breaks and the quenching moiety is no longer in close proximity to the fluorescent label and fluorescence increases. Hairpin quenched primers are typically used as primers for nucleic acid synthesis and are therefore incorporated into synthetic or amplified nucleic acids. Examples of hairpin quenching primers are Amplifluor primers and Scorpion primers.

  A cleavage activation primer is similar to a cleavage activation probe except that it is a primer that is incorporated into the replication strand and then cleaved later.

Labels Labels can be incorporated directly into nucleotides and nucleic acids or coupled to detection molecules such as probes and primers to aid in the detection and quantification of nucleic acids produced using the disclosed methods. Can do. As used herein, a label is any molecule that can be directly or indirectly associated with a nucleotide or nucleic acid and that directly or indirectly provides a measurable and detectable signal. Many such labels for incorporation into nucleotides and nucleic acids or for binding to nucleic acid probes are known to those skilled in the art. Examples of labels suitable for use in the disclosed methods are radioisotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands. Fluorescent labels, particularly in the context of fluorescent change probes and primers, are useful for real-time detection of amplification.

  Examples of suitable fluorescent labels include fluorescein isothiocyanate (FITC), 5,6-carboxymethylfluorescein, Texas Red, nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride , Rhodamine, amino-methylcoumarin (AMCA), eosin, erythrosine, BODIPY (registered trademark), CASCADE BLUE (registered trademark), OREGON GREEN (registered trademark), pyrene, lissamine, xanthene, acridine, oxazine, phycoerythrin, quantum dye ( Macrocyclic chelates of lanthanide ions such as TM), fluorescent energy transfer dyes such as thiazole orange-ethidium heterodimers, and cyanine dyes Cy3, Cy3.5, Cy5, Cy5 5, and a Cy7. Examples of other specific fluorescent labels include 3-hydroxypyrene 5,8,10-trisulfonic acid, 5-hydroxytryptamine (5-HT), acid fuchsin, alizarin complexone, alizarin red, allophycocyanin, aminocoumarin. , Anthroyl stearate, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7GLL, Atabulin, Auramin, Aurophosphine, Aurophosphine G, BAO9 (Bisaminophenyloxadiazole), BCECF, Sulfuric Acid Berberine, Bisbenzamide, Blancoform FFG solution, Blancofor SV, Bodipy F1, Brilliant sulfoflavin FF, Calcien blue, Calcium green, Calcoflor RW solution, Ca Coflor white, Calcohol white ABT solution, Calcohol white standard solution, carbostyril, cascade yellow, catecholamine, quinacrine, coliphosphine O, coumarin-phalloidin, CY3.18, CY5.18, CY7, dance (1- Dimethylaminonaphthalin 5 sulfonic acid), dancer (diaminonaphthyl sulfonic acid), dansyl NH-CH3, diaminophenyloxadiazole (DAO), dimethylamino-5-sulfonic acid, dipyrromethene boron difluoride, diphenyl brilliant flavin 7GFF, Dopamine, erythrosine ITC, euclicin, FIF (formaldehyde-induced fluorescence), furazo orange, fluor 3, fluorescamine, fura-2, genacryl brilliant red B, Acrylic Brilliant Yellow 10GF, Genacrylic Pink 3G, Genacrylic Yellow 5GF, Glucaric Acid, Granular Blue, Hematoporphyrin, India-1, Intra White Cf Liquid, Leukophor PAF, Leukophor SF, Leukophor WS, Lisamin Rhodamine B200 (RD200) , Lucifer Yellow CH, Lucifer Yellow VS, Magdara Red, Marina Blue, Maxilon Brilliant Flavin 10GFF, Maxilon Brilliant Flavin 8GFF, MPS (Methyl Green Pyroninstilbene), Mitromycin, NBD amine, Nitrobenzoxazidole, Noradrenaline , Nuclea First Red, Nuclea Yellow, Niro San Brilliant Flavin E8G, Oxadiazole, Passif Dick Blue, Pararosaniline (Woilgen), Holwit AR Solution, Holwit BKL, Holwit Rev, Holwit RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, Phycoerythrin B, Polyazaindenepontochrome Blue Black, Porphyrin, Primulin, Procion Yellow, Pyronin , Pyronin B, pyrozal brilliant flavin 7GF, quinacrine mustard, rhodamine 123, rhodamine 5GLD, rhodamine 6G, rhodamine B, rhodamine B200, rhodamine B extra, rhodamine BB, rhodamine BG, rhodamine WT, serotonin, cebron brilliant red 2B, cebron brilliant Red 4G, Sebron Brilliant Red B, Sebron Orange, Sebron Yellow L, SITS (primulin), SITS (stilbene isothiosulfonic acid), stilbene, snarf 1, sulforhodamine B Can C, sulforhodamine G extra, tetracycline, thiazine red R, thioflavin SCN, thioflavin TCN, thioflavine 5, thiolite, thio Zol Orange, Chinopol CBS, True Blue, Ultralight, Uranine B, Ubitex SFC, Xylene Orange, and XRITC.

  The respective absorption and emission maxima in some of these phosphors are 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 simultaneous detection. Other examples of fluorescein dyes include 6-carboxyfluorescein (6-FAM), 2 ', 4', 1,4, -tetrachlorofluorescein (TET), 2 ', 4', 5 ', 7', 1 2,4-hexachlorofluorescein (HEX), 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7 ', 8'-condensation Phenyl-1,4-dichloro-6-carboxyfluorescein (NED) and 2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) are included. Fluorescent labels can be obtained from a variety of commercial suppliers including Amersham Pharmacia Biotech, Piscataway, NJ, Molecular Probes, Eugene, OR, and Research Organics, Cleveland, Ohio.

  Additional labels of interest include those that provide a signal only when the probe to which they are associated is specifically bound to the target molecule, such labels include Tyagi & Kramer, Nature Biotechnology (1996). ) 14: 303 and European Patent No. 0 070 685 B1 include “molecular beacons”. Other labels of interest include those described in US Pat. No. 5,563,037, which is incorporated herein by reference.

  Labeled nucleotides are forms of labels that can be incorporated directly into the amplification product during synthesis. Examples of labels that can be incorporated into amplified nucleic acids include nucleotide analogs such as nucleotides modified with BrdUrd, aminoallyldeoxyuridine, 5-methylcytosine, bromouridine, and biotin, or with appropriate heptenes such as digoxigenin. Contains the body. Suitable fluorescently labeled nucleotides are fluorescein-isothiocyanate dUTP, cyanine-3-dUTP and cyanine-5-dUTP. An example of nucleotide analog labeling of DNA is BrdUrd (bromodeoxyuridine, BrdUrd, BrdU, BUdR, Sigma-Aldrich Co). Other examples of nucleotide analogs for incorporation of labels into DNA are AA-dUTP (aminoallyl-deoxyuridine triphosphate, Sigma-Aldrich Co.) and 5-methyl-dCTP (Roche Molecular Biochemicals). An example of a nucleotide analog for incorporation of a label into RNA is biotin-16-UTP (biotin-16-uridine-5'-triphosphate, Roche Molecular Biochemicals). Fluorescein, Cy3, and Cy5 can be bound to dUTP for direct labeling. Cy3.5 and Cy7 are available as avidin or anti-digoxigenin conjugates for secondary detection of biotin- or digoxigenin labeled probes.

Labels that are incorporated into amplified nucleic acids, such as biotin, can later be detected using sensitive methods well known in the art. For example, biotin can be detected using a streptavidin-alkaline phosphatase conjugate (Tropix, Inc.) that is conjugated to biotin, which can then be detected using a suitable substrate (eg, chemiluminescent substrate CSPD: 3- (4 -Methoxyspiro- [1,2, -dioxetane-3-2 '-(5'-chloro) tricyclo [3.3.1.1 ^3,7 ] decan] -4-yl) phenyl phosphate disodium; Tropix , Inc.). The label can be detected, for example, by chemical signal amplification, or by using an enzyme that produces light (eg, a chemiluminescent 1,2-dioxetane substrate) or a substrate for an enzyme that produces a fluorescent signal, alkaline phosphatase, soybean peroxidase, whole It can also be an enzyme such as radish peroxidase and polymerase.

  Molecules that combine these two or more labels are also considered labels. Any of the known labels can be used with the disclosed probes, tags, and methods to label and detect nucleic acids amplified using the disclosed methods. Methods of detecting and measuring the signal generated by the label are also known to those skilled in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization, fluorescent molecules can be detected by fluorescence spectrophotometer, and phosphorescent molecules can be detected by direct visualization by spectrophotometer or camera The enzyme can be detected by detection or visualization of the product of the reaction catalyzed by the enzyme, and the antibody can be detected by detecting a secondary label bound to the antibody. As used herein, a detection molecule is a molecule that interacts with an amplified nucleic acid and a molecule to which one or more labels are bound.

  It is understood that one method of assessing whether a specific mRNA increase or mRNA expression has occurred or whether a specific mRNA is present is by comparison with a control and is considered herein. The Accordingly, a method of diagnosing cancer in a subject comprising performing real-time PCR, RT-PCR, or other PCR reaction on mRNA in a tissue sample of the subject, wherein the polymerase chain reaction comprises one or more ROS1 sequences At least one reverse primer that can specifically hybridize with, and at least one forward method primer, an increase in the amount of amplification product relative to the control indicates the presence of ROS1-related cancer, Methods obtained from non-cancerous tissue are contemplated herein. It will be further appreciated that for ROS1-related cancers, the use of non-cancerous tissue controls can be utilized, but not necessarily because cancerous tissue from non-ROS1-related cancers can also be used. Accordingly, a method of diagnosing ROS1-related cancer in a subject comprising performing a real-time PCR or reverse transcription-PCR (RT-PCR) reaction on mRNA in the subject test sample, wherein one or more polymerase chain reactions An increase in the amount of amplification product relative to the control indicates the presence of a ROS1-related cancer, wherein the control tissue comprises a reverse primer capable of specifically hybridizing to a ROS1 sequence of at least one and a forward primer Methods obtained from non-ROS1-related cancerous tissue are disclosed herein.

  The disclosed methods can be used to diagnose any disease that results in uncontrolled cell growth, referred to herein as “cancer”. A non-limiting list of different types of ROS1-related cancers is as follows: Lymphoma (Hodgkin and non-Hodgkin), leukemia, carcinoma, solid tissue carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high grade glioma, blastoma, neuroblastoma, plasmacytoma, Histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or general cancer.

  A representative but non-limiting list of cancers that can be diagnosed using the disclosed methods 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, small cell lung cancer And lung cancer such as non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, oral cavity, throat, pharynx, and lung squamous cell carcinoma, colon Cancer, bile duct cancer, colorectal cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colon cancer, hematopoietic cancer, ovarian cancer, Colon and rectal cancer, prostate cancer, or pancreatic cancer.

  Therefore, a method for diagnosing cancer, wherein the cancer is non-small cell lung cancer, diffuse large B cell lymphoma, systemic histiocytosis, breast cancer, colorectal cancer, esophageal squamous cell carcinoma, undifferentiated large cell Disclosed herein is a method selected from the group consisting of lymphoma, neuroblastoma, cholangiocarcinoma, renal cancer, colorectal cancer, glioblastoma, and inflammatory myofibroblast tumor (IMT) Yes. For example, a method of diagnosing ROS1-related cancer in a subject, comprising performing real-time PCR, RT-PCR, or PCR reaction on mRNA in a subject tissue sample, wherein the polymerase chain reaction comprises one or more ROS1 sequences A reverse primer that can specifically hybridize with and at least one forward primer, an increase in the amount of amplification product compared to the control indicates the presence of ROS1-related cancer, and the cancer is a non-small cell Lung cancer, diffuse large B cell lymphoma, systemic histiocytosis, breast cancer, colorectal cancer, esophageal squamous cell carcinoma, anaplastic large cell lymphoma, neuroblastoma, bile duct cancer, renal cancer, colorectal cancer, Disclosed herein is a method selected from the group consisting of glioblastoma, and inflammatory myofibroblast tumor, (IMT).

Methods for assessing the suitability of ROS1-directed therapy While not being bound by current theory, inhibition of these forms of RTK genes by small molecule drug candidates prevents the proliferation of associated abnormal cells, and neuroblasts In addition, both preclinical animal models and early clinical experience with these inhibitors are believed to promote apoptosis in cell lines of tumors and other RTK-related tumors. It not only has significant anti-tumor activity, but is also well tolerated without being restricted by target-related toxicity.

  These findings highlight the need for specialized diagnostic tests for ROS1 mutations. For example, such an assay can be used to screen family children suffering from hereditary neuroblastoma to help facilitate the detection of tumors in the early stages where treatment is more acceptable. Accordingly, a method for assessing the suitability of ROS1 inhibitor treatment in a subject comprising measuring nucleic acid in a tissue sample of the subject, wherein an increase in the amount of ROS1 sequence mRNA relative to a control is treated with a ROS1 inhibitor Methods for indicating cancer that can be performed are disclosed herein.

  It is understood and contemplated herein that any of the techniques disclosed herein for measuring the disclosed mRNA can be used in these methods. Thus, for example, a method of assessing the suitability of ROS1 inhibitor treatment in a subject comprising performing real-time PCR, RT-PCR, or other PCR reactions on mRNA or DNA in a subject tissue sample, The reaction comprises a reverse primer capable of specifically hybridizing with one or more ROS1 sequences, and at least one forward primer, and an increase in the amount of amplification product compared to the control is treated by the ROS1 inhibitor Methods for indicating cancer that can be performed are disclosed herein. It is further understood that the disclosed methods can further include any of the primers disclosed herein and can utilize the disclosed multiplexed PCR techniques.

  Accordingly, in one aspect, in a subject, assessing the susceptibility or risk of a disease or condition, monitoring disease progression, determining susceptibility or resistance to treatment of a therapeutic ROS1 inhibitor, or nucleic acid mutation, cleavage Or a method for determining the suitability of a cancer associated with a ROS1 fusion to ROS1 inhibitor treatment, detecting the presence of a level in a subject tissue sample, or an expression level of DNA, cDNA, or mRNA, or An increase in the amount of amplification product of ROS1 kinase relative to a control, wherein the corresponding increase is absent in wild-type ROS1, indicates the presence of a ROS1-related cancer and thus a cancer sensitive to ROS1 inhibitor treatment Such methods, as disclosed herein, include PCR, real-time PCR, RT-P R, or by amplification methods such as real-time RT-PCR or may be achieved by the practice of in situ hybridization, such as FISH,.

  In one aspect, a method for determining the sensitivity or resistance of a cancer to a therapeutic treatment for a ROS1 inhibitor or the suitability of a ROS1 inhibitor therapy in a subject having cancer, wherein the presence of ROS1 kinase activity is detected. A method comprising is disclosed herein. Thus, for example, in a subject having cancer, a method of diagnosing susceptibility or resistance to therapeutic treatment of ROS1-related cancer, or suitability for cancer treated with a ROS1 inhibitor, wherein a tissue sample is obtained from the subject Isolating the nucleic acid from the tissue sample; performing RT-PCR, real-time PCR, or real-time RT-PCR on the nucleic acid; and presence of nucleic acids associated with wild-type ROS1 and ROS1 kinase domains in the tissue sample Or RT-PCR or real-time PCR reaction is a wild-type ROS1 sequence (extracellular domain sequence of ROS1, such as SEQ ID NOS: 13, 14, 20, and 21). Forward and reverse primer pairs and / or fields that specifically hybridize with Including the use of forward and reverse primer pairs that specifically hybridize with type ROS1 kinase domain sequences (eg, SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18), and tissue samples Detecting the presence or measuring the amount of a nucleic acid associated with one or a combination of both wild-type ROS1 and ROS1 kinase domains in an increased amplicon relative to a normal control; Or disclosed herein is a method comprising the presence of an amplicon indicating that the subject has a ROS1-related cancer and is therefore susceptible to treatment with a ROS1 inhibitor. Absence of amplicon, or amplicon levels equal to normal controls, indicate that the cancer is not sensitive to ROS1 treatment and resistant to such treatment. A method for determining sensitivity or resistance to therapeutic treatment of a ROS1-related cancer in a subject having cancer, or suitability of a cancer to be treated with a ROS1 inhibitor, comprising obtaining a tissue sample from the subject, Disclosed is a method comprising isolating nucleic acid from a sample, wherein the nucleic acid of the tissue sample is RNA, wherein the method comprises synthesizing cDNA from the RNA sample and PCR the cDNA. And detecting the presence or measuring the amount of nucleic acid associated with one or a combination of both wild-type ROS1 and ROS1 kinase domains in a tissue sample, and with a normal control An increase in amplicon or the presence of an amplicon compared to indicates that the subject has ROS1-related cancer. In further embodiments, the disclosed methods can utilize probes complementary to sequences of real-time RT-PCR products (eg, SEQ ID NOs: 6, 8, 19, and 22) or Determining the cycle threshold (Ct) value of wild-type ROS1 and wild-type ROS1 kinase, wherein a high (Ct) value of wild-type ROS1 compared to ROS1 kinase indicates the presence of the fusion and thus It indicates that the cancer is sensitive to treatment with a ROS1 inhibitor or that treatment with a ROS1 inhibitor is suitable for that cancer. When a probe is used, it is understood that the probe can include a reporter dye at its end and a quencher dye at its other end. Also disclosed is a method further comprising administering a ROS1 inhibitor to a subject having a cancer susceptible to ROS1 inhibitor treatment when the cancer is determined to be compatible or suitable for treatment with a ROS1 inhibitor. Has been. Conversely, if the cancer is not sensitive to ROS1-related inhibitors, the method can further comprise treating the patient with cancer using a form of treatment other than the ROS1-inhibitor.

  In a further alternative method of determining the susceptibility or resistance to therapeutic treatment of a ROS1-related cancer or the suitability of a cancer to be treated with a ROS1 inhibitor in a subject having cancer, the ROS1 fusion is a ROS1 fusion. Only the forward primer that binds to is used in the first reaction, and in the second reaction following the first reaction, the amplicon of the first reaction is subject to second amplification, probe-based detection, or sequencing. The probe or primer used and used as a template in the reaction is specific for ROS1 at the fusion breakpoint 3 ', and detection of ROS1 in the amplicon of the second reaction results in the ROS1 fusion and thus ROS1 Detected by qPCR using two-step detection, indicating ROS1-related cancers that are sensitive to inhibitor treatment. Also, in an embodiment having cancer, a method for determining the sensitivity or resistance to therapeutic treatment of an RO1-related eye, or the suitability of a cancer to be treated with a ROS1 inhibitor, comprising obtaining a tissue sample from a subject, Isolating nucleic acid from a tissue sample and performing a first amplification reaction using a forward primer that binds to the ROS1 fusion partner, wherein the amplicon of the first reaction is a second amplification A primer specific for the ROS1 sequence at 3 ′ of the fusion breakpoint is used to serve as a template for the reaction and to perform a second amplification reaction after the first reaction. Being used in an amplification reaction (eg, SEQ ID NOs: 4, 5, 7, 8, 15, 16, 17, and 18) and detecting the presence of ROS1 in the amplicon of the second reaction, Detecting the presence of ROS1 in the amplicon of the second reaction indicates the presence of a ROS1 fusion and detecting the presence of a nucleic acid associated with the ROS1 kinase domain in the tissue sample, wherein the presence of the amplicon is Wherein the subject is susceptible to treatment with a ROS1 inhibitor, and wherein the absence of an amplicon indicates that the cancer is resistant or insensitive to ROS1 inhibitor treatment. It is disclosed in the document. Also disclosed is a method further comprising administering a ROS1 inhibitor to a subject having a cancer susceptible to ROS1 inhibitor treatment when the cancer is determined to be compatible or suitable for treatment with a ROS1 inhibitor. Has been. Conversely, if the cancer is not sensitive to ROS1-related inhibitors, the method can further comprise treating the patient with cancer using a form of treatment other than the ROS1-inhibitor.

Methods for Screening The ROS1 fusions disclosed herein are targets for cancer therapy. Accordingly, a method of screening for an agent that inhibits ROS1-related cancer in a subject comprising:
a) obtaining a tissue sample from a subject having ROS1-related cancer;
b) extracting mRNA from the tissue sample;
c) contacting the tissue sample with the drug;
d) performing real-time PCR, RT-PCR, or other PCR reactions on the mRNA in the tissue sample;
Including
An amplification product comprising a reverse primer capable of specifically hybridizing with one or more ROS1 sequences, and at least one forward primer, wherein the RT-PCR reaction exhibits a ROS1 fusion compared to an untreated control Disclosed herein are methods wherein a decrease in amount indicates an agent that can inhibit a ROS1-related cancer. The measure of effective treatment with the drug is understood to be a reduction in the ROS1 amplicon cycle threshold 5 ′ of the fusion break point compared to the untreated control and is considered herein. It is understood that the process of performing the RT-PCR reaction involves synthesizing cDNA from the isolated RNA and performing PCR on the cDNA, and is contemplated herein.

Nucleic acids The disclosed methods and compositions use a variety of nucleic acids. In general, any nucleic acid can be used in the disclosed methods. For example, the disclosed nucleic acids of interest and the disclosed reference nucleic acids can be selected based on the desired analysis and information obtained or evaluated. The disclosed method also produces new altered nucleic acids. The nature and structure of such nucleic acids is established by the way they are produced and manipulated in the method. Thus, for example, extension products and hybridizing nucleic acids are produced by the disclosed methods. As used herein, a hybridizing nucleic acid is a hybrid of an extension product and a second nucleic acid.

  It will be understood and contemplated herein that the nucleic acid of interest can be any nucleic acid in which determination of the presence or absence of nucleotide mutations is desirable. Thus, for example, the nucleic acid of interest can include a sequence corresponding to the wild-type sequence of the reference nucleic acid. In the disclosed method, the first nucleic acid is a reference nucleic acid and the second nucleic acid is a target nucleic acid, or the first nucleic acid is a target nucleic acid and the second nucleic acid is a reference nucleic acid It is further disclosed herein that it can be implemented in some cases.

  It is understood that the reference nucleic acid can be any nucleic acid to which the nucleic acid of interest is compared and is contemplated herein. Typically, the reference nucleic acid has a known sequence (and / or is known to have the sequence of interest as a reference). Although not required, it is useful that the reference sequence has a known or presumed close relationship with the nucleic acid of interest. For example, if it is desired that a single nucleotide variation be detected, the reference sequence may be a nucleic acid of interest, such as a nucleic acid derived from the same gene or genetic element from the same or related organism or individual. It may be useful to be selected to be a sequence that is homologous or closely matched. Thus, for example, the reference nucleic acid can comprise a wild-type sequence, or alternatively can comprise a known mutation, eg, a mutation whose presence or absence is associated with resistance to a disease or therapeutic treatment, Considered herein. Thus, for example, the disclosed method compares a nucleic acid of interest with a reference nucleic acid comprising a wild-type sequence (ie, known to have no mutation) and the presence of a mutation in the nucleic acid of interest. Or it is contemplated that the absence can be used to detect or diagnose the presence of a known mutation in the nucleic acid of interest by examining the absence and indicating the absence of the mutation. Alternatively, the reference nucleic acid can have a known mutation. Thus, for example, the disclosed method compares a nucleic acid of interest with a reference nucleic acid containing a known mutation that indicates susceptibility to the disease, tests for the presence or absence of the mutation, and the presence of the mutation is the disease It is contemplated that can be used to detect susceptibility to a disease or condition.

  Here, the term “nucleotide mutation” means any change or difference in the nucleotide sequence of the nucleic acid of interest compared to the nucleotide sequence of the reference nucleic acid. Thus, a nucleotide variation is said to occur when the sequence between the reference nucleic acid and the nucleic acid of interest (or its complement, where appropriate in the context) is different. Thus, for example, substitution of adenine (A) to guanine (G) at a particular position in a nucleic acid is a nucleic acid mutation as long as the reference nucleic acid contains A at the corresponding position. It is understood that the determination of the mutation is based on the reference nucleic acid and does not indicate whether the sequence is wild-type and is considered herein. Thus, for example, if a nucleic acid with a known mutation is used as a reference nucleic acid, a nucleic acid without a mutation (including a wild-type nucleic acid) is considered to have a nucleotide variation (compared to a reference nucleic acid). The

Nucleotides The disclosed methods and compositions use a variety of nucleotides. Throughout this application and the methods disclosed herein, reference is made to the nucleotide base type. When reference is made to a base type, this is understood to mean a base that allows nucleotides in a nucleic acid strand to hybridize (combine) with a defined set of one or more critical bases. Considered in the book. Thus, for example, when reference is made to an extension product extended in the presence of three nuclease resistant nucleotides rather than in the presence of a nucleotide comprising the same type of base as the modified nucleotide, this is, for example, When the base is adenine (A), it means that the nuclease resistant nucleotide can be, for example, guanine (G), thymine (T), and cytosine (C). Each of these bases (representing the four limit bases) can hybridize with a different one of the four limit bases and are therefore suitable as different types of bases, Defined in As another example, inosine bases are predominantly paired with adenine and cytosine (in DNA) and are therefore considered adenine and base pairs with tyrosine and guanine, respectively, and a different kind of base from cytosine. Can not be considered as a different kind of base from guanine or thymine, which base pairs with cytosine and adenine, respectively, because guanine and thymine base pairing overlaps with the inosine hybridization pattern (i.e. different Not).

  Any type of modification or alternative base can be used in the disclosed methods and compositions that are generally limited only by the ability of the enzyme used to use such a base. Many modifications and alternative nucleotides and bases are known, some of which are described below and elsewhere herein. The type of base represented by such modifications and alternative bases can be determined by the base pairing pattern of the base, as described herein. Thus, for example, if the modified nucleotide is adenine, any analog, derivative, modified, or variant base primarily based on a pair with thymine is considered the same type of base as adenine. In other words, as long as the analog, derivative, modified, or variant has the same pattern of base pairing with another base, it can be considered the same type of base. Modifications can be made to the sugar or phosphate group of the nucleotide. In general, such modifications do not change the base-pairing pattern of the base. However, the nucleotide base pairing pattern in the nucleic acid strand determines the type of base in the nucleotide.

  The modified nucleotide incorporated into the extension product by the disclosed method and selectively removed by the disclosed agent can be any modified nucleotide that functions as needed in the disclosed method; It is described elsewhere in this specification. Modified nucleotides can also be produced in existing nucleic acid strands such as extension products, eg, by chemical modification, enzymatic modification, or a combination.

  Many types of nuclease resistant nucleotides are known and can be used in the disclosed methods. For example, a nucleotide can have a modified phosphate group and / or a modified sugar group and be resistant to one or more nucleases. Nuclease resistance is defined herein as being resistant to removal from a nucleic acid by any one or more nucleases. In general, the nuclease resistance of a particular nucleotide can be defined with respect to the relevant nuclease. Thus, for example, if a particular nuclease is used in the disclosed method, the nuclease resistant nucleotide needs to be resistant only to that particular nuclease. Examples of useful nuclease resistant nucleotides include thio modified nucleotides and borano modified nucleotides.

  There are a variety of molecules disclosed herein that are based on nucleic acids. Non-limiting examples of these and other molecules are discussed herein. For example, a nucleotide is understood to be a molecule that contains a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides bind together through these phosphate and sugar moieties to create an internucleoside bridge. The base part of the nucleotide is adenine-9-yl (adenine, A), cytosine-1-yl (cytosine, C), guanine-9-yl (guanine, G), uracil-1-yl (uracil, U), And thymin-1-yl (thymine, T). The sugar moiety of the nucleotide is ribose or deoxyribose. The phosphate portion of the nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides are 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

  Nucleotide analogs are nucleotides that contain certain modifications in the base, sugar or phosphate moieties. Modifications to the base moiety include natural and synthetic modifications of A, C, G, and T / U, and different purine or pyrimidine bases, such as uracil-5-yl (Ψ), hypoxanthine-9- Yl (inosine), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanidine, and alkyl derivatives thereof, adenine and guanidine. 2-propyl and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, 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, especially 5-bromo, 5- Trifluoromethyl and other 5-substituted ura Le and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3- deazaguanine and 3-deazaadenine include, but are not limited to. Additional base modifications can be found, for example, in US Pat. No. 3,687,808, the teachings of which are incorporated herein by reference in their entirety. Certain such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine Nucleotide analogues. 5-methylcytosine can increase the stability of duplex formation. In many cases, base modifications can be combined with sugar modifications, such as 2'-O-methoxyethyl, to achieve unique properties such as increased duplex stability.

  Nucleic acid analogs can also include modifications of the sugar moiety. Modifications of the sugar moiety include natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2 ′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where Alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl, or C2-C10 alkenyl, and alkynyl. Also, 2 'sugar modifications include -O [(CH2) nO] mCH3, -O (CH2) nOCH3, -O (CH2) nNH2, -O (CH2) nCH3, -O (CH2) n-ONH2, and- O (CH2) nON [(CH2) nCH3)] 2 is included, but is not limited thereto, where n and m are from 1 to about 10.

Other modifications at the 2 'position, C1 -C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O- alkaryl or O- _{aralkyl, SH, SCH 3, OCN,} Cl, Br, CN, CF3, OCF3 , SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterosilalkalyl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interventional substance, pharmacokinetic properties of oligonucleotide , Or groups that improve the pharmacodynamic properties of the oligonucleotide, and other substituents that have similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3 'position of the sugar of the 3' terminal nucleotide or 2'-5 'linked oligonucleotide and the 5' position of 5 'terminal nucleotide. Modified sugars also include those containing modifications at the bridging ring oxygen such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

  Nucleotide analogs can also be modified with a phosphate moiety. In modified phosphate moieties, the bridge between two nucleotides is phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates, chiral Phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates, aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates This includes, but is not limited to, those that may be modified to contain. These phosphate or modified phosphate bridges between two nucleotides can be via 3'-5 'bridges or -2'-5' bridges, where bridges are 3'-5 'to -5'-3. It is understood that reverse orientations such as', or 2'-5 'to -5'-2' can be included. Various salt, mixed salt and free salt forms are also included.

  Nucleotide analogs need only contain a single modification, but it is understood that multiple modifications can be contained between one or different parts.

  Nucleotide substitutes are molecules that have functionality similar to nucleotides, but do not contain a phosphate moiety, such as peptide nucleic acids (PNA). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but bind together through moieties other than the phosphate moiety. Nucleotide substitutes can conform to a double helix type structure when interacting with the appropriate target nucleic acid.

  Nucleotide substitutes are nucleotides or nucleotide analogs in which the phosphate and / or sugar moieties are replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substituted phosphates are, for example, short chain alkyl or cycloalkyl internucleoside bridges, mixed heteroatoms, and alkyl or cycloalkyl internucleoside bridges, or one or more short chain heteroatoms or heterocyclic internucleoside bridges Can do. These include morpholino bridges (partially formed at the sugar portion of the nucleoside); siloxane backbone; sulfide, sulfoxide and sulfone backbone; formacetyl and thioformacetyl backbone; methyleneformacetyl and thioformacetyl. Main chain; alkene-containing main chain; sulfamic acid main chain; methyleneimino and methylenehydrazino main chain; sulfonic acid and sulfonamide main chain; having amide main chain, and mixed N, O, S and CH 2 component parts Others are included.

  It will also be understood in nucleic acid substitutions that both sugar and phosphate moieties of a nucleotide can be replaced, for example, with an amide type bridge (aminoethylglycine) (PNA). US Pat. Nos. 5,539,082, 5,714,331, and 5,719,262 teach how to make and use PNA molecules, see each As incorporated herein.

  Other types of molecules (conjugates) can be attached to other nucleotides or nucleotide analogs. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di- Hexadecyl-rac-glycerine or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chain, or adamantaneacetic acid, palmityl moiety, or octadecylamine or hexylamino-carbonyl -Include but are not limited to oxycholesterol moieties.

  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 nucleotides, nucleotide analogs, or nucleotide substitutes includes purine-based nucleotides, nucleotide analogs, or nucleotide substituents at C2, N1, and C6 positions, and pyrimidine-based nucleotides, nucleotide analogs Or the C2, N3, C4 position of the nucleotide substitution.

  A Hoogsteen interaction is an interaction that occurs at the Hoogsteen face of a nucleotide or nucleotide analog that is exposed to the main groove of double-stranded DNA. The Hoogsteen face includes a reactive group (NH2 or O) at the N7 position and the C6 position of the purine nucleotide.

Hybridization / Selective Hybridization The term hybridization typically refers to a sequence that induces an interaction between at least two nucleic acid molecules, such as a primer or probe, and a gene. Sequence-inducible interaction means an interaction that occurs between two nucleotides, or nucleotide analogs or nucleotide derivatives, in a nucleotide-specific manner. For example, G that interacts with C or A that interacts with T is a sequence-induced interaction. Typical sequence-induced interactions occur at the Watson-Crick or Hoogsteen face of nucleotides. Hybridization of two nucleic acids is affected by a number of conditions and parameters known to those skilled in the art. For example, the salt concentration, pH, and temperature of the reaction all affect how the nucleic acid molecule hybridizes.

  Parameters for selective hybridization between two nucleic acid molecules are well known to those skilled in the art. For example, in some embodiments, selective hybridization conditions are defined as stringent hybridization conditions. For example, the stringency of hybridization is controlled by both temperature and salt concentration in either or both of the hybridization and washing steps. For example, hybridization conditions to achieve selective hybridization include strong ionic strength solutions (6 × SSC or 6 ×) at temperatures about 12-25 ° C. below Tm (melting temperature at which half molecules dissociate from hybridization partners). SSPE) followed by washing with a combination of temperature and salt concentration selected such that the washing temperature is about 5 ° C. to 20 ° C. below Tm. The temperature and salt concentration are readily determined empirically in preliminary experiments in which a sample of reference DNA immobilized on a filter is hybridized with the labeled nucleic acid of interest and then washed under conditions of different stringency. . Hybridization temperatures are typically high in DNA-RNA and RNA-RNA hybridization. Conditions can be used as described above to achieve stringency, or are known to those skilled in the art. Strict hybridization conditions preferred for DNA: DNA hybridization may be about 68 ° C. in 6 × SSC or 6 × SSPE (in aqueous solution) followed by a 68 ° C. wash. The stringency of hybridization and washing can be reduced, if desired, as the desired complementarity is reduced and further depending on the GC or AT enrichment in any region where variability is sought. Similarly, stringency of hybridization and washing increases as desirable homology increases if desired, and further depending on the abundance of GC or AT in any region where high homology is sought. All known in the art.

Another way to define selective hybridization is to look at the amount (rate) at which one nucleic acid is bound to the other nucleic acid. For example, in some embodiments, the selective hybridization conditions are at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, This is the case when 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the restriction nucleic acids are bound to non-restricted nucleic acids. Typically, non-restricted primers are in excess of, for example, 10, or 100, or 1000 times. This type of assay has both restricted and non-restricted primers, for example 10 times, 100 times, or 1000 times lower than their k _d , or one of the nucleic acid molecules is 10 times, 100 times, or 1000 times. Or one or both nucleic acid molecules can be performed under conditions above these k _d .

  Another way to define selective hybridization is to look at the rate of primers that are enzymatically engineered at conditions that facilitate the desired manipulation of hybridization. For example, in some embodiments, the selective hybridization conditions are at least about 60, 65, 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 percent of the primers are enzymatically engineered under conditions that facilitate enzymatic manipulation. For example, if the enzymatic manipulation is DNA extension, the selective hybridization conditions are at least about 60, 65, 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 percent extended primer molecules Is a case to be. Conditions include those suggested by the manufacturer as being suitable for performing the operation enzymatically, and those indicated in the art.

  As with homology, it is understood that there are a variety of methods disclosed herein for determining the level of hybridization between two nucleic acid molecules. While it is understood that these methods and conditions can result in different rates of hybridization between two nucleic acid molecules, it is sufficient to meet the parameters of either method, unless otherwise indicated. For example, if 80% hybridization is required and hybridization occurs within the parameters required in any of these methods, it is considered disclosed herein.

  Those skilled in the art will appreciate that if a composition or method meets any one of the criteria for determining hybridization collectively or alone, this is the composition or method disclosed herein. Understanding is understood.

Kits Disclosed herein are kits from which reagents that can be used to perform the methods disclosed herein are derived. In particular, the kit can include any reagent or combination of reagents understood or necessary or beneficial for performing the methods discussed herein or disclosed. For example, the kit uses one or more primers disclosed herein that perform the extension, replication, and amplification reactions discussed in a particular embodiment of the method, as well as the primers as intended. Buffers and enzymes necessary to do so can be included.

  It will be appreciated that a reverse primer that hybridizes to wild type ROS1 can be used to detect ROS1-related fusions. Accordingly, kits comprising at least one reverse primer that hybridizes with a portion of wild type ROS1 such as the kinase domain of wild type ROS1 are disclosed herein. In addition, it is understood that the kits disclosed herein can include one or more forward primers that specifically hybridize to a fusion partner of ROS1 and / or wild type ROS1. Thus, for example, a forward primer can be a wild-type ROS1 such as a ROS1 sequence outside the ROS1 fusion breakpoint region (ie, 3 ′ of the fusion breakpoint), CD74, FIG, SLC34A2, or other fusion partners. And can be hybridized. Those skilled in the art include more than a single primer or primer pair, including, for example, a single reverse primer such as SEQ ID NO: 5, 8, 12, 14, 15, 18, or 21, and a plurality of forward primers. It can be appreciated that it is suitable to have a kit to obtain. Alternatively, the kit can include a primer pair for wild-type ROS1 and a primer pair for ROS1 kinase outside the fusion breakpoint (ie, 3 'of the fusion breakpoint). The kit can further comprise one or more forward primers and / or one or more reverse primers that specifically hybridize with wild type ROS1, CD74, FIG, SLC34A2.

  Accordingly, in one embodiment, (a) a first primer labeled with a first detection reagent, wherein the first primer is a reverse primer, and the reverse primer is an amino acid sequence of SEQ ID NO: 1. Or a first primer that is one or more polynucleotides that hybridize to a first polynucleotide encoding its complement, and (b) at least one second primer, said second primer ROS1-related cancer comprising: a forward primer, wherein the forward primer is one or more polynucleotides that hybridize to a second polynucleotide encoding wild-type ROS1. Disclosed herein are kits for determining the suitability of a cancer for treatment or the suitability of treatment for cancer. It has been.

  Also disclosed herein are kits that include one or more forward primers (eg, SEQ ID NOs: 13 and 20) that specifically bind to the ECD of ROS1. Also disclosed herein are kits comprising one or more forward primers (eg, SEQ ID NO: 4, 7, 15, or 17) that specifically bind to the ROS1 kinase domain. It will be appreciated that the kit may include one or more forward primers specific for the ECD and kinase domain of ROS1. Also disclosed are kits that further comprise one or more reverse primers specific for ROS1 ECD (eg, SEQ ID NOs: 14 and 21). Also a kit comprising one or more reverse primers that specifically bind to the ROS1 kinase domain (eg, SEQ ID NOs: 5, 8, 12, 16, and 18), and also specific for the ECD and kinase domain of ROS1 A kit comprising a combination of one or more reverse primers is disclosed herein. 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, for example, one or more forward primers such as ROS1-specific forward primer SEQ ID NO: 4, 7, 13, 15, 17, or 20) and / or one or more ROS1-specific reverse primer SEQ ID NO: Kits comprising 5, 8, 12, 14, 16, 18, and 21) are disclosed herein.

  Also comprising one or more polynucleotide probes, said probes being about 20 to about 30 nucleotides in length, comprising a reporter dye at one end and a quenching dye at the other end, eg Kits that are number 6, 9, 19, or 22 are disclosed herein.

It is understood that the disclosed kits can also include controls to ensure that the methods disclosed herein function correctly and to normalize results between assays. Thus, for example, a positive cDNA control, a negative cDNA control, and a control primer pair are disclosed herein. For example, the disclosed kits include homosapiens ATP synthase, H + transport, mitochondrial F1 complex, O subunit (ATP5O), nucleogene-encoded mitochondrial protein mRNA; homosapiens NADH dehydrogenase (ubiquinone) 1 alpha partial complex, 2 , 8 kDa (NDUFA2), mRNA; homosapiens glyceraldehyde-3 phosphate dehydrogenase (GAPDH), mRNA; homosapiens H3 histone, family 3A (H3F3A), mRNA; homosapiens proteasome (prosome, macropain) subunit, beta Type 4 (PSMB4), mRNA; Homo sapiens ribosomal protein S27a (RPS27A), transcript variant, 1, mRNA; Homo sapiens eukaryotic translation Factor 4A, isoform 2 (EIF4A2), mRNA; homosapiens ribosomal protein L18 (RPL18), mRNA; homosapiens adenosine deaminase, RNA specific (ADAR), transcript variant 1, mRNA; or homosapiens cytochrome c oxidase sub Unit Vb (COX5B), a control primer pair can be included to detect mRNA. Examples of primer pairs include, but are not limited to, primer pairs found in Table 1.

  In addition, the disclosed kits can include such other reagents and materials for performing the disclosed methods, such as enzymes (eg, polymerases), buffers, sterile water, reaction tubes, etc. That is understood. In addition, the kit can also include modified nucleotides, nuclease resistant nucleotides, and / or labeled nucleotides. In addition, the disclosed kits can include instructions for performing the methods disclosed herein, and software that can calculate the presence of ROS1 mutations.

  In one aspect, the disclosed kit uses a single assay to perform a method of determining whether a use contains wild-type ROS1, a known ROS1 fusion, or a previously unidentified nROS1 fusion. Enough material can be included to be performed simultaneously or separately. The kit can also contain sufficient material to perform a control reaction. Thus, in one embodiment, a positive cDNA control reaction tube, a negative cDNA control reaction tube, a control primer reaction tube, a reaction tube for detecting a known ROS1 fusion, and / or a reaction tube for detecting wild-type ROS1, Also disclosed herein is a kit comprising a reaction tube for detecting ROS1 kinase activity.

  In another form, the disclosed kit determines the status of ROS1, which is either wild type expression, kinase domain overexpression, or fusion mutation overexpression, based on measurements relative to an internal control gene. Can be used to Internal control genes are described elsewhere in this disclosure and are understood to be stably and constitutively expressed regardless of cell cycle, development, or environmental factors. Thus, in one embodiment, the status of ROS1 can be determined by the ROS1 (numerator) / internal control (denominator) equation and the resulting quotient is that the test tissue, cell line, or other sample is ROS1 positive or ROS1 negative It is the range of the result which shows that it is either. With the understanding that a particular tissue expresses internal control transcripts at different levels, the determined ratio and quotient indicating a ROS1 positive or negative status is established separately for each tissue and specimen type.

  The compositions disclosed herein and the compositions necessary to practice the disclosed methods are subject to any method known to one of ordinary skill in the art for a particular reagent or compound, unless otherwise indicated. Can be made using.

Nucleic Acid Synthesis The disclosed nucleic acids, such as oligonucleotides used as primers, can be made using standard chemical synthesis methods, or can be produced using enzymatic methods or any other method. Such methods range from standard enzymatic digestion to subsequent nucleotide fragment isolation, pure synthesis methods, eg, Milligen or Beckman System 1 Plus DNA synthesizers (eg, Model 8700 automated from Milligen-Biosearch, Burlington, MA). It may be within the scope of the cyanoethyl phosphoramide method using a synthesizer, or ABI Model 380B).
Example

  The following examples provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods claimed herein are made and evaluated. It is described for the purpose of providing and is intended to be purely illustrative and not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Centigrade, or is ambient temperature, and pressure is at or near atmospheric.

Example 1: Development and validation of a FISH assay to detect ROS1 fusions As indicated above, three different ROS1 partners, RIG, CD74, and SLC34A2, are involved in cancer-associated ROS1 fusions. Have been identified. Although currently unknown, these three ROS1 partners are not the only genes that form fusions with kinases in cancer based on experience with other fusion kinases, for example> Fifteen different genes (some of which are shown in FIG. 1) are currently fused with a truncated ALK receptor tyrosine kinase (RTK) to form a chimeric kinase that induces cancer development. Are known. Although the ROS1 fusion FISH assay can be specifically designed for each of the three known fusion parts involved, such an approach can fail to find ROS1 fusions involving fusion partners that have not yet been identified. In addition, the clinical use of three different FISH assays to detect ROS1 fusions in tumor specimens is extremely time and labor inefficient.

  The ROS1 fusion assay uses large genomic clones (typically BAC and / or PAC clones) adjacent to positions within the ROS1 locus where cleavage occurs during cancer-related chromosomal rearrangements. Such a “breakapart” FISH assay uses adjacent genomic clones labeled with differently colored fluorescent dyes. In normal cells, different colors (eg, red and green, sometimes appearing yellow when colors overlap) are physically paired because the locus is intact, in contrast, In cancer cells containing organization, the colors are physically separated. As shown in FIG. 4, the ROS1 breakapart FISH assay works in normal and ROS1 fusion-containing cells, as expected.

Example 2: Development of a PCR-based assay to detect ROS1 fusions The ROS1 breakapartFISH assay detects the majority of ROS1 fusions regardless of the partner gene involved. However, additional ROS1 fusion clinical diagnostics are still significant: 1) diagnostic platforms other than FISH (which may be somewhat technically demanding) in some countries and certain situations As well as 2) produced by microdeletions in the ROS1 locus that are not brought about by cleavage of adjacent probes (FIG gene is actually embedded in the intron of the ROS1 gene, and as a result of the microdeletion in the locus, FIG. To address these problems, which are because the FIG-ROS1 fusion cannot be detected by FISH because it is juxtaposed adjacent to the ROS1 exon that encodes a portion of ROS1 found in ROS1. Based assay is a complementary protocol to FISH to detect ROS1 fusion mutations. It was developed as platforms.

  PCR diagnostics for ROS1 (branded as Insight ROS1 Screen ™) is a real-time PCR diagnostic that utilizes different RT primers that selectively amplify ROS1 fusions in lung cancer specimens. The basic design of Insight ROS1 Screen is illustrated in FIGS.

  In contrast to oncogenic ALK expression in NSCLC, ROS1 is essentially expressed in both NSCLC and normal lung tissue. Therefore, an effective ROS1 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 iterative optimization for newly discovered fusion partners and variants using a mixture of detection primers that continues to expand. Insight ROS1 Fusion Screen circumvents these problems by maintaining a comprehensive PCR detection method for the ROS1 kinase domain. Oncogenic ROS1 fusions are targeted in a first strand synthesis step that selects for amplification of basal full-length ROS1 expression in tumor lung tissue. In this method, the detection phase of the assay remains constant regardless of the fusion partner.

  The assay utilizes two separate PCR primer sets, one amplifying a region of the ROS1 gene that encodes the ROS1 extracellular domain found only in normal RTKs (not the ROS1 fusion) and the other primer set Amplifies the ROS1 gene segment encoding the kinase domain found in both normal and fused ROS1. This assay design can detect not only the presence of the ROS1 fusion, but also the overexpression of the intact ROS1 gene (most frequently correlated with DNA amplification of the locus). Normal ROS1 RTK mRNA expression correlates with the internal control, and normal ROS1 overexpression is indicated by a lower threshold Ct value than the internal control, while the presence of ROS1 fusions indicates that wild-type and kinase real-time PCR This is evident by the difference in threshold Ct values between the reactions (Ct of the kinase domain reaction is lower than the wild type reaction). As shown, this sophisticated and simple design identifies all ROS1 derivatives regardless of the fusion partner involved, and in addition identifies and quantifies wild-type ROS1 overexpression. Although the incidence and significance of ROS1 amplification / overexpression in cancer remains unknown, it is known that ROS1 amplification / overexpression results in constitutive kinase activation that induces tumor growth when crossed over a certain threshold. It is contemplated herein to follow the amplification / overexpression pattern of other RTKs that have been identified (eg, ALK and EGFR), and importantly, cancers with such RTK amplification / overexpression are induced Sensitive to pharmacological inhibition of sex kinases.

Example 3: Development of Insight ROS1 Fusion Screen The methodology described above is based on fusion-specific oligonucleotides (to target ROS1 fusions), random hexamer mix (to estimate total ROS1), or It was established by testing different reverse transcriptase enzymes stimulated without primer (to confirm mock stimulation) at different reaction temperatures. These conditions were initially optimized in SLC34A2-ROS1 fusion expressing cell lines or normal lung templates (Table 2). When the signal was detected in the absence of RT primer, the reaction conditions were found not to be rigorous enough to prevent indiscriminate RT activity (see Superscript III @ 56 ° C). To address this problem, a thermostable enzyme (Thermoscript, Invitrogen) that has activity at temperatures above 60 ° C. was used. Performing the reaction at high temperature reduces RNA secondary structure, but also reduces the specificity of the FS-RT primer. If signal is detected in normal lung using FS-RT primer, the response is not specific to distinguishing full length ROS1 from ROS1 fusion (see Thermoscript with 57 ° C. Tm primer). . The loss of specificity was addressed by redesigning a series of primers at a melting temperature of approximately extended temperature (ie, 63-65 ° C.). High melting temperature primers eliminated non-specific cDNA synthesis of full length ROS1 transcripts in normal lung samples (Table 2).

The same design parameters in the SLC34A2-ROS1 fusion assay were then applied to the detection of an additional ROS1 fusion, a CD74-ROS1 fusion transcript available from the Insight catalog. Another set of FS-RT primers designed at melting temperatures above 63 ° C., template RNA derived from Ba / F3 cell line expressing CD74-ROS1 fusion and lacking full length ROS1 expression Was used to stimulate first strand synthesis by Thermoscript enzyme. As shown in Table 3, specific detection of the ROS1 kinase domain was only detected in fusion expressing cell lines using the FS-RT CD74-ROS1 fusion primer. Again, normal lung was used as a control to identify any non-specific amplification of full length ROS1 (Table 2).

Routine PCR testing of ROS1 fusions is currently performed using allele specific primers adjacent to the fusion junction. In contrast, Insight ROS1 Fusion Screen utilizes a comprehensive PCR primer set for detection of the ROS1 kinase domain after fusion-specific first strand synthesis. In order to compare and contrast both methods for PCR efficiency, cDNA generated using FS-RT primers and random hexamers were individually subjected to both types of post-detection methods. Table 4 shows that ROS1 kinase specific assays have similar crossing point (Ct) values when compared to allele specific primers for specific samples using the same concentration of starting total RNA. In addition, the ROS1 kinase specific assay incorporates the ability to detect differences in fusion transcripts based on amplification / detection of common gene regions across diverse fusion points. In contrast, allele-specific assays require additional template input for each reaction and / or complex multiplexing for the detection phase of the reaction.

In order to evaluate the sensitivity of the FS-RT method, the CD74-ROS1-expressing Ba / F3 cell line was diluted in RNA extracted from normal lungs. Normal lungs express only full-length ROS1 and Ba / F3 cell lines express only CD74-ROS1 fusion translocation, so normal lung RNA is used to Ba / until quench to determine the limit of detection. It can be diluted in F3 RNA. Each RNA dilution series was subjected to fusion-specific first strand synthesis and amplification by ROS1 kinase qPCR assay (ie, Insight ROS1 Fusion Screen). The results in Table 5 indicate that the sensitivity of the assay is 1:10 or has a detection level of 10%.

Example 4: Optimization of Insight ROS1 Screen in FFPE To identify ROS1 fusions within the IGI Biobank repository, the 169FFPE lung adenocarcinoma test for diverse ROS1 fusions using traditional allele-specific detection methods Screened the pieces. Two ROS1 fusions were detected (frequency rate 1.2%) and one of these samples was identified as having a CD74-ROS1 fusion. Second-site RT-PCR and Sanger sequencing confirmed the presence of the CD74-ROS1 fusion in two independent isolated samples. CD74-ROS1-positive FFPE was then tested by in vitro confirmation Insight ROS1 Fusion Screen. The results show that the reverse transcriptase high temperature parameters optimized in the cell line were successfully translated into FFPE samples. However, PCR reactions that detect ROS1 kinase expression required a larger amount of cDNA template input than when tested in cell lines (Table 6). Finally, confirmation of CD74-ROS1-specific amplification by high temperature RT reaction is determined by testing the cDNA for ROS1 ECD expression and detects full-length ROS1 expression (and promiscuous RT amplification). As can be seen from Table 6, full length ROS1 was not detected in these samples, confirming that Insight ROS1 Fusion Screen was optimized to specifically detect ROS1 fusions in FFPE samples.

Example 5: Development of Insight ROS1 Screen ™ Version 2 The high background level of wild-type RO1 constitutively expressed in lung tissue is due to the wild-type and kinase described in the Insight ROS1 Screen PCR-based assay. Any detectable difference in the threshold Ct value of the domain real-time PCR reaction may be masked. If a detectable difference is masked, an alternative qPCR approach could be used for NSCLC cell lines, or three ROS1 fusions currently reported in patient tumors, SLC34A2-ROS (long form), SLC34A2-ROS (short Form), and exclusive detection of CD74-ROS. The qPCR assay utilizes two different primers for the multiplex cDNA synthesis reaction, one cDNA primer can be specific for a region present in both long and short forms of SLC34A2, and another cDNA primer can be C74 Present exclusively in genes. Both cDNA primers are strategically placed just 5 'of each fusion break point for ease of cDNA extension through the translocation region of three different ROS1 fusions.

The second qPCR Insight ROS1 Fusion Screen ™ described herein is referred to herein as Insight ROS1 Fusion Screen ™ v2. In summary, Insight ROS1 Fusion Screen ™ v2 is the SLC34A2-ROS1 when present in the cDNA synthesis phase that generates three possible cDNAs, cleaved wild type SLC34A2, cleaved wild type CD74, and certain patient specimens. Or consists of a potential extension and synthesis of the immediate translocation region of the CD74-ROS1 fusion. To prevent carryover of the reaction's one-step phase to the detection phase, all cDNA primers have a low Tm (to prevent annealing during high temperature real-time reactions) and reverse transcriptase is Inactivated at 65 ° C. for 20 minutes. The nascent cDNA can then be used in qPCR detection reactions using primers specific for the ROS1 region juxtaposed to the translocation point in both SLC34A2 or CD74 ROS1 fusions (FIG. 7). Again, the distance between the start site of cDNA synthesis and the nucleotide separating the position of the primer that mediates the actual qPCR detection reaction is kept to a minimum to circumvent the problem with degraded FFPE RNA. The ROS1-specific qPCR kinase reaction then amplifies the ROS1 cDNA region present only in the fusion form translocated to SLC34A2-ROS1 or CD74-ROS1. The wild type ROS1 kinase region is absent in each sample because it lacks any ROS1-specific cDNA primer present in the reverse transcription phase of the assay (FIG. 7). Specific sequences are listed in Table 7.

Example 6: Determination of sensitivity and specificity of both Insight ROS1 FISH and qPCR-based assays Both the Insight ROS1 FISH assay and the RT-qPCR assay were evaluated by use of various cancer cell lines or tissue samples. For example, Table 8 shows two types of cell lines, one cell line expressing the FIG-ROS derivative and one expressing both SLC34A2-ROS fusions (long and short) at various expression levels. Expressed in Normal lung total RNA consisting of full-length ROS1 only at the wild-type level can be used as a background control.
RNA extraction and one-step qPCR procedures follow well established experimental protocols.
References
Aquaviva J, Wong R, Charest A. The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer.Biochimica et Biophysica Acta 1795: 37-52, 2009.

Bergethon K, Shaw AT, Ou SH, Katayama R, Lovly CM, McDonald NT, Massion PP, Siwak-Tapp C, Gonzalez A, Fang R, Mark EJ, Batten JM, Chen H, Wilner KD, Kwak EL, Clark JW, Carbone DP, Ji H, Engelman JA, Mino-Kenudson M, Pao W, Iafrate AJ.ROS1 rearrangements define a unique molecular class of lung cancers J Clin Oncol. 2012 Mar 10; 30 (8): 863-70. Epub 2012 Jan 3.

Bilous M, Morey A, Armes J, Cummings M, Francis G. Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridization and immunohistochemistry. Pathology 38: 120-124, 2006.

Bolzer A, Craig JM, Cremer T, Speicher MR. A complete set of repeat-depleted, PCR-amplifiable, human chromosome-specific painting probes. Cytogenet Cell Genet 84: 233-240, 1999.

Camidge DR, Hirsch FR, Varella-Garcia M, Franklin WA. Finding ALK-positive lung cancer: what are we really looking for? J Thorac Oncol 6: 411-413, 2011.

Camidge DR, Kono SA, Flacco A, Tan AC, Doebele RC, Zhou Q, Crino L, Franklin WA, Varella-Garcia M. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res 16: 5581-5590, 2010.

Carbone A, Botti G, Gloghini A, Simone G, Truini M, Curcio MP, Gasparini P, Mangia A, Perin T, Salvi S, Testi A, Verderio P. Delineation of HER2 gene status in breast carcinoma by silver in situ hybridization is reproducible among laboratories and pathologists. J Mol Diagn 10: 527-536, 2008.

Carlson RW, Moench SJ, Hammond ME, Perez EA, Burstein JH, Allred DC, Vogel CL, Goldstein LJ, Somlo G, Gradishar WJ, Hudis CA, Jahanzeb M, Stark A, Wolff AC, Press MF, Winer EP, Paik S , Ljung BM; NCCN HER2 Testing in Breast Cancer Task Force.HER2 testing in breast cancer: NCCN Task Force report and recommendations.J Natl Compr Canc Netw 4 Suppl 3: S1-22, 2006.

Charest A, Kheifets V, Park J, Lane K, McMahon K, Nutt CL, Housman D. Oncogenic targeting of an activated tyrosine kinase to the Golgi apparatus in a glioblastoma.Proc Natl Acad Sci USA 100: 916-921, 2003.

Charest A, Lane K, McMahon K, Park J, Preisinger E, Conroy H, Housman D. Fusion of FIG to the receptor tyrosine kinase ROS in a glioblastoma with an interstitial del (6) (q21q21). Genes Chromosomes Cancer 37: 58 -61, 2003.

Charest A, Wilker EW, McLaughlin ME, Lane K, Gowda R, Coven S, McMahon K, Kovach S, Feng Y, Yaffe MB, Jacks T, Housman D. ROS fusion tyrosine kinase activates a SH2 domain-containing phosphatase-2 / phosphatidylinositol 3-kinase / mammalian target of rapamycin signaling axis to form glioblastoma in mice.Cancer Res 66: 7473-7481, 2006.

Craig JM, Kraus J, Cremer T. Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography. Hum Genet 100: 472-476, 1997.

Dandachi N, Dietze O, Hauser-Kronberger C. Chromogenic in situ hybridization: a novel approach to a practical and sensitive method for the detection of HER2 oncogene in archival human breast carcinoma.Lab Invest 82: 1007-1014, 2002.

Dugan LC, Pattee MS, Williams J, Eklund M, Sorensen K, Bedford JS, Christian AT. Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH. Chromosome Res 13: 27-32, 2005.

El-Deeb IM, Park BS, Jung SJ, Yoo KH, Oh CH, Cho SJ, Han DK, Lee JY, Lee SH.Design, synthesis, screening and molecular modeling study of a new series of ROS1 receptor tyrosine kinase inhibitors. Med Chem Lett 19: 5622-5626, 2009.

El-Deeb IM, Yoo KH, Lee SH.ROS receptor tyrosine kinase: a new potential target for anticancer drugs. Med Res Rev (2011) 31: 794-818.

Francis GD, Jones MA, Beadle GF, Stein SR.Bright-field in situ hybridization for HER2 gene amplification in breast cancer using tissue microarrays: correlation between chromogenic (CISH) and automated silver-enhanced (SISH) methods with patient outcome. Pathol 18: 88-95, 2009.

Gallegos Ruiz MI, Floor K, Vos W, Grunberg K, Meijer GA, Rodriquez JA, Giaccone G. 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.

Garcia-Garcia E, Gomez-Martin C, Angulo B, Conde E, Suarez-Gauthier A, Adrados M, Perna C, Rodriguez-Peralto JL, Hidalgo M, Lopez-Rios F. Hybridization for human epidermal growth factor receptor 2 testing in gastric carcinoma: a comparison of fluorescence in-situ hybridization with a novel fully automated dual-colour silver in-situ hybridization method.Histopathology 59: 8-17, 2011.

Gruver AM, Peerwani Z, Tubbs RR.Out of the darkness and into the light: bright field in situ hybridisation for delineation of ERBB2 (HER2) status in breast carcinoma.J Clin Pathol 63: 210-219, 2010.

Gu TL, Deng X, Huang F, Tucker M, Crosby K, Rimkunas V, Wang Y, Deng G, Zhu L, Tan Z, Hu Y, Wu C, Nardone J, MacNeill J, Ren J, Reeves C, Innocenti G , Norris B, Yuan J, Yu J, Haack H, Shen B, Peng C, Li H, Zhou X, Liu X, Rush J, Comb MJ.Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.PLoS ONE 6 (1): e15640, 2011.

Hirsch FR, Herbst RS, Olsen C, Chansky K, Crowley J, Kelly K, Franklin WA, Bunn PA Jr, Varella-Garcia M, Gandara DR. Increased EGFR gene copy number detected by fluorescent in situ hybridization predicts outcome in non-small -cell lung cancer patients treated with cetuximab and chemotherapy. J Clin Oncol 26: 3351-3357, 2008.

Hout D, Xue L, Choppa P, Handshoe J, Bouman D, Schmidt K, Bloom K, Ross D, Nickols J, Morris SW.Insight ALK Screen ^TM , a highly sensitive and specific RT-qPCR first-line screening assay for the comprehensive detection of all oncogenic anaplastic lymphoma kinase fusions. AACR Annual Meeting 2011

Kim H, Yoo SB, Choe JY, Paik JH, Xu X, Nitta H, Zhang W, Grogan TM, Lee CT, Jheon S, Chung JH.Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.J Thorac Oncol 6: 1359-1366, 2011.

Kwak EL, Bang YJ, Camnidge DR, Shaw AT, Solomon B, Maki RG, Ou SH, Dezube BJ, Janne PA, Costa DB, Varella-Garcia M, Kim WH, Lynch TJ, Fidias P, Stubbs H, Engleman JA, Sequist LV, Tan W, Gandhi L, Mino-Kenudson M, Wei GC, Shreeve SM, Ratain MJ, Settleman J, Christensen JG, Haber DA, Wilner K, Slagia R, Shapiro GI, Clark JW, Iafrate AJ. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 363: 1693-1703, 2010.

Lauter G, Soll I, Hauptmann G. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.Neural Dev 6: 10, 2011.

Lauter G, Soll I, Hauptmann G. Two-color fluorescent in situ hybridization in the embryonic zebra fish brain using differential detection systems.BMC Dev Biol 11: 43, 2011.

Leong AS, Haffajee Z. Microwaves for chromogenic in situ hybridization.Method Mol Biol 724: 79-89, 2011.

Linton K, Hey Y, Dibben S, Miller C, Freemont A, Radford J, Pepper S. Biotechniques. 2009 Jul; 47 (1): 587-96.Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.

Lovly CM, Heuckmann JM, de Stanchina E, Chen H, Thomas RK, Liang C, Pao W. Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors. Cancer Res 71 (14): 4920-4931, 2011 .

Lucas JN, Wu X, Guo E, Chi LE, Chen Z. An efficient chemical method to generate repetitive sequences depleted DNA probes. Am J Med Genet 140: 2115-2120, 2006.

Mathew P, Sanger WG, Weisenburger DD, Valentine M, Valentine V, Pickering D, Higgins C, Hess M, Cui X, Srivastava DK, Morris SW.Detection of the t (2; 5) (p23; q35) and NPM- ALK fusion in non-Hodgkin's lymphoma by two-color fluorescence in situ hybridization.Blood 89: 1678-1685, 1997.

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, 2011.

Mosquera JM, Dal Cin P, Mertz KD, Perner S, Davis IJ, Fisher DE, Rubin MA, Hirsch MS. Validation of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinomas.Diagn Mol Pathol 20: 129- 137, 2011.

Newkirk HL, Knoll JH, Rogan PK.Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect.Nucleic Acids Res 33: e191, 2005.

Nitta H, Hauss-Wegrzyniak B, Lehrkamp M, Murillo AE, Gaire F, Farrell M, Walk E, Penault-Llorca F, Kurosumi M, Dietel M, Wang L, Loftus M, Pettay J, Tubbs RR, Grogan TM. of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH). Diagn Pathol 3: 41, 2008 .

Passoni L, Longo L, Collini P, Coluccia AM, Bozzi F, Podda M, Gregorio A, Gambini C, Garaventa A, Pistoia V, Del Grosso F, Tonini GP, Cheng M, Gambacorti-Passerini C, Anichini A, Fossati- Bellani F, Di Nicola M, Luksch R. Mutation-independent anaplastic lymphoma kinase overexpression in poor prognosis neuroblastoma patients.Cancer Res 69: 7338-7346, 2009.

Pavlekovic M, Schmid MC, Schmider-Poignee N, Spring S, Pilhofer M, Gaul T, Fiandaca M, Loffler FE, Jetten M, Schleifer KH, Lee NM. Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment. J Microbiol Methods 78: 119-126, 2009.

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 Immunohistochem Mol Morphol 12: 259-265, 2004.

Powell RD, Pettay JD, Powell WC, Roche PC, Grogan TM, Hainfeld JF, Tubbs RR. Metallographic in situ hybridization. Hum Pathol 38: 1145-1149, 2007.

Qian X, Lloyd RV. Recent developments in signal amplification methods for in situ hybridization.Diagn Mol Pathol 12: 1-13, 2003.

Rauch J, Wolf D, Craig JM, Hausmann M, Cremer C. Quantitative microscopy after fluorescence in situ hybridization-a comparison between repeat-depleted and non-depleted DNA probes. J Biochem Biophys Methods 44: 59-72, 2000.

Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J , Ren JM, Yuan J, Bakalarski CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J, Comb MJ.Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131: 1190-1203, 2007.

Rimkunas V, Crosby K, Kelly M, Silver M, Hincman K, Polakiewicz R, Li D, Zhou X, Haack H. Frequencies of ALK and ROS in NSCLC FFPE tumor samples utilizing a highly specific immunohistochemistry-based assay and FISH analysis. Clin Oncol 28: 15s (suppl; abstr 10536), 2010.

Rogan PK, Cazcarro PM, Knoll JH. Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization.Genome Res 11: 1086-1094, 2001.

Ruhe JE, Streit S, Hart S, Wong CH, Specht K, Knyazev P, Knyazeva P, Knyazeva T, Tay LS, Loo HL, Foo P, Wong W, Pok S, Lim SJ, Ong H, Luo M, Ho HK , Peng K, Lee TC, Bezler M, Mann C, Gaertner S, Hoefler H, Iacobelli S, Peter S, Tay A, Brenner S, Venkatesh B, Ullrich A. Genetic alterations in the tyrosine kinase transcriptome of human cancer cell lines. Cancer Res 67: 11368-11376, 2007.

Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L, Gray NS, Wilner K, Christensen JG, Demetri G, Shapiro GI, Rodig SJ, Eck MJ, Janne PA.The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers.Cancer Res. 2010 Dec 15; 70 (24): 10038-43.

Sequist LV, Gettinger S, Senzer NN, Martins RG, Janne PA, Lilenbaum R, Gray JE, Iafrate AJ, Katayama R, Hafeez N, Sweeney J, Walker JR, Fritz C, Ross RW, Grayzel D, Engelman JA, Borger DR , Paez G, Natale R. Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in patients with molecularly defined non-small-cell lung cancer.J Clin Oncol 28: 4953-4960, 2010.

Sholl LM, Iafrate JA, Chou YP, Wu MT, Goan YG, Su L, Huang YT, Christiani DC, Chirieac LR.Validation of chromogenic in situ hybridization for detection of EGFR copy number amplification in nonsmall cell lung carcinoma.Mod Pathol 20: 1028-1035, 2007.

Sonnenberg-Riethmacher E, Walter B, Riethmacher D, Godecke S, Birchmeier C. The c-ros tyrosine kinase receptor controls regionalization and differentiation of epithelial cells in the epididymis. Genes Dev 10: 1184-1193, 1996.

Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, Asaka R, Hamanaka W, Ninomiya H, Uehara H, Choi YL, Satoh Y, Okumura S, Nakagawa K, Mano H, Ishikawa Y. RET, ROS1 and ALK fusions in lung cancer.Nat Medicine 18 (3): 378-381, 2012.

Terry J, Barry TS, Horsman DE, Hsu FD, Gown AM Huntsman DG, Nielsen TO.Fluorescence in situ hybridization for the detection of t (X; 18) (p11.2; q11.2) in a synovial sarcoma tissue microarray using a breakapart-style probe. Diagn Mol Pathol 14: 77-82, 2005.

Van de Vijver M, Bilous M, Hanna W, Hofmann M, Kristel P, Penault-Llorca F, Ruschoff J. Chromogenic in situ hybridisation for the assessment of HER2 status in breast cancer: an international validation ring study.Breast Cancer Res 9: R68, 2007.

Ventura RA, Martin-Subero JI, Jones M, McParland J, Gesk S, Mason DY, Siebert R. FISH analysis for the detection of lymphoma-associated chromosome abnormalities in paraffin-embedded tissue.J Mol Diagn 8: 141-151, 2006 .

Verrant JA. 2008. Methods and composition to generate unique sequence DNA probes labeling of DNA probes and the use of these probes. US 2009/0220955 A1, filed September 20, 2006.

Walter J, Joffe B, Bolzer A, Albiez H, Benedetti PA, Muller S, Speicher MR, Cremer T, Cremer M, Solovei I. Towards many colors in FISH on 3D-preserved interphase nuclei. Cytogenet Genome Res 114: 367-378 , 2006.

Webb TR, Slavish J, George RE, Look AT, Xue L, Jiang Q, Cui X, Rentrop WB, Morris SW.Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy.Expert Rev Anticancer Ther 9: 331-356, 2009.

Wlodarska I, De Wolf-Peeters C, Falini B, Verhoef G, Morris SW, Hagemeijer A, Van den Berghe H. The cryptic inv (2) (p23q35) defines a new molecular genetic subtype of ALK-positive anaplastic large-cell lymphoma Blood 92: 2688-2695, 1998.

Yan J, Guilbault E, Masse J, Ronsard M, DeGrandpre P, Forest JC, Drouin R. Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei.Clin Genet 58: 309 -318, 2000.

Yoo SB, Lee HY, Park JO, Choe G, Chung DH, Seo JW, Chung JH. Reliability of chromogenic in situ hybridization for epidermal growth factor receptor gene copy number detection in non-small-cell lung carcinomas: a comparison with fluorescence in situ hybridization study. Lung Cancer 67: 301-305, 2010.

Yoshida A, Tsuta K, Nitta H, Hatanaka Y, Asamura H, Sekine I, Grogan TM, Fukayama M, Shibata T, Furuta K, Kohno T, Tsuda H. Bright-field dual-color chromogenic in situ hybridization for diagnosing echinoderm microtubule -associated protein-like 4 anaplastic lymphoma kinase-positive lung adenocarcinomas. J Thorac Oncol 6: 1677-1686, 2011.

Zhang W, McElhinny A, Nielsen A, Wang M, Miller M, Singh S, Rueger R, Rubin BP, Wang Z, Tubbs RR, Nagle RB, Roche P, Wu P, Pestic-Dragovich L. Automated brightfield dual-color in situ hybridization for detection of mouse double minute 2 gene amplification in sarcomas.Appl Immunohistochem Mol Morphol 19: 54-61, 2011.

Zordan A. Fluorescence in situ hybridization on formalin-fixed, paraffin-embedded tissue sections.Method Mol Biol 730: 189-202, 2011.

Claims (34)

  A method of diagnosing ROS1-related cancer in a subject having cancer, obtaining a tissue sample from the subject, isolating nucleic acid from the tissue sample, and performing a nucleic acid amplification process on the nucleic acid; Detecting the presence of or measuring the amount of nucleic acid associated with one or a combination of one or both of the wild type ROS1 and ROS1 kinase domains in the tissue sample, wherein the amplicon is compared to a normal control. An increase, or presence of an amplicon, indicates that the subject has ROS1-related cancer.   2. The nucleic acid from the tissue sample is RNA, and the method further comprises synthesizing cDNA from the RNA sample, and the method comprises performing PCR on the cDNA. the method of.   The method of claim 1, wherein the nucleic acid amplification process is reverse transcription polymerase chain reaction (RT-PCR).   The method according to claim 2, wherein the RT-PCR is real-time RT-PCR.   The real-time RT-PCR uses a probe that is complementary to the product sequence of the real-time RT-PCR, and the probe has a reporter dye at its end and a quencher dye at the other end. Method.   6. The method of claim 5, wherein the probe is selected from the group consisting of SEQ ID NO: 6, 9, 19, or 22.   The method of claim 1, wherein the nucleic acid amplification process is real-time PCR.   The PCR, RT-PCR, or real-time PCR reaction forward and reverse specifically hybridizes with the wild type ROS1 sequence, and forward and reverse specifically hybridizes with the wild type ROS1 kinase domain sequence. 8. A method according to claim 2, 3 or 7, comprising the use of directional primer pairs.   9. The method of claim 8, wherein the at least one reverse primer comprises SEQ ID NO: 5, 8, 12, 14, 15, 18, or 21.   The method of claim 9, wherein the at least one forward primer comprises SEQ ID NO: 4, 7, 13, 15, 17, or 20.   9. The method of claim 8, wherein the at least one forward primer hybridizes to the extracellular region of wild type ROS1.   13. The method of claim 12, wherein the forward primer is SEQ ID NO: 13 or 20.   9. The method of claim 8, wherein the RT-PCR or real-time PCR reaction comprises a forward use capable of specifically hybridizing with the ROS1 kinase domain.   14. The method of claim 13, wherein the forward primer is that of SEQ ID NO: 4, 7, 15, or 17.   9. The method of claim 8, wherein the reverse primer or the wild type ROS1 and ROS1 kinase are the same primer.   9. The method of claim 8, further comprising determining a cycle threshold (Ct) value of wild type ROS1 and wild type ROS1 kinase, wherein a high (Ct) value of wild type ROS1 compared to ROS1 kinase indicates the presence of the fusion. The method described.   A forward primer that binds to ROS1 fusion partner at 5 ′ of the fusion breakpoint and a reverse primer that binds to ROS1 at 3 ′ of the fusion breakpoint, said primer extending throughout the fusion, ROS1 and The method of claim 1, wherein detection of the presence of an amplicon having both fusion partner nucleic acids indicates the presence of the fusion. a) a first amplification reaction using a forward primer that binds to the ROS1 fusion partner, wherein the amplicon from said first reaction is used as a template in a second amplification reaction;
b) a second amplification reaction after the first reaction, wherein a primer specific for the ROS1 sequence 3 ′ of the fusion breakpoint is used for the second amplification reaction; ,
c) detecting the presence of ROS1 in the amplicon from the second reaction, wherein detection of ROS1 in the amplicon from the second reaction indicates the presence of a ROS1 fusion;
The method of claim 1 comprising:   The cancer is neuroblastoma, breast cancer, ovarian cancer, colorectal cancer, non-small cell lung cancer, diffuse large B cell lymphoma, squamous cell carcinoma of the esophagus, anaplastic large cell lymphoma, neuroblastoma, inflammatory 2. The method of claim 1, wherein the method is selected from the group consisting of a myofibroblastic tumor, malignant histiocytosis, bile duct cancer, renal cancer, and glioblastoma.   2. The method of claim 1, further comprising subsequently treating a subject identified as having a ROS1-related cancer by administering to the subject a ROS1 inhibitor.   A method of diagnosing ROS1-related cancer in a subject, wherein a nucleic acid in a cell hybridizes with a first probe that hybridizes with ROS1 kinase and a ROS1 sequence 3 'of said fusion breakpoint of ROS1. Detection of the disrupted locus indicated by the differentially labeled probe, the isolated probe indicates the presence of a ROS1 fusion, which is in contact with a ROS1-related cancer The method of indicating the presence of   (A) a first primer labeled with a first detection reagent, wherein the first primer is a reverse primer, and the reverse primer encodes the amino acid sequence of SEQ ID NO: 1 or its complement A first primer that is one or more polynucleotides that hybridize to the first polynucleotide to be, and (b) at least one second primer, wherein the second primer is a forward primer A kit for diagnosing ROS1-related cancer, wherein the forward primer is one or more polynucleotides that hybridize with a second polynucleotide encoding wild-type ROS1.   23. The kit of claim 22, wherein the forward primer specifically hybridizes with the extracellular region of wild type ROS1.   24. The kit of claim 23, wherein the forward primer is SEQ ID NO: 13 or 20.   23. The kit of claim 22, wherein the forward primer specifically hybridizes with the ROS1 kinase domain.   26. The kit of claim 25, wherein the forward primer is that of SEQ ID NO: 4, 7, 15, or 17.   24. The kit of claim 22, further comprising second forward and reverse primers.   23. The kit of claim 22, wherein the reverse primer is SEQ ID NO: 5, 8, 12, 14, 16, 18, or 21.   The first forward and reverse primers specifically hybridize with the kinase domain of wild-type ROS1, and the second forward and reverse primers specifically hybridize with wild-type ROS1. The kit according to 22.   24. The kit of claim 22, further comprising a control primer pair.   32. The kit of claim 30, wherein the control primer pair specifically hybridizes with COX5B.   23. The kit of claim 22, wherein the first and second primers are labeled with first and second detection reagents, respectively.   23. The kit of claim 22, further comprising a polynucleotide probe, the probe being about 20 to about 30 nucleotides in length, including a reporter dye at one end and a quenching dye at the other end.   34. The kit of claim 33, wherein the polynucleotide probe comprises the sequence set forth in SEQ ID NO: 6, 9, 19, or 22.