MDM2-Containing Double Minute Chromosomes And Methods Therefore

Abstract

Contemplated systems and methods allow for computational genomic analysis using paired-end sequence analysis and split read refinement to thereby identify high-confidence breakpoints associated with high copy numbers and orientation of rearrangements, which is then the basis for full reconstruction of double minutes (DM). In especially preferred aspects, the DM will also include an oncogene or tumor suppressor gene, and/or may be found in blood or blood derived fluids.

What is claimed is: 
     
         1 . A method of analyzing genomic data, comprising:
 determining a relative copy number between a tumor genomic sequence and a matched normal genomic sequence;   identifying putative breakpoints in the tumor genomic sequence and the matched normal genomic sequence;   refining the putative breakpoints to identify a breakpoint location and an orientation of the tumor genomic sequence;   using a read support threshold to confirm the breakpoint as a significant breakpoint;   using the relative copy number, the significant breakpoint, and the orientation to determine a genomic arrangement having a circular solution.   
     
     
         2 . The method of  claim 1  wherein the step of determining the relative copy number is performed using dynamic windowing and/or wherein the step of identifying putative breakpoints is performed using discordant paired reads. 
     
     
         3 . The method of  claim 1  wherein the genomic arrangement is determined by generating a breakpoint graph and solving the breakpoint graph to arrive at the circular solution. 
     
     
         4 . The method of  claim 1  wherein the step of refining the putative breakpoints is performed using fragmenting the tumor genomic sequence and comparing the fragments with a reference database. 
     
     
         5 . The method of  claim 1  wherein the read support threshold is user-determined. 
     
     
         6 . The method of  claim 1  wherein the circular solution is indicative of a double minute chromosome as the genomic arrangement. 
     
     
         7 . The method of  claim 1  wherein the tumor genomic sequence is from a tumor that is a solid tumor, and wherein the tumor genomic sequence is isolated from genetic material present in a biological fluid. 
     
     
         8 . The method of  claim 7  wherein the biological fluid is blood. 
     
     
         9 . The method of  claim 8  wherein the solid tumor is glioblastoma multiforme or non-small cell lung cancer. 
     
     
         10 . A method of analyzing genomic data, comprising:
 associating a copy number of a tumor genomic sequence with a breakpoint in the tumor genomic sequence upon reaching a read support threshold for the breakpoint;   determining orientation of the tumor genomic sequence; and   determining genomic arrangement using the copy number, position of the breakpoint, and orientation of the tumor genomic sequence.   
     
     
         11 . The method of  claim 10  wherein the read support threshold is a user-defined read support threshold. 
     
     
         12 . The method of  claim 10  wherein the step of determining genomic arrangement is performed by generating a breakpoint graph using the copy number of the tumor genomic sequence, the position of the breakpoint within a genome, and the orientation of the tumor genomic sequence, wherein in the breakpoint graph the copy number is expressed as an edge and wherein the breakpoint position is expressed as a vertex. 
     
     
         13 . A method of analyzing genomic data of a solid tumor, comprising:
 identifying the solid tumor as a tumor of which at least a portion of a tumor genome is present in a biological fluid;   obtaining from a patient the biological fluid and isolating the at least portion of the tumor genome; and   using the isolated at least portion of the tumor genome to analyze the genomic data according to a method of  claim 1  or  claim 10 .   
     
     
         14 . The method of  claim 13  wherein the at least portion of the tumor genome is present as a double minute chromosome. 
     
     
         15 . The method of  claim 13  further comprising a step of identifying an oncogene or a tumor suppressor gene within the isolated at least portion of the tumor genome. 
     
     
         16 . The method of  claim 15  further comprising a step of treating the patient using a pharmaceutical regimen that targets the oncogene or tumor suppressor gene. 
     
     
         17 . A method of analyzing genomic data of a solid tumor, comprising:
 obtaining from a patient a biological fluid and isolating at least a portion of a tumor genome from the biological fluid; and   determining if a region surrounding an oncogene exhibit a clustered pattern of breakpoints indicative of an amplified double minute.   
     
     
         18 . The method of  claim 17  wherein oncogene is a wild type or mutant form of EGFR, c-Myc, or MDM2. 
     
     
         19 . The method of  claim 17  wherein the step of determining comprises use of a method according to  claim 1  or  claim 10 . 
     
     
         20 . The method of  claim 17  wherein the solid tumor is glioblastoma multiforme or non-small cell lung cancer, and wherein the biological fluid is blood. 
     
     
         21 . A method of de-novo diagnosing a neoplastic disease, comprising:
 obtaining a biological sample from a patient and isolating a nucleic acid from the sample;   analyzing the nucleic acid for a copy number of a genomic sample and a breakpoint in the genomic sample;   associating the copy number of the genomic sequence with the breakpoint in the genomic sequence upon reaching a read support threshold for the breakpoint;   determining orientation of the genomic sequence; and   determining genomic arrangement using the copy number, position of the breakpoint, and the orientation of the genomic sequence; and   using the genomic arrangement to determine likelihood for the neoplastic disease.   
     
     
         22 . The method  claim 21  further comprising a step of identifying the genomic arrangement as a double minute. 
     
     
         23 . The method  claim 22  further comprising a step of identifying an oncogene or tumor suppressor gene in the genomic arrangement. 
     
     
         24 . The method  claim 21  wherein the neoplastic disease is a gastric cancer, a colon cancer, a prostate cancer, a lung cancer, a leukemia, or a breast cancer.