Personalized tumor biomarkers

Abstract

Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers revealed an average of nine rearranged sequences (range 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints were able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.

1 . A method of identifying a personalized tumor marker for a cancer patient, comprising:
 making a mate-paired library from tumor DNA of the patient, wherein mate pairs of said library comprise two genomic tags that are co-linear but not contiguous in a segment of the tumor DNA;   determining sequence of a plurality of mate pairs of the library;   determining regions of copy number differences among regions in the tumor DNA of the patient;   identifying mate paired tags which map within a region of copy number difference or spanning a boundary of copy number difference as potential markers of a tumor-specific DNA rearrangement in the cancer patient.   
     
     
         2 . The method of  claim 1  further comprising:
 identifying a breakpoint between, within, or spanning the regions of copy number differences and designing amplification primers that hybridize to priming sites that flank the breakpoint, wherein the priming sites are separated by between 20 and 200 basepairs in the tumor DNA. 
 
     
     
         3 . The method of  claim 1  further comprising:
 testing identified paired tags by comparing to non-tumor DNA or to sequence of non-tumor DNA, and confirming a tumor-specific DNA rearrangement if the two genomic tags of a mate paired tag are at different locations or in a different orientation within a chromosome or on different chromosomes of non-tumor DNA compared to tumor DNA. 
 
     
     
         4 . The method of  claim 3  wherein the non-tumor DNA is from the cancer patient. 
     
     
         5 . The method of  claim 3  further comprising:
 identifying a breakpoint in the DNA rearrangement and designing amplification primers that hybridize to priming sites that flank the breakpoint, wherein the priming sites are separated by less than 200 basepairs in the tumor DNA. 
 
     
     
         6 . The method of  claim 2  or  5 , further comprising:
 amplifying a DNA fragment using a template from the patient's tissues or body fluids and said amplification primers; 
 determining the amount or proportion of amplified DNA fragment in the patient's tissue or body fluid, wherein said amount is an indication of tumor burden. 
 
     
     
         7 . A method of assessing or detecting tumor in a patient, comprising:
 amplifying a DNA fragment using a template from the patient's tissues or body fluids and primers that span a patient-specific, tumor-specific rearrangement breakpoint, wherein the rearrangement breakpoint is between genes or gene loci involved in rearrangements in <1% of tumors of patients with the same type of tumor;   determining the amount or proportion of amplified DNA fragment in the patient's tissue or body fluid.   
     
     
         8 . The method of  claim 7  wherein the rearrangement breakpoint is between genes or gene loci involved in rearrangements in <0.1% of tumors of patients with the same type of tumor. 
     
     
         9 . The method of  claim 7  wherein the rearrangement breakpoint is not associated with a leukemia, lymphoma, sarcoma, or prostate cancer. 
     
     
         10 . The method of  claim 7  wherein the step of determining is performed with samples obtained from the cancer patient at a plurality of times. 
     
     
         11 . The method of  claim 10  wherein the plurality of times are before and during an anti-tumor therapy. 
     
     
         12 . The method of  claim 10  wherein the plurality of times are during an anti-tumor therapy. 
     
     
         13 . The method of  claim 10  wherein the plurality of times are before and after surgery. 
     
     
         14 . The method of  claim 10  wherein the plurality of times are to monitor a patient in remission for relapse or recurrence. 
     
     
         15 . The method of  claim 7  wherein presence of an amount of amplified DNA fragment indicates residual disease. 
     
     
         16 . The method of  claim 7  wherein presence of an amount of amplified DNA fragment in a tumor margin indicates incomplete surgical resection. 
     
     
         17 . The method of  claim 7  wherein the template is from the patient's lymph nodes. 
     
     
         18 . The method of  claim 7  wherein the template is from the patient's blood. 
     
     
         19 . A method of identifying a personalized tumor marker for a cancer patient, comprising:
 determining sequence of two ends of each of a plurality of fragments of DNA from the cancer patient;   determining regions of copy number differences among regions in the tumor DNA of the patient;   identifying fragments of the plurality of fragments which map within a region of copy number difference or spanning a boundary of copy number difference as potential markers of a tumor-specific DNA rearrangement in the cancer patient.   
     
     
         20 . The method of  claim 19  further comprising:
 identifying a breakpoint between, within, or spanning the regions of copy number differences and designing amplification primers that hybridize to priming sites that flank the breakpoint, wherein the priming sites are separated by between 20 and 200 basepairs in the tumor DNA. 
 
     
     
         21 . The method of  claim 19  further comprising:
 testing two ends of a fragment by comparing to non-tumor DNA or to sequence of non-tumor DNA; and 
 identifying a tumor-specific DNA rearrangement if the two ends are at different locations or in a different orientation within a chromosome or on different chromosomes of non-tumor DNA compared to tumor DNA. 
 
     
     
         22 . The method of  claim 21  wherein the non-tumor DNA is from the cancer patient. 
     
     
         23 . The method of  claim 21  further comprising:
 identifying a breakpoint in the DNA rearrangement and designing amplification primers that hybridize to priming sites that flank the breakpoint, wherein the priming sites are separated by less than 200 basepairs in the tumor DNA. 
 
     
     
         24 . The method of  claim 20  or  23  further comprising:
 amplifying a DNA fragment using a template from the patient's tissues or body fluids and said amplification primers; 
 determining the amount or proportion of amplified DNA fragment in the patient's tissue or body fluid, wherein said amount is an indication of tumor burden. 
 
     
     
         25 . A method of identifying a personalized tumor marker for a cancer patient, comprising:
 testing a plurality of mate paired tags of a library of mate paired tags by comparing to non-tumor DNA or to sequence of non-tumor DNA, wherein each of said mate paired tags comprises two genomic tags that are co-linear but not contiguous in a segment of tumor DNA of the cancer patient; and   identifying a tumor-specific DNA rearrangement if the two genomic tags of a mate paired tag are at different locations or in a different orientation within a chromosome or on different chromosomes of non-tumor DNA compared to tumor DNA.   
     
     
         26 . The method of  claim 25  further comprising:
 identifying a breakpoint in the DNA rearrangement and designing amplification primers that hybridize to priming sites that flank the breakpoint, wherein the priming sites are separated by less than 200 basepairs in the tumor DNA. 
 
     
     
         27 . The method of  claim 25  wherein prior to the step of testing the mate paired tags are identified as being within regions of copy number difference relative to adjacent regions of the genome. 
     
     
         28 . The method of  claim 25  wherein prior to the step of testing the mate paired tags are enriched for tags from regions of copy number difference relative to adjacent regions of the genome. 
     
     
         29 . A method of identifying a personalized tumor marker for a cancer patient, comprising:
 testing two ends of a plurality of fragments of tumor DNA of the cancer patient by comparing to non-tumor DNA or to sequence of non-tumor DNA; and   identifying a tumor-specific DNA rearrangement if the ends of a fragment are at different locations or in a different orientation within a chromosome or on different chromosomes of non-tumor DNA compared to tumor DNA.   
     
     
         30 . The method of  claim 29  further comprising:
 identifying a breakpoint in the DNA rearrangement and designing amplification primers that hybridize to priming sites that flank the breakpoint, wherein the priming sites are separated by less than 200 basepairs in the tumor DNA. 
 
     
     
         31 . The method of  claim 29  wherein prior to the step of testing the plurality of fragments are identified as being within regions of copy number difference relative to adjacent regions of the genome. 
     
     
         32 . The method of  claim 29  wherein prior to the step of testing the plurality of fragments are enriched for tags from regions of copy number difference relative to adjacent regions of the genome. 
     
     
         33 . A method of screening for a cancer in a human, comprising:
 testing a plurality of mate paired tags of a library of mate paired tags by comparing to normal DNA or to sequence of normal DNA, wherein each of said mate paired tags comprises two genomic tags that are co-linear but not contiguous in a segment of DNA in the blood of the human; and   identifying DNA rearrangement if the two genomic tags of a mate paired tag are at different locations or in a different orientation within a chromosome or on different chromosomes of normal DNA compared to blood DNA, wherein the presence of a DNA rearrangement suggests the presence of a cancer in the human.   
     
     
         34 . A method of screening for a cancer in a human, comprising:
 testing two ends of a fragment of blood DNA of the human by comparing to normal DNA or to sequence of normal DNA; and   identifying a DNA rearrangement if the ends are at different locations or in a different orientation within a chromosome or on different chromosomes of normal DNA compared to blood DNA, wherein the presence of a DNA rearrangement suggests the presence of a cancer in the human.   
     
     
         35 . The method of  claim 33  or  34  wherein the normal DNA is lymphocyte DNA. 
     
     
         36 . The method of  claim 33  or  34  wherein the normal DNA is buccal DNA. 
     
     
         37 . The method of  claim 25  or  29  wherein the patient has a cancer that is not a leukemia, lymphoma, sarcoma, or prostate cancer. 
     
     
         38 . A kit for monitoring presence or amount of a breakpoint in a somatic DNA rearrangement in tumor DNA of a patient, comprising:
 one or more pairs of amplification primers, wherein each pair is complementary to priming sites on opposite sides of a breakpoint, wherein the priming sites are separated by less than 200 basepairs in the tumor DNA, and wherein the DNA rearrangement occurs in <1% of tumors of patients with the same type of tumor.   
     
     
         39 . The kit of  claim 38  wherein the tumor DNA is not from a leukemia, lymphoma, sarcoma, or prostate cancer. 
     
     
         40 . The kit of  claim 38  wherein the DNA rearrangement occurs in <0.1% of tumors of patients with the same type of tumor. 
     
     
         41 . The kit of  claim 38  wherein the kit comprises a plurality of pairs of amplification primers and the plurality of pairs are complementary to priming sites on opposite sides of a plurality of breakpoints in tumor DNA of the patient. 
     
     
         42 . The kit of  claim 38  wherein the kit further comprises a DNA polymerase for amplification. 
     
     
         43 . The kit of  claim 38  wherein the kit further comprises deoxyribonucleotides for amplification substrates. 
     
     
         44 . The kit of  claim 38  wherein the kit further comprises reagents for preparing template from tumor DNA of the patient.