Multiplexed Assay Using Differential Fragment Size to Identify Cancer Specific Cell-Free DNA
AssigneeCadex Genomics, Corp.
InventorsSudhir SINHA, Gary SPITZER, Hiromi BROWN, Patrick HALL
Life of this patent
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Abstract
A retrotransposable element-based multiplexed quantitative polymerase chain reaction (qPCR) assay system to quantitate and distinguish cell free DNA integrity and concentration in blood, plasma, and serum as a measure of minimum residual disease, therapeutic effectiveness, neoadjuvant effectiveness in a patient having stage I, stage II, stage III, or stage IV cancer, and disease progression, thereby improving patient outcomes.
Claims30
A method to quantitate the integrity of circulating cell free human DNA and implement a treatment of a patient, said method comprising:
- (a) providing a first and second sample of serum, plasma, urine, or other biological fluid from a subject wherein the first and second samples are obtained at least one week apart, at least 2 weeks apart, at least 3 weeks apart or at least 4 weeks apart, the first and second samples comprising cell free human DNA (cfDNA), the cfDNA comprising (i) a first and second short retrotransposable interspersed element (RE) target sequence having a length of between about 60 base pairs to about 135 base pairs and (ii) a long RE target sequence having a length of between 200 bp and 300 bp, wherein the first and second short targets differ in length;
- (b) quantitating each of the short and long RE targets in the first and second samples using a quantitative polymerase chain reaction (qPCR) method;
- (c) obtaining for each of the quantitated RE targets in the first and second samples a threshold cycle number;
- (d) comparing the threshold cycle number of each quantitated RE target with a standard curve to determine an amount of each of the quantitated RE targets that were present in the samples, wherein the amount of short RE targets in the second samples is indicative of the integrity of the circulating cell free DNA;
- (e) determining an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level, and
- (f) implement a treatment of a patient having an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level.
A multiplexed system for identifying an ineffective neoadjuvant or cancer treatment, or for characterizing cancer or MRD in humans, the system comprising:
- a. a sample of serum, plasma, urine, or other biological fluid from a human, the sample comprising cell free DNA, the cell free DNA comprising two short RE targets and a long RE target, the short RE targets having a length between 60 bp and 135 bp with the proviso that the two short RE targets differ in size sufficiently to distinguish their amplicons, the long RE target having a length between 200 and 300 bp, between 207 bp and 270 bp, or between 260 and 265 bp, the short RE targets and the long RE target being independent of each other, the sample further comprising an internal positive control comprising synthetic DNA;
- b. a TaqMan® probe corresponding to each of the short RE targets, the long RE target and the IPC, each probe comprising a detectable label that is distinct from the labels incorporated into the other probes;
- c. a forward primer and a reverse primer for amplifying each of the short RE target, the long RE target and the IPC;
- d. a DNA standard for generating standard curves for RE targets;
- e. a qPCR system for simultaneously amplifying the short RE targets, the long RE target and the IPC and for producing a threshold cycle number for each target;
- and
- f. a qPCR data analysis system for producing DNA quantitation values for each RE target by interpolation using threshold cycle numbers and standard curves and for using the DNA quantitation values to produce an indication of the integrity of the cell free DNA.
Dependencies are inferred from the claim text.