USE OF CIRCULATING CELL-FREE RNA FOR DIAGNOSIS AND/OR MONITORING CANCER
The invention provides compositions, methods, and systems for using cell-free RNA for improved detection of rare cells and/or species that are useful for diagnosing and/or monitoring cancer. The invention also provides for compositions, methods, and systems for early detection of cells that are resistant and/or developing resistance to cancer therapies.
1 . A method for detecting mRNA of a biomarker in a biological sample from an individual, the method comprising:
a. isolating cfRNA from the biological sample on a solid support, wherein the blood sample has been interacted with RNA stabilizer;
b. digesting DNA from the biological sample while the cfRNA is on the solid support;
c. eluting cfRNA at least once from the solid support;
d. reverse transcribing the cfRNA to cDNA;
e. reacting the cDNA with at least one primer that is specific for
i. a gene mutation specific to the biomarker, or
ii. a fusion transcript of the biomarker, or
iii. gene expression of the biomarker; and
wherein the biomarker is selected from the group consisting of ERCC1, EGFR, TS, AREG, EREG, VEGFR2, EML4ALK, ROS1, RET, c-Met, FGFR1, KRAS, BRAF, NRAS, Her-2, PIK3CA, KIT, GNAQ, and GNA11.
2 . The method of claim 1 wherein the eluate of step (c) is passed over the same column for a dual elution prior to the reverse transcribing the cfRNA to cDNA.
3 . The method of claim 1 wherein the individual has or is suspected of having cancer.
4 . The method of claim 1 , wherein the biological sample is a blood sample or a plasma sample.
5 . The method of claim 4 , wherein the plasma sample is processed within 7 days of interacting with the RNA stabilizer.
6 . The method of claim 1 wherein random hexamers are used in step (d) to reverse transcribe the RNA to cDNA.
7 . The method of claim 1 , further comprising sequencing the cDNA.
8 . The method of claim 7 , wherein the sequencing comprises polymerase chain reaction (PCR)-based sequencing.
9 . The method of claim 8 , wherein the PCR-based sequencing is next generation sequencing.