DNA methylation and mutational analysis methods for bladder cancer surveillance
The present disclosure relates to methods of monitoring bladder cancer patients and analyzing patient samples for presence of methylated DNA and optionally particular gene mutations. In some embodiments, analysis results are correlated with clinical outcome measures such as risk of bladder cancer recurrence.
1. A method of characterizing a urine sediment sample obtained from a patient with bladder cancer, comprising:
obtaining the urine sediment sample from the patient;
bisulfite converting DNA from the sample;
performing methylation-specific quantitative PCR on the bisulfite converted DNA to determine the amount and/or the concentration of methylated DNA in the sample from at least four genes selected from MEIS1, NKPD1, ONECUT2, KLF2, OSR1, SOX1, EOMES, DDX25, and TMEM106A, wherein the at least four genes comprise NKPD1 and/or KLF2, and from at least one reference gene.
2. The method of claim 1 , wherein the patient has been diagnosed with non-muscle-invasive bladder cancer.
3. The method of claim 1 , wherein the sample is obtained from the patient prior to cystoscopy or after a negative cystoscopy.
4. The method of claim 1 , wherein the at least four genes comprise each of MEIS1, NKPD1, ONECUT2, and KLF2.
5. The method of claim 1 , wherein the at least one reference gene comprises one or more of CTNS, TOP3A, COL2A and SLC24A3.
6. The method of claim 1 , wherein the genes further comprise at least one gene selected from EPHX3, IRX5, NID2, VIM, and ITPKB.
7. The method of claim 1 , wherein the genes comprise one of the following gene sets:
(a) MEIS1, NKPD1, ONECUT2, KLF2;
(b) MEIS1, NKPD1, ONECUT2, OSR1;
(c) MEIS1, NKPD1, KLF2, SOX1;
(d) MEIS1, NKPD1, ONECUT2, OSR1, TMEM106A; EPHX3;
(e) MEIS1, NKPD1, ONECUT2, KLF2, TMEM106A, IRX5;
(f) MEIS1, NKPD1, ONECUT2, KLF2, SOX1, TMEM106A; or
(g) MEIS1, NKPD1, ONECUT2, KLF2, TMEM106A, ITPKB.
8. The method of claim 1 , wherein the method further comprises analyzing the bisulfate converted DNA for mutations in one or both of FGFR3 and TERT.
9. The method of claim 1 , wherein the genes comprise each of NKPD1 and KLF2.