US 2026/0269010 A1 · CELL-FREE DETECTION OF METHYLATED BREAST TUMOR

Abstract

Provided herein is a method for detecting a tumour that can be applied to cell-free samples, to cell-free detect circulating tumour DNA. The method utilizes detection of adjacent methylation signals within a single sequencing read as the basic positive tumour signal, thereby decreasing false positives. The method comprises extracting DNA from a cell-free sample obtained from a subject, bisulphite converting the DNA, amplifying regions methylated in cancer (CpG islands, CpG shores, and/or CpG shelves), generating sequencing reads, and detecting tumour signals. To increase sensitivity, biased primers designed based on bisulphite converted methylated sequences can be used. Target methylated regions can be selected from a pre-validated set according to the specific aim of the test. Absolute number, proportion, and/or distribution of tumour signals may be used for tumour detection or classification. The method is also useful in, predicting, prognosticating, and/or monitoring response to treatment, tumour load, relapse, cancer development, or risk.

A method of detecting the presence of breast cancer, comprising:

amplifying multiple regions of tumour DNA extracted from a cell-free sample obtained from a human subject with a plurality of PCR primer pairs;
wherein the multiple regions are either
(I) the regions amplified by a primer pair from each of (1) to (19) below:
(1) SEQ ID NOs:
166 and 167, SEQ ID NOs:
168 and 169, or SEQ ID NOs:
168 and 170,
(2) SEQ ID NOs:
171 and 172, SEQ ID NOs:
171 and 173, SEQ ID NOs:
174 and 175, SEQ ID NOs:
174 and 177, SEQ ID NOs:
176 and 177, SEQ ID NOs:
176 and 175, SEQ ID NOs:
178 and 179, or SEQ ID NOs:
919 and 920,
(3) SEQ ID NOs:
184 and 185, SEQ ID NOs:
186 and 187, SEQ ID NOs:
186 and 189, SEQ ID NOs:
188 and 189, or SEQ ID NOs:
188 and 187,
(4) SEQ ID NOs:
199 and 200, or SEQ ID NOs:
201 and 202,
(5) SEQ ID NOs:
213 and 214, SEQ ID NOs:
215 and 216, or SEQ ID NOs:
217 and 218,
(6) SEQ ID NOs:
233 and 235, SEQ ID NOs:
234 and 235, SEQ ID NOs:
236 and 237, SEQ ID NOs:
236 and 238, SEQ ID NOs:
239 and 240, SEQ ID NOs:
239 and 242, SEQ ID NOs:
241 and 242, or SEQ ID NOs:
241 and 240,
(7) SEQ ID NOs:
243 and 244, SEQ ID NOs:
245 and 246, or SEQ ID NOs:
245 and 247,
(8) SEQ ID NOs:
254 and 255, or SEQ ID NOs:
256 and 257,
(9) SEQ ID NOs:
317 and 318,
(10) SEQ ID NOs:
652 and 653, SEQ ID NOs:
654 and 655, SEQ ID NOs:
656 and 657, or SEQ ID NOs:
658 and 659,
(11) SEQ ID NOs:
712 and 713, or SEQ ID NOs:
714 and 715,
(12) SEQ ID NOs:
736 and 737, SEQ ID NOs:
738 and 740, or SEQ ID NOs:
739 and 740,
(13) SEQ ID NOs:
787 and 788,
(14) SEQ ID NOs:
803 and 804,
(15) SEQ ID NOs:
789 and 190,
(16) SEQ ID NOs:
567 and 568,
(17) SEQ ID NOs:
429 and 430,
(18) SEQ ID NOs:
779 and 780, or SEQ ID NOs:
781 and 782, and
(19) SEQ ID NOs:
797 and 798, or
(II) the regions amplified by a primer pair from a plurality of (20) to (124) below:
(20) SEQ ID NOs:
1 and 2, SEQ ID NOs:
3 and 4, SEQ ID NOs:
3 and 5, SEQ ID NOs:
6 and 7, SEQ ID NOs:
8 and 9, SEQ ID NOs:
10 and 11, SEQ ID NOs:
12 and 13, or SEQ ID NOs:
14 and 15,
(21) SEQ ID NOs:
16 and 18, SEQ ID NOs:
17 and 18, SEQ ID NOs:
19 and 20, SEQ ID NOs:
19 and 21, SEQ ID NOs:
22 and 23, SEQ ID NOs:
24 and 25, SEQ ID NOs:
26 and 27, SEQ ID NOs:
28 and 29, SEQ ID NOs:
30 and 31, SEQ ID NOs:
32 and 33, SEQ ID NOs:
34 and 35, or SEQ ID NOs:
36 and 37,
(22) SEQ ID NOs:
56 and 57, SEQ ID NOs:
58 and 59, SEQ ID NOs:
60 and 61, SEQ ID NOs:
62 and 63, SEQ ID NOs:
64 and 65, or SEQ ID NOs:
66 and 67,
(23) SEQ ID NOs:
38 and 39, SEQ ID NOs:
40 and 41, SEQ ID NOs:
42 and 43, SEQ ID NOs:
44 and 45, SEQ ID NOs:
46 and 47, SEQ ID NOs:
48 and 49, SEQ ID NOs:
50 and 51, SEQ ID NOs:
52 and 53, or SEQ ID NOs:
54 and 55,
(24) SEQ ID NOs:
154 and 155, SEQ ID NOs:
156 and 157, SEQ ID NOs:
158 and 159, SEQ ID NOs:
160 and 161, SEQ ID NOs:
162 and 163, or SEQ ID NOs:
164 and 165,
(25) SEQ ID NOs:
68 and 69, SEQ ID NOs:
70 and 71, SEQ ID NOs:
72 and 73, SEQ ID NOs:
72 and 74, SEQ ID NOs:
75 and 76, SEQ ID NOs:
77 and 78, SEQ ID NOs:
79 and 80, or SEQ ID NOs:
81 and 82,
(26) 83 and 84, SEQ ID NOs:
85 and 86, SEQ ID NOs:
87 and 88, SEQ ID NOs:
87 and 89, SEQ ID NOs:
90 and 91, SEQ ID NOs:
92 and 93, SEQ ID NOs:
94 and 95, SEQ ID NOs:
96 and 97, or SEQ ID NOs:
98 and 99,
(27) 100 and 102, SEQ ID NOs:
101 and 102, SEQ ID NOs:
103 and 104, SEQ ID NOs:
105 and 106, SEQ ID NOs:
105 and 107, SEQ ID NOs:
108 and 109, SEQ ID NOs:
110 and 112, SEQ ID NOs:
111 and 112, SEQ ID NOs:
113 and 114, SEQ ID NOs:
115 and 116, SEQ ID NOs:
117 and 118, SEQ ID NOs:
119 and 120, SEQ ID NOs:
575 and 576, or SEQ ID NOs:
577 and 578,
(28) SEQ ID NOs:
121 and 123, SEQ ID NOs:
122 and 123, SEQ ID NOs:
124 and 125, SEQ ID NOs:
126 and 127, SEQ ID NOs:
128 and 129, SEQ ID NOs:
130 and 131, SEQ ID NOs:
132 and 133, SEQ ID NOs:
134 and 135, SEQ ID NOs:
136 and 137, or SEQ ID NOs:
829 and 830,
(29) SEQ ID NOs:
138 and 139, SEQ ID NOs:
140 and 141, SEQ ID NOs:
142 and 143, SEQ ID NOs:
144 and 145, SEQ ID NOs:
146 and 147, SEQ ID NOs:
148 and 149, SEQ ID NOs:
150 and 151, or SEQ ID NOs:
152 and 153,
(30) SEQ ID NOs:
166 and 167, SEQ ID NOs:
168 and 169, or SEQ ID NOs:
168 and 170,
(31) SEQ ID NOs:
171 and 172, SEQ ID NOs:
171 and 173, SEQ ID NOs:
174 and 175, SEQ ID NOs:
174 and 177, SEQ ID NOs:
176 and 177, SEQ ID NOs:
176 and 175, SEQ ID NOs:
178 and 179, or SEQ ID NOs:
919 and 920,
(32) SEQ ID NOs:
180 and 181, or SEQ ID NOs:
182 and 183,
(33) SEQ ID NOs:
184 and 185, SEQ ID NOs:
186 and 187, SEQ ID NOs:
186 and 189, SEQ ID NOs:
188 and 189, or SEQ ID NOs:
188 and 187,
(34) SEQ ID NOs:
190 and 191, SEQ ID NOs:
192 and 194, or SEQ ID NOs:
193 and 194,
(35) SEQ ID NOs:
195 and 196, or SEQ ID NOs:
197 and 198,
(36) SEQ ID NOs:
199 and 200, or SEQ ID NOs:
201 and 202,
(37) SEQ ID NOs:
203 and 204, or SEQ ID NOs:
205 and 206,
(38) SEQ ID NOs:
207 and 208, SEQ ID NOs:
209 and 210, SEQ ID NOs:
209 and 211, SEQ ID NOs:
211 and 212, or SEQ ID NOs:
211 and 210,
(39) SEQ ID NOs:
213 and 214, SEQ ID NOs:
215 and 216, or SEQ ID NOs:
217 and 218,
(40) SEQ ID NOs:
219 and 220, SEQ ID NOs:
219 and 221, or SEQ ID NOs:
222 and 223,
(41) SEQ ID NOs:
224 and 225, or SEQ ID NOs:
226 and 227,
(42) SEQ ID NOs:
228 and 229, SEQ ID NOs:
228 and 230, or SEQ ID NOs:
231 and 232,
(43) SEQ ID NOs:
233 and 235, SEQ ID NOs:
234 and 235, SEQ ID NOs:
236 and 237, SEQ ID NOs:
236 and 238, SEQ ID NOs:
239 and 240, SEQ ID NOs:
239 and 242, SEQ ID NOs:
241 and 242, or SEQ ID NOs:
241 and 240,
(44) SEQ ID NOs:
243 and 244, SEQ ID NOs:
245 and 246, or SEQ ID NOs:
245 and 247,
(45) SEQ ID NOs:
248 and 249, SEQ ID NOs:
250 and 251, SEQ ID NOs:
250 and 253, SEQ ID NOs:
252 and 253, or SEQ ID NOs:
252 and 251,
(46) SEQ ID NOs:
254 and 255, or SEQ ID NOs:
256 and 257,
(47) SEQ ID NOs:
258 and 259, SEQ ID NOs:
258 and 261, SEQ ID NOs:
260 and 261, SEQ ID NOs:
260 and 259, SEQ ID NOs:
262 and 264, or SEQ ID NOs:
263 and 264,
(48) SEQ ID NOs:
265 and 266, SEQ ID NOs:
267 and 268, SEQ ID NOs:
269 and 271, or SEQ ID NOs:
270 and 271,
(49) SEQ ID NOs:
272 and 274, SEQ ID NOs:
273 and 274, or SEQ ID NOs:
275 and 276,
(50) SEQ ID NOs:
277 and 278, SEQ ID NOs:
279 and 280, SEQ ID NOs:
279 and 281, SEQ ID NOs:
281 and 282, or SEQ ID NOs:
281 and 280,
(51) SEQ ID NOs:
283 and 284, or 285 and 286,
(52) SEQ ID NOs:
287 and 288, SEQ ID NOs:
287 and 290, SEQ ID NOs:
289 and 291, or SEQ ID NOs:
289 and 288,
(53) SEQ ID NOs:
291 and 292, SEQ ID NOs:
293 and 294, SEQ ID NOs:
295 and 297, SEQ ID NOs:
296 and 297, SEQ ID NOs:
298 and 299, SEQ ID NOs:
298 and 300, or SEQ ID NOs:
301 and 302,
(54) SEQ ID NOs:
303 and 304, or SEQ ID NOs:
305 and 306,
(55) SEQ ID NOs:
307 and 308,
(56) SEQ ID NOs:
309 and 310, SEQ ID NOs:
309 and 312, SEQ ID NOs:
311 and 312, SEQ ID NOs:
311 and 310, SEQ ID NOs:
313 and 314, SEQ ID NOs:
313 and 316, SEQ ID NOs:
315 and 316, or SEQ ID NOs:
315 and 314,
(57) SEQ ID NOs:
317 and 318,
(58) SEQ ID NOs:
319 and 320, SEQ ID NOs:
319 and 322, SEQ ID NOs:
321 and 322, or SEQ ID NOs:
321 and 320,
(59) SEQ ID NOs:
323 and 324,
(60) SEQ ID NOs:
325 and 326,
(61) SEQ ID NOs:
327 and 328, or SEQ ID NOs:
893 and 894,
(62) SEQ ID NOs:
329 and 330, SEQ ID NOs:
329 and 322, SEQ ID NOs:
331 and 332, or SEQ ID NOs:
331 and 330,
(63) SEQ ID NOs:
335 and 336, SEQ ID NOs:
337 and 338, SEQ ID NOs:
337 and 340, SEQ ID NOs:
339 and 340, SEQ ID NOs:
340 and 339, SEQ ID NOs:
341 and 342, SEQ ID NOs:
341 and 344, SEQ ID NOs:
343 and 344, or SEQ ID NOs:
343 and 342,
(64) SEQ ID NOs:
333 and 334,
(65) SEQ ID NOs:
345 and 346, SEQ ID NOs:
345 and 348, SEQ ID NOs:
347 and 348, or SEQ ID NOs:
347 and 346,
(66) SEQ ID NOs:
349 and 350, SEQ ID NOs:
349 and 352, SEQ ID NOs:
351 and 352, SEQ ID NOs:
351 and 350, or SEQ ID NOs:
353 and 354,
(67) SEQ ID NOs:
355 and 356, SEQ ID NOs:
355 and 358, SEQ ID NOs:
357 and 358, or SEQ ID NOs:
357 and 356,
(68) SEQ ID NOs:
359 and 360, or SEQ ID NOs:
361 and 362,
(69) SEQ ID NOs:
613 and 614, SEQ ID NOs:
615 and 616, or SEQ ID NOs:
617 and 618,
(70) SEQ ID NOs:
619 and 620, or 621 and 622,
(71) SEQ ID NOs:
623 and 624, SEQ ID NOs:
625 and 626, or SEQ ID NOs:
625 and 627,
(72) SEQ ID NOs:
628 and 629, SEQ ID NOs:
628 and 631, SEQ ID NOs:
630 and 631, SEQ ID NOs:
630 and 629, or SEQ ID NOs:
632 and 633,
(73) SEQ ID NOs:
634 and 635, or SEQ ID NOs:
636 and 637,
(74) SEQ ID NOs:
638 and 639, or SEQ ID NOs:
640 and 641,
(75) SEQ ID NOs:
642 and 643, SEQ ID NOs:
642 and 645, SEQ ID NOs:
644 and 645, SEQ ID NOs:
644 and 643, SEQ ID NOs:
646 and 647, SEQ ID NOs:
646 and 649, SEQ ID NOs:
648 and 649, or SEQ ID NOs:
650 and 651,
(76) SEQ ID NOs:
652 and 653, SEQ ID NOs:
654 and 655, SEQ ID NOs:
656 and 657, or SEQ ID NOs:
658 and 659,
(77) SEQ ID NOs:
660 and 661, or SEQ ID NOs:
865 and 866,
(78) SEQ ID NOs:
662 and 663, or SEQ ID NOs:
664 and 665,
(79) SEQ ID NOs:
666 and 667, SEQ ID NOs:
666 and 669, SEQ ID NOs:
668 and 669, SEQ ID NOs:
668 and 667, or SEQ ID NOs:
670 and 671,
(80) SEQ ID NOs:
672 and 673, SEQ ID NOs:
672 and 675, SEQ ID NOs:
674 and 675, SEQ ID NOs:
674 and 673, SEQ ID NOs:
676 and 677, or SEQ ID NOs:
678 and 679,
(81) SEQ ID NOs:
680 and 682, SEQ ID NOs:
681 and 682, or SEQ ID NOs:
683 and 684,
(82) SEQ ID NOs:
685 and 686, SEQ ID NOs:
685 and 688, SEQ ID NOs:
687 and 688, SEQ ID NOs:
687 and 686, SEQ ID NOs:
689 and 690, or SEQ ID NOs:
691 and 692,
(83) SEQ ID NOs:
694 and 694, or SEQ ID NOs:
695 and 696,
(84) SEQ ID NOs:
697 and 698, SEQ ID NOs:
697 and 700, SEQ ID NOs:
699 and 700, SEQ ID NOs:
699 and 698, SEQ ID NOs:
701 and 702, SEQ ID NOs:
701 and 704, SEQ ID NOs:
703 and 704, SEQ ID NOs:
703 and 702, SEQ ID NOs:
705 and 706, or SEQ ID NOs:
705 and 707,
(85) SEQ ID NOs:
708 and 709, or SEQ ID NOs:
710 and 711,
(86) SEQ ID NOs:
712 and 713, or SEQ ID NOs:
714 and 715,
(87) SEQ ID NOs:
716 and 717, SEQ ID NOs:
716 and 719, SEQ ID NOs:
718 and 719, SEQ ID NOs:
718 and 717, or SEQ ID NOs:
720 and 721,
(88) SEQ ID NOs:
722 and 723, or SEQ ID NOs:
724 and 725,
(89) SEQ ID NOs:
726 and 727, SEQ ID NOs:
726 and 729, SEQ ID NOs:
728 and 729, SEQ ID NOs:
728 and 727, or SEQ ID NOs:
730 and 731,
(90) SEQ ID NOs:
732 and 733, or SEQ ID NOs:
734 and 735,
(91) SEQ ID NOs:
736 and 737, SEQ ID NOs:
738 and 740, or SEQ ID NOs:
739 and 740,
(92) SEQ ID NOs:
741 and 742, or SEQ ID NOs:
743 and 744,
(93) SEQ ID NOs:
745 and 746, SEQ ID NOs:
745 and 748, SEQ ID NOs:
747 and 748, SEQ ID NOs:
747 and 746, SEQ ID NOs:
749 and 750, SEQ ID NOs:
749 and 752, SEQ ID NOs:
751 and 752, or SEQ ID NOs:
751 and 750,
(94) SEQ ID NOs:
753 and 754, or SEQ ID NOs:
755 and 756,
(95) SEQ ID NOs:
787 and 788,
(96) SEQ ID NOs:
335 and 336, SEQ ID NOs:
337 and 338, SEQ ID NOs:
337 and 340, SEQ ID NOs:
339 and 340, SEQ ID NOs:
339 and 338, SEQ ID NOs:
341 and 342, SEQ ID NOs:
341 and 344, SEQ ID NOs:
343 and 344, or SEQ ID NOs:
343 and 342,
(97) SEQ ID NOs:
783 and 784, or SEQ ID NOs:
785 and 786,
(98) SEQ ID NOs:
602 and 604,
(99) SEQ ID NOs:
759 and 760,
(100) SEQ ID NOs:
773 and 774,
(101) SEQ ID NOs:
801 and 802,
(102) SEQ ID NOs:
397 and 398,
(103) SEQ ID NOs:
813 and 814,
(104) SEQ ID NOs:
815 and 816,
(105) SEQ ID NOs:
803 and 804,
(106) SEQ ID NOs:
811 and 812,
(107) SEQ ID NOs:
789 and 190,
(108) SEQ ID NOs:
431 and 432
(109) SEQ ID NOs:
809 and 810,
(110) SEQ ID NOs:
791 and 792, or SEQ ID NOs:
793 and 794,
(111) SEQ ID NOs:
567 and 568,
(112) SEQ ID NOs:
479 and 480,
(113) SEQ ID NOs:
775 and 776,
(114) SEQ ID NOs:
429 and 430,
(115) SEQ ID NOs:
765 and 766, or SEQ ID NOs:
767 and 768,
(116) SEQ ID NOs:
777 and 778,
(117) SEQ ID NOs:
795 and 796,
(118) SEQ ID NOs:
363 and 364,
(119) SEQ ID NOs:
563 and 564,
(120) SEQ ID NOs:
486 and 486,
(121) SEQ ID NOs:
779 and 780, or SEQ ID NOs:
781 and 782,
(122) SEQ ID NOs:
797 and 798,
(123) SEQ ID NOs:
378 and 379, and
(124) SEQ ID NOs:
799 and 800,
wherein individual primer pairs of the plurality of PCR primer pairs are modified to include non-native DNA sequences corresponding to methylation specific versions of the multiple regions by selecting C residues to be replaced with T residues according to their methylation status within individual regions of the multiple selected regions;
generating sequencing reads from each of the amplified regions;
aligning the sequencing reads with a reference sequence for each region, wherein the reference sequence is obtained from a cell-free sample obtained from a healthy human subject and the aligning is performed by a computer;
and
detecting at least two adjacent CpG residues that are methylated in each of the two or more selected regions in the DNA extracted from the cell-free sample;
determining that a level of methylation within the two or more selected regions is greater than a level of methylation within the two or more selected regions in subjects without breast cancer, which is indicative of the human subject having breast cancer.

550 more elements

Cell-free detection of methylated breast tumor

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Cell-free detection of methylated breast tumor

Life of this patent

Abstract

Claims2