US 20200263256A1 INI ( 19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2020/0263256 A1 ZHANG et al . ( 43 ) Pub . Date : Aug. 20 , 2020 ( 54 ) METHYLATION MARKERS FOR ( 60 ) Provisional application No. 62 / 466,328 , filed on Mar. DIAGNOSING HEPATOCELLULAR 2 , 2017 , provisional application No. 62 / 466,329 , filed CARCINOMA AND LUNG CANCER on Mar. 2 , 2017 , provisional application No. 62/569 , 462 , filed on Oct. 6 , 2017 . ( 71 ) Applicants: You Health Oncotech , Limited , Grand Cayman (KY ); The Regents of the Publication Classification University of California , Oakland, CA ( 51 ) Int. Cl . ( US ) C12Q 1/6886 ( 2006.01 ) ( 72 ) Inventors: Kang ZHANG , San Diego , CA ( US ) ; ( 52 ) U.S. CI . Rui HOU , Shenyang ( CN ) ; Lianghong CPC C12Q 1/6886 ( 2013.01 ) ; C12Q 2600/154 ZHENG , Shenyang ( CN ) ( 2013.01 ) ; C12Q 2600/118 ( 2013.01 ) ( 57 ) ABSTRACT ( 21 ) Appl. No .: 16 / 672,365 Disclosed herein , in certain embodiments , are methods and kits for diagnosing a subject as having hepatocellular car cinoma ( HCC ) or lung cancer. In some instances, also ( 22 ) Filed : Nov. 1 , 2019 described herein are methods of determining the prognosis of the subject having HCC or lung cancer . In additional Related U.S. Application Data instances, described herein are methods of determining the ( 63 ) Continuation of application No. 15 / 910,857 , filed on specific staging of HCC or lung cancer in a subject. Mar. 2 , 2018 , now Pat . No. 10,513,739 . Specification includes a Sequence Listing . Patent Application Publication Aug. 20 , 2020 Sheet 1 of 66 US 2020/0263256 A1 FIG . IA Simputation . ... " LISTENT FIG . 1B Karimo base.com on Sat Sample 3 Patent Application Publication Aug. 20 , 2020 Sheet 2 of 66 US 2020/0263256 A1 FIG . 2A ARALLISISSA Truepositiverate MA False positive rate FIG . 2B Composite Patent Application Publication Aug. 20 , 2020 Sheet 3 of 66 US 2020/0263256 A1 FIG . 2C Normal ' ' . ' ' . ? Patent Application Publication Aug. 20 , 2020 Sheet 4 of 66 US 2020/0263256 A1 FIG . 2D Tata Patent Application Publication Aug. 20 , 2020 Sheet 5 of 66 US 2020/0263256 A1 FIG . 3A Compositescore H Healthy control Liver disease HCC FIG . 3B AFPng/ml(Log) ++ Healthy control sease HCC Patent Application Publication Aug. 20 , 2020 Sheet 6 of 66 US 2020/0263256 A1 FIG . 3C LUNC p = 0.04 p < 0.001 Compositescore 1 www Non smoking Smoking LUNC Healthy control Healthy control Patent Application Publication Aug. 20 , 2020 Sheet 7 of 66 US 2020/0263256 A1 FIG . 4A - FIG . 4D FIG . 4A FIG . 4B --- + Compositescore Sefore treatment Response Progression FIG . 4C FIG . 4D Patent Application Publication Aug. 20 , 2020 Sheet Bof 66 US 2020 / 0263256A1 FIG.4E - FIGH FIG . 4E FIG . 4F P14? 11 ? || ?? HT ? ? ??? ?? ? ???? ?? ? ? FIG.4G FIG . 4H Patent Application Publication Aug. 20 , 2020 Sheet 9 of 66 US 2020/0263256 A1 FIG . 41 - FIG . 4L FIG . 41 FIG . 4J LULU wwwwwwwwwww Airte Turepositiverate AFPngimi(Log) ?????? False positive rate Voodoo.pno AFPngimi(Log) AFPng/ml(Log) Mwd FIG . 4K FIG . 4L Patent Application Publication Aug. 20 , 2020 Sheet 10 of 66 US 2020/0263256 A1 FIG . 5 FIG . 5A FIG . 5B wwws FIG . 5C FIG . 5D $% 3.X FIG . 5E FIG . 5F Wybierajinekologiyalarinitieghuliwa ? ? itirsimittarithimpiantiitinutiktu mitihani 20 Patent Application Publication Aug. 20 , 2020 Sheet 11 of 66 US 2020/0263256 A1 FIG . 6A FIG . 6B we Patent Application Publication Aug. 20 , 2020 Sheet 12 of 66 US 2020/0263256 A1 FIG . 7 FIG . 7A ?? VissWwiiwasi Bilder FIG . 7B Patent Application Publication Aug. 20 , 2020 Sheet 13 of 66 US 2020/0263256 A1 FIG . 8 FIG . 8A LUNC WHCC FIG . 8B tiam Tinano Patent Application Publication Aug. 20 , 2020 Sheet 14 of 66 US 2020/0263256 A1 SØSXS3 WXSQ vongipusosoggenstossXL ***** SWEX Was SC $ % : $ YOX . FIG.9 ? ???? *** Xasax XXX8 18733XS $ SHAD $$$ VYYS : * 3 V. $ X883 * XX Patent Application Publication Aug. 20 , 2020 Sheet 15 of 66 US 2020/0263256 A1 FIG . 10 FIG . 10A FIG . 10B Patent Application Publication Aug. 20 , 2020 Sheet 16 of 66 US 2020/0263256 A1 FIG . 11 NANFIG . 11A . FIG . 11B LO Patent Application Publication Aug. 20 , 2020 Sheet 17 of 66 US 2020/0263256 A1 il 02 FIG.12A N W WS wwire Patent Application Publication Aug. 20 , 2020 Sheet 18 of 66 US 2020/0263256 A1 su FIG.12B . Uno Wait Patent Application Publication Aug. 20 , 2020 Sheet 19 of 66 US 2020/0263256 A1 Yesus 232,33DE Fase63%. MS23 433H200W TUBESHIKOPESEBOK Aixmixabink265% FIG.13 XeryDenny ng FaxExtisticaXXX M3 SantosGKC Zekeriyasi WUMVIXX3 vo SSBS3,33333433X316SSSSS Patent Application Publication Aug. 20 , 2020 Sheet 20 of 66 US 2020/0263256 A1 FIG . 14 FIG . 14A FIG . 14B I I FIG . 14C FIG . 14D LUTA XX Patent Application Publication Aug. 20 , 2020 Sheet 21 of 66 US 2020/0263256 A1 21.708 controlSmoking IS P w FIG.15B FIG.15D 17 H MO H 800-134Out HH HE HE NonsmokingSmokingcontrol $ FIG.15A FIG.15C Patent Application Publication Aug. 20 , 2020 Sheet 22 of 66 US 2020/0263256 A1 FIG . 16 FIG . 16A FIG . 16B dowwwwwwwwwwwww wwwwwwwwwwww 1 cdscore FIG . 16C FIG . 16D S FIG . 16E FIG . 16F cdscore FIG . 16G FIG . 16H W Patent Application Publication Aug. 20 , 2020 Sheet 23 of 66 US 2020/0263256 A1 FIG . 16 cont . FIG . 161 FIG . 16J CERA30m FIG . 16K FIG . 16L FIG . 16M FIG . 16N he FIG . 160 FIG . 16P Patent Application Publication Aug. 20 , 2020 Sheet 24 of 66 US 2020/0263256 A1 FIG . 160 Truepositiverate cd - score (AUC :0.977 ) n : norma = 2247, LUNG = 599 WWW o normal956 , LUNC - 205 False positive rate FIG . 16R Truepositiverate n : normat= 2247 , HCC = 889 AFP ( AUC :0.835 ) False positive rate Patent Application Publication Aug. 20 , 2020 Sheet 25 of 66 US 2020/0263256 A1 FIG . 17 FIG . 17A HCC FIG . 17B ??. FIG . 17C FIG . 17D ??? ??? ???? ??? ?????? ????? ?????? ?????? FIG . 17E FIG . 17F "!***tip 0. Patent Application Publication Aug. 20 , 2020 Sheet 26 of 66 US 2020/0263256 A1 FIG . 18 FIG . 18A Stage I LUNC : n = 94 Benign nodules : n = 126 29191m FIG . 18B Stage I LUNC 27 Specificity ( % ) Patent Application Publication Aug.20 , 2020 Sheet 27 of 66 US 2020/0263256 A1 ?? ? ? ? ? ? ?? ? FIG.19B FIG.19D ? ?? ? ? ? ? FIG.19A FIG190. Patent Application Publication Aug. 20 , 2020 Sheet 28 of 66 US 2020/0263256 A1 FIG . 20A FIG . 20B Patent Application Publication Aug. 20 , 2020 Sheet 29 of 66 US 2020/0263256 A1 FIG . 20C HCC Patent Application Publication Aug. 20 , 2020 Sheet 30 of 66 US 2020/0263256 A1 wy 12:2 IT'S tyring 224722727222277 72 3 3 3 ?? ? ? } } 3 3 ?& WMWWW 262 3.12 Patent Application Publication Aug. 20 , 2020 Sheet 31 of 66 US 2020/0263256 A1 32 WS ???? FIG.22 WiKAWXHLWEBWE $ 2 Patent Application Publication Aug. 20 , 2020 Sheet 32 of 66 US 2020/0263256 A1 FIG . 23A I T colore FIG . 23B ????? Patent Application Publication Aug. 20 , 2020 Sheet 33 of 66 US 2020/0263256 A1 FIG . 24 23 TotalBettylationCOB(kg) TotalMethylationof TotalMotinylationcopy min F F B 6 * 3 3 22 CEADom! 34 - - - ~ 23 $ ten ? ta 28 & po vor k RE Patent Application Publication Aug. 20 , 2020 Sheet 34 of 66 US2020 / 0263256 A1 FG 25 - ;??%?daspg#** PS ?? g Patent Application Publication Aug. 20 , 2020 Sheet 35 of 66 US 2020/0263256 A1 Votiskionaset 369HIC1* Hiponosisanesis Pramurydataset HOROKO Maskassaamiot BruwataCox LASSOC0x Prognosispredictivemodelmarkers 8hy FIG.26 HCC(ICGANormatblandGSEL aristhywion marksis100selecttogla Targetedbisulfatesequencing USUONAandamCENA 40tablewu8kers Targetedsisutfstesequisiting inB6mstientsStununtalmod az1933(10984CCMamal} Voittistinaset 3-658038382791ormai Proiasdesign markers)24 DiBgnosisandygis 715HCC560Normalrm1275( akasselestion modelpredictiveDiagnosismarkers overlapping@by 30markers) Patent Application Publication Aug. 20 , 2020 Sheet 36 of 66 US 2020/0263256 A1 FIG . 27A FIG . 27B Patent Application Publication Aug. 20 , 2020 Sheet 37 of 66 US 2020/0263256 A1 FIG . 27C FIG . 27D 28 Patent Application Publication Aug. 20 , 2020 Sheet 38 of 66 US 2020/0263256 A1 FIG . 27E 2000 Trueposiverate AUCCIO !! . SO False positive rate FIG . 27F Truepostiverate auc 80.96 02 False positive rate Patent Application Publication Aug. 20 , 2020 Sheet 39 of 66 US 2020/0263256 A1 FIG . 27G FIG . 27H *** Patent Application Publication Aug. 20 , 2020 Sheet 40 of 66 US 2020/0263256 A1 FIG . 28A p = 0.85 10 cd-score o Healthy controls Liver disease HCC FIG . 28B le 05 1e +03 AFP(Log) + Healthy controls Liver disease HCC Patent Application Publication Aug. 20 , 2020 Sheet 41 of 66 US 2020/0263256 A1 FIG . 28C 20 10 cd-score T Normal HCC HCOWA FIG . 28D 20 1 88 CHA HCC Before HCC with HCC treatment Response Progression Patent Application Publication Aug. 20 , 2020 Sheet 42 of 66 US 2020/0263256 A1 FIG . 28E cd-score surgery Recurrence FIG . 28F 10 cd-score Normal HCC of HCC of HCC of HCC of controls Stage / Stage || Stage III Stage IV Patent Application Publication Aug. 20 , 2020 Sheet 43 of 66 US 2020/0263256 A1 FIG . 28G Truepositiverate AUC of cd score : 0.969 ( 0.926-0.977 ) X2 To False positive rate FIG . 28H 20 7e36 220 cd-score Initial diagnosis Progression Response Recurrence Patent Application Publication Aug. 20 , 2020 Sheet 44 of 66 US 2020/0263256 A1 FIG . 281 5.8 AFP(Log) with HCC HEO Initial diagnosis Progression Response Recurrence FIG . 28J +0.005 cd-score HCC of HCC or Stage 1 Stage II Stage Ill Stage IV Patent Application Publication Aug. 20 , 2020 Sheet 45 of 66 US 2020/0263256 A1 FIG . 28K ** 0 p = 0.001 AFP(Log) Stagel Stage || Stage Ill Stage IV FIG . 29A Survivalprobability(%) Log - rank test o < 0.0001 Hazard Ratio = 0.16 ( 0.09 0.26 ) 200 300 500 600 Overall survival (Days ) Patent Application Publication Aug. 20 , 2020 Sheet 46 of 66 US 2020/0263256 A1 FIG . 29B Survivalprobability(%) Log - rank test p = 0.0014 Hazard Ratio = 0.32 (0.16-0.61 ) 200 300 500 600 Overall survival (Days ) Patent Application Publication Aug.