USOO9689041B2

(12) United States Patent (10) Patent No.: US 9,689,041 B2 Ye et al. (45) Date of Patent: Jun. 27, 2017

(54) METHOD AND KIT FOR DETERMINING IN 2005/0287544 A1 12/2005 Bertucci et al. VTRO THE PROBABILITY FOR AN 2008/0311574 A1 12/2008 Manne et al. 2011/O117083 A1 5, 2011 Bais et al. INDIVIDUAL TO SUFFER FROM COLORECTAL CANCER FOREIGN PATENT DOCUMENTS (75) Inventors: Xun Ye, Shanghai (CN); Fei Wu, CN 101.068936 A 11/2007 Shanghai (CN); Qinghua Xu, Zhejiang CN 101111604. A 1, 2008 EP O 201 184 A2 12, 1986 (CN); Fang Liu, Shanghai (CN); Xia EP 2 169 O78 A1 3, 2010 Meng, Shanghai (CN); Bruno Mougin, EP 2 177 615 A1 4/2010 Lyons (FR) FR 2 780 059 A1 12/1999 FR 2 816 711 A1 5, 2002 FR 2816958 A1 5, 2002 (73) Assignee: BIOMERIEUX, Marcy 1 Etoile (FR) WO WO 89.10977 A1 11, 1989 WO WO 90/01069 A1 2, 1990 (*) Notice: Subject to any disclaimer, the term of this WO WO 90.03382 A1 4f1990 patent is extended or adjusted under 35 WO WO 9006995 A1 6, 1990 WO WO 91 (02818 A1 3, 1991 U.S.C. 154(b) by 610 days. WO WO 91/19812 A1 12, 1991 WO WO 94,12670 A1 6, 1994 (21) Appl. No.: 14/007,439 WO 97,452O2 A1 12/1997 WO WO 99/15321 A1 4f1999 (22) PCT Filed: Mar. 23, 2012 WO 99,355OO A1 7, 1999 WO WO 99,53304 A1 10, 1999 WO WO 99,65926 A1 12, 1999 (86). PCT No.: PCT/CN2012/072931 WO WOOO/O5338 A1 2, 2000 S 371 (c)(1), WO WOOO,71750 A1 11, 2000 (2), (4) Date: Oct. 18, 2013 WO WO O1/44506 A1 6, 2001 (Continued) (87) PCT Pub. No.: WO2012/130103 OTHER PUBLICATIONS PCT Pub. Date: Oct. 4, 2012 Xu et al. (Cancer Biology & Therapy, 2011, vol. 11, No. 2, p. (65) Prior Publication Data 188-195).* Nov. 19, 2015 Office Action issued in U.S. Appl. No. 13/698.219. US 2014/OO578O2 A1 Feb. 27, 2014 Jul. 9, 2015 Office Action issued in U.S. Appl. No. 13/698.219. Abstract of ComBat: 'Combatting Batch Effects. When Combining (30) Foreign Application Priority Data Batches of Expression Microarray Data, obtained from http:// www.bu.edu/lab/wp-assets/ComBat/Abstract.html on Feb. 14. Mar. 25, 2011 (CN) ...... PCT/CN2011/072155 2013. Tachibana, T., et al., “Increased Intratumor Vol.24-Positive Natural (51) Int. Cl. Killer T Cells: A Prognostic Factor for Primary Colorectal Carci CI2O I/68 (2006.01) nomas,” Clin. Cancer Res., Oct. 15, 2005, pp. 7322-7327, vol. 11. (52) U.S. Cl. Liu, J. et al., “ profiling for nitric oxide prodrug CPC ..... CI2O 1/6886 (2013.01); C12O 2600/158 JS-K to kill HL-60 myeloid leukemia cells.” Genomics, 2009, pp. (2013.01) 32-38, vol. 94. (58) Field of Classification Search (Continued) None See application file for complete search history. Primary Examiner — Stephanie K Mummert (74) Attorney, Agent, or Firm — Oliff PLC (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT 4,672,040 A 6/1987 Josephson The present invention provides a method for determining in 4,683, 195 A 7, 1987 Mullis et al. vitro, in a peripheral blood sample, the probability for an 4,683.202 A 7, 1987 Mullis individual to Suffer from a colorectal cancer, using the 4,800,159 A 1, 1989 Mullis et al. 4,981,783 A 1/1991 Augenlicht comparison of the amount of expression products of nucleic 5,234,809 A 8, 1993 Boom et al. acids of of the individual to be tested with the amount 5,399,491 A 3, 1995 Kacian et al. of expression products of nucleic acids of the same genes 5,445,934 A 8, 1995 Fodor et al. obtained from a CRC group of patients constituting the 5,700,637 A 12/1997 Southern positive control and with the amount of expression products 5,744,305 A 4, 1998 Fodor et al. 5,750,338 A 5, 1998 Collins et al. of nucleic acids of the same genes obtained from a CNC 5,807,522 A 9, 1998 Brown et al. group of individuals constituting the negative control; and a 2004.0002082 A1 1/2004 Feinberg kit comprising specific binding partners for said expression 2004/0033516 A1 2/2004 Mougin products. 2004/0265230 A1* 12/2004 Martinez ...... A61K 39.0011 424,149 2005/O130170 A1 6/2005 Harvey et al. 27 Claims, No Drawings US 9,689,041 B2 Page 2

(56) References Cited Tyagi et al., “Molecular Beacons: Probes that Fluoresce Upon Hybridization.” Nature Biotechnology, Mar. 1996, vol. 14, pp. 303.308. FOREIGN PATENT DOCUMENTS Kricka, "Nucleic Acid Detection Technologies—Labels, Strategies, WO WO O1/44507 A1 6, 2001 and Formats.” Clinical Chemistry, 1999, vol. 45, No. 4, pp. 453 WO WO O2/4O711 A1 5, 2002 458. WO WO O2/O903.19 A1 11, 2002 Keller et al., “Non-Radioactive Labeling Procedures,” and “Hybrid WO WO O2/O90584 A2 11/2002 ization Formats and Detection Procedures.” DNA Probes, 1993, pp. WO 2005/O54508 A2 6, 2005 V and 173-253. WO WO2O08063414 * 5, 2008 ...... C12O 1/68 Chee et al., “Accessing Genetic Information with High-Density WO 2009/049228 A2 4/2009 DNA Arrays,” Science, Oct. 5, 1996, vol. 274, No. 5287, pp. WO 2009.126804 A2 10/2009 610-614. WO 2010/040571 A2 4/2010 Pease et al., “Light-Generated Oligonucleotide Arrays for Rapid WO 2010/0563.74 A2 5, 2010 DNA Sequence Analysis.” Proc. Natl. Acad. Sci. USA, May 1994, vol. 91, pp. 5022-5026. OTHER PUBLICATIONS Cheng et al., “Preparation and Hybridization Analysis of DNA Clemson, C.M. et al., “An Architectural Role for a Nuclear Noncod RNA from E. coli on Microfabricated Bioelectronic Chips,” Nature ing RNA: NEAT1 RNA is Essential for the Structure of Biotechnology, Jun. 1998, vol. 16, pp. 541-546. Paraspeckles.” Molecular Cell, Mar. 27, 2009, pp. 717-726, vol. 33. Livache et al., “Preparation of a DNA Matrix Via an Electrochemi Sheu, B., et al., “Up-regulation of Inhibitory Natural Killer Recep cally Directed Copolymerization of Pyrrole and Oligonuclieotides tors CD94/NKG2A with Suppressed Intracellular Perforin Expres Bearing a Pyrrole Group.” Nucleic Acids Research, 1994, vol. 22. sion of Tumor-Infiltrating CD8+ T Lymphocytes in Human Cervical No. 15, pp. 2915-2921. Carcinoma,” Cancer Res., Apr. 1, 2005, pp. 2921-2929, vol. 65, No. Ginot, “Oligonucleotide Micro-Arrays for Identification of 7. Unknown Mutations: How Far From Reality?” Human Mutation, McGilvray, R.W., et al., “NKG2D Ligand Expression in Human 1997, vol. 10, pp. 1-10. Colorectal Cancer Reveals Associations with Prognosis and Evi Cheng et al., “Microchip-Based Devices for Molecular Diagnosis of dence for Immunoediting.” Clin. Cancer Res., Nov. 15, 2009, pp. Genetic Diseases.” Molecular Diagnosis, 1996, vol. 1, No. 3, pp. 6993-7002, vol. 15, No. 22. 183-200. Apr. 1, 2011 International Search Report issued in International Bustin, “Quantification of mRNA Using Real-Time Reverse Tran Application No. PCT/EP2010/057843. scription PCR (RT-PCR): Trends and Problems,” Journal of Apr. 1, 2011 Written Opinion of the International Searching Author Molecular Endocrinology, 2002, vol. 29, p. 23-39. ity issued in International Application No. PCT/EP2010/057843. Giulietti et al., “An Overview of Real-Time Quantitative PCR: Dec. 19, 2014 Office Action issued in U.S. Appl. No. 13/698.219. Applications to Quantify Cytokine Gene Expression.” Methods, Apr. 16, 2014 Office Action issued in U.S. Appl. No. 13/698.219. 2001, vol. 25, pp. 386–401. U.S. Appl. No. 13/698.219, filed Nov. 15, 2012 in the name of Ye Irizarry et al., “Exploration, Normalization and Summaries of High et al. Density Oligonucleotide Array Probe Level Data.” Biostatistics, Killer Cell Lectin-Like Receptor Subfamily K. Member 1. 2003, vol. 4, No. 2, pp. 249-264. Datasheet online. Weizmann Institute of Science, Oct. 23, 2013 Maglottet al., “ Gene: Gene-Centered Information at NCBI.” retrieved on Apr. 4, 2014). Retrieved from the Internet:

Gene SEQ ID Mean t-test P Fold Direction Probeset id: Symbol: * NOS: signal ** value Change (in CRC) 227062 at NEAT1 1 621 3.84 10' 1.46 up 223204 at FAM198B 2, 3, 4 97 3.56 10-2 1.52 up 205785 at ITGAM 5, 6 95 1.35 107 1.32 up 213906 at MYBL1 7, 8 139 3.51 108 1.38 down 209339 at SIAH2 9 252 8.06 106 1.25 up 1553589 a at PDZK1IP1 10 407 9.32 10 1.37 down 1553991 s at VSIG10 11 65 1.47 10- 1.41 up Up: means that the mean signal for the CRC group is higher than in the CNC group Down means that the mean signal for the CRC group is lower than for CNC group * means Probeset id according to Affymetrix annotation version in 2010 (https://www.affymetrix.com analysisinetaffx.xmlauery, affx?netaffx=netaffx4 annot& requested=403680) means the identified gene and its variants or related sequences to said gene or variants *** means average signals observed for 100 CRCs and 100 CNCs array experiments

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 11

<21 Os SEQ ID NO 1 &211s LENGTH: 22743

US 9,689,041 B2 37 38 - Continued t cct taaaat ttgttctato tacttatatt taatttgatt taatggittac togcc.cagata 294 O ttgaga attg gttcaaat at tdagt.gtgtt toaatatatt atctggctta tittcaacatg 3 OOO agtaatatga gcaaaataag ttaaaacctg cqtctgatca attitt cotca tdactagaac 3 O 6 O taaaac agta aatttggaca at attaagcc ticaaataatc atcto caaac toctitctaac 312 O actittittaaa toagattgga agacatggac aaatcaggitt catgtgttgc atc.tt tatgt 318O c ctittgccaa tat coaagat catca catat gigtagatatt cacatggagt ttcaaattica 324 O gaatagatta ccattacctt cct gcc.ctta cacat cotac toctitattta aaagttctat 33 OO ttgttgactitt to attt cotgaaagtttalaa aatacaattt gagaatgttt ataatacatt 3360 citct cotgtc. ttitt cacggit tacgtctgtt attgctgaaa tacaccacat tittctttgtt 342O ctgg to aagg ttaact caat atctgttgttga aagagaacta ctaacaacgt tacaatagag 3480 gctagatttgaaaaaaaaaa totatagatc taattgatac aattgtagaa caaaatgtca 354 O aaataatgtt ttalagtataa gagaagatgg accaaggaga gagagat cat ttgaaaatct 36OO aattgtagct tttctaggct cacatt catg tact acttitt agcacccitta tdggctgtgc 366 O tcqc coccitg gacagttgag ctittggatta t ct tcct citt caattitt coc totattgacc 372 O cgagtgtc.t.c cct ctdct to tacagattta tag tact cott toggct cittitt gag to tccac 378 O ttitt acticac tdt citctggg atttittaaga t cottitt citt ct cittataaa totatic ct citt 384 O aatgaaaatt agcctaacaa aagtttggag actggaatcc tactittgagc cactgacttg 3900 aaataact ct tttggcaagt togcctgacat cotgtc.ttac caaggtggca tatttgcatt 396 O tttact.gctt aaaacatttt tttittittitta c catctittat c caaattitat cat attgatg 4 O2O gtaggactaa Caggctttitt agaagctggc tittaactittg agt ct caagc tacaatgctg 4 O8O ttgggcagcc tdgtott.ccc acgtgagggit tta actttgt ttatttgcct c cagttatt c 414 O caaaatgctt attaaatgaa agt cocagga acatgttitat tittagt cacc tittgctttitt 42OO aacaattittgttttgtaatc aatgagtaat t catgatgaa ttatttittga ctaatggata 426 O gcc.gaaggcc aggcttittaa ttctaatagg taatgttctt ctitttgtc.tt attgaaacaa 432O tgagaatact ctdtgcattt caaatgcact c cqattatgc tigtggittitta t t cacataag 438 O cacaatatgt gttittattta taactitcata acaaactitat aatataataa tttacct tag 4 44 O caga catgca aaa.gct tatt Cttgttgttgac titactitt citt taagctaata atataaaaat 4500 aaatatgtat cittaaaaatc tataataaaa cattagaaat taaagatatg togctttittat 456 O tittgcagatg agttcatttg Cttttgtaga tigtgtttitca gagct aggta Cagaggaatg 462O tittgctacct ttagcggtga aaaaagaaag agagt caaga attttgttgg attgttgtttg 468O tgttgtgcata tatttgatat cat cattata tttgtaatct ttggacttgt aat catago c 474. O tgtttatt ct actgtgcc at taaatatact ttaccttata cataacgaat aaaataccta 48OO gaagtagatt tatttacaaa aaaaaaaaaa aaa 4833

<210s, SEQ ID NO 3 &211s LENGTH: 4854 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 3 gtttittaaaa gotttgtatic ticittaaaacc atgcagoagt cagtttccaa gttittgctitt 6 O gcaatcagta gttittcaagg gagcttittaa agctgaactgaaatgtttga aatgtggaac 12 O actic ttgacc atgaaatatgttctacttac atgcct cago ctittaaaagt totttgcatt 18O

US 9,689,041 B2 41 42 - Continued gagcatcaga gct gaccata catttaaa.ca gaaatgctgg tittatttgca aaatcaccag 2580 tatattitt ct attgttgttcta taaaaaatca gtcatttaag tacaagaatc at attitt coa 264 O titcc tttitta gaaatttatt ttgttgtc.cc tatggaaatc attca catct gacaattitat 27 OO atgttaaaga gttt tact ct citctattittg gtccaatttg tat citagtgg ctgagaaatt 276 O aaataattct aaagtatgaa gttacct atc tdaaaatgta cittacagagt at cattittaa 282O aatggatgtc. tctittaaaaa ttttgttact tttaccalaca atgtaatata atttatgtat 288O attitt attaa taatagtgaa titcCttaaaa tttgttctat gtact tatat ttaatttgat 294 O ttaatggitta citgcc.cagat attgagaatt ggttcaaata ttgagtgttgt ttcaatatat 3 OOO tatctggctt atttcaa.cat gagtaatatg agcaaaataa gttaaaacct gcgtctgat c 3 O 6 O aattitt cotc atgact agaa ctaaaacagt aaatttggac aat attaagc citcaaataat 312 O catcto caaa citccttctaa cactttittaa atcagattgg aagacatgga caaat caggit 318O t catgtgttg catctt tatgtcc tittgcca at atccaaga t catcacata togg tagatat 324 O t cacatggag tittcaaattic agaatagatt accattacct tcc togcc citt acacatccta 33 OO cticcittattt aaaagttcta tttgttgactt tt cattt cott gaaagtttaa aaatacaatt 3360 tgagaatgtt tataatacat t ct ct cotgt cittitt cacgg ttacgtctgt tattgctgaa 342O atacaccaca ttittctttgt totggtcaag gttaact caa tatctgtgttgaaagagaact 3480 actaacaacg ttacaataga gqctagattt gaaaaaaaaa atctatagat ctaattgata 354 O

Caattgtaga acaaaatgtc. aaaataatgt tittaagtata agagaagatg gaccalaggag 36OO agagagat catttgaaaatc taattgtagc titt to taggc t cacatt cat gtact actitt 3660 tagcaccctt atgggctgtg citcqc cocct gga cagttga gctittggatt atct tcc tot 372 O t caattitt co citctattgac ccgagtgtct c cct c togctt ctacagattt at agtacticc 378 O ttggct ctitt tagt citcca ctitt tactica citgtc.tctgg gatttittaag atccttittct 384 O t ct cittataa atcatcct ct taatgaaaat tagcc talaca aaagtttgga gactggaatc 3900 c tactittgag ccactgactt gaaataactic titttggcaag ttgcc tigaca toctdtctta 396 O c caaggtggc at atttgcat titt tactgct taaaacattt tttitttittitt accatctitta 4 O2O tccaaattta t catattgat gig taggacta acaggcttitt tagaa.gctgg ctitta actitt 4 O8O gagt ct caag ctacaatgct gttgggcago Ctggit ct tcc cacgtgaggg tttalactittg 414 O tittatttgcc ticcagttatt coaaaatgct tattaaatga aagtic cc agg aacatgttta 42OO ttittagt cac ctittgcttitt taacaattitt gttttgtaat caatgagtaa tt catgatga 426 O attatttittg actaatggat agc.cgaaggc caggcttitta attctaatag gtaatgttct 432O t ctitttgtct tattgaaaca atgagaatac totgtgcatt toaaatgcac toccattatg 438 O ctgtggittitt attcacataa goacalatatgtgttt tattt ataactt cat aacaaactta 4 44 O taatataata atttacctta gcaga catgc aaaagctitat t cittgttgttga citt actittct 4500 ttaa.gctaat aatataaaaa taaatatgta t cittaaaaat citataataaa acattagaaa 456 O ttaaagatat gtgctttitta ttittgcagat gagttcattt gcttttgtag atgtgttitt c 462O agagct aggt acagaggaat gtttgctacc tittagcggtgaaaaaagaala gagagt caag 468O aattttgttg gattgttgttt gtgtgtgcat at atttgata t catcattat atttgtaatc 474. O tittggactitg taat catago citgtttatt c tactgtgcca ttaaatatac tttacctitat 48OO acataacgaa taaaatacct agaagtag at ttatttacaa aaaaaaaaaa aaaa 4854

US 9,689,041 B2 45 46 - Continued act acttgag aaatatttgt ttatatttitt citctaac atc atgctatgtg toagtctgaa 222 O catctgacaa cagaaattitc agittattatt ctagotaagt tittgaaaa.ca tttgt catgc 228O tgtttaatag aaaactgcaa accagaga.ca citgactic cat taataaacca tattttgtgc 234 O cgttittgact gttctgacca aatactaatg ggaacaattic ttgacgttitt totgttgctg 24 OO attgttaa.ca tagagcagtic tictacactac cct gaggcaa citctacattg gaacact gag 246 O gcttacagcc tdcaagagca totagagctga ccatacattt aaacagaaat gctggittitat 252O ttgcaaaatc accagtatat tittctattgt gtctataaaa aat cagt cat ttaagtacaa 2580 gaat catatt titc cattcct ttittagaaat ttattttgtt gtc.cc tatgg aaatcattca 264 O catctgacaa tittatatgtt aaagagttitt act ct ct cta ttittggit coa atttgtatict 27 OO agtggctgag aaattaaata attctaaagt atgaagttac citatctgaaa atgtact tac 276 O agagtat cat tittaaaatgg atgtc.t.ctitt aaaaattittgttactitttac caacaatgta 282O atataattta totatattitt attaataata gtgaatticct taaaatttgt totatgtact 288O tatatttaat ttgatttaat gigt tactgcc cagatattga gaattggttcaaatattgag 294 O tgttgtttcaa tat attatct ggct tatttic aacatgagta atatgagcaa aataagttaa 3 OOO aacctg.cgt.c tdatcaattt to ct catgac tagaactaaa acagtaaatt toggacaatat 3 O 6 O taagcct caa ataatcatct c caaactic ct tctaacactt tittaaat cag attggaagac 312 O atggacaaat caggttcatg togttgcatct titatgtc.ctt togccaatatic caagat catc 318O acatatggta gatatt caca toggagttt ca aattcagaat agattaccat tacct tcctg 324 O c cct tacaca t cc tactic ct tatttaaaag ttctatttgt gacttitt cat ttic ctgaaag 3300 tittaaaaata caatttgaga atgtttataa tacattct ct c ct gttcttitt cacggittacg 3360 tctgttattg ctgaaataca cca catttitc tttgttctgg toaaggittaa citcaatat ct 342O gtgtgaaaga gaactact aa caacgttaca atagaggcta gatttgaaaa aaaaaatcta 3480 tagatctaat tdatacaatt gtagaacaaa atgtcaaaat aatgttittaa gtataagaga 354 O agatggacca aggaga.gaga gat catttga aaatctaatt gtagcttitt C taggcticaca 36OO tt catgtact acttittagca ccct tatggg ctgtgct cqc ccc.ctggaca gttgagctitt 366 O ggattatctt cotcttcaat titt coct cita ttgaccc.gag tdt ct coctic togcttctaca 372 O gatttatagt act cottggc ticttittgagt citccacttitt act cactgtc. tctgggattit 378 O ttaagatcct titt cittct ct tataaatcat cotcttaatgaaaattagcc taacaaaagt 384 O ttggag actg gaatcc tact ttgagccact gactitgaaat aactic ttittg gcaagttgcc 3900 tgacatcc td tottaccalag gtggcatatt togcatttitta citgcttaaaa catttitttitt 396 O tttittaccat ctittatccaa attitat cata ttgatgg tag gactaac agg ctittittagaa 4 O2O gctggCttta actittgagtic tica agctaca atgctgttgg gcagcctggit Ctt Cocacgt 4 O8O gagggitttaa citttgttt at ttgcct coag titatt coaaa atgct tatta aatgaaagtic 414 O c caggaacat gtttattitta gttcacctittg citttittaa.ca attttgttitt gtaat caatg 42OO agtaattcat gatgaattat ttittgactaa tdgatagc.cg aaggc.caggc titttaattct 426 O aatagg taat gttcttcttt tdt cittattgaaacaatgag aatactctgt gcatttcaaa 432O tgcact coga t tatgctgtg gttittatt ca cataagcaca atatgtgttt tatttataac 438 O tt cataacaa acttataata taataattta cct tagcaga catgcaaaag cittatt cittg 4 44 O tgttgacttac titt ctittaag ctaataatat aaaaataaat atgitat citta aaaatctata 4500 ataaaacatt agaaattaaa gatatgtgct ttittattittg cagatgagtt catttgctitt 456 O

US 9,689,041 B2 57 58 - Continued aaaagaaaat aaaggaactt gagatgct tc titatgtcagc tigagaatgala gttagaagaa 26 O agcgaatt CC at Cacagcct ggaagtttitt Ctagctggtc tigtagttt C ct catggatg 32O ataa catgtc. taatacticta aatagccttg acgagcacac tagtgagttt tacag tatgg 38O atgaaaatca gcctgtgtct gct cagoaga attcacccac aaagttcct g gcc.gtggagg 44 O caaacgctgt gttatcct ct ttgcagacca toccagaatt togcagagact citagaactta SOO ttgaatctga t cotgtagca toggagtgacg ttaccagttt tdatatttct gatgctgctg 560 cittct c citat caaatccacc ccagttaa at taatgagaat t cago acaat gaaggagc.ca 62O tggaatgc.ca atttalacgt.c agt cttgtac ttgaagggala aaaaaac act totaatggtg 68O gcaa.ca.gtga agctgttcct ttaa.catc cc caaatatago caagtttagc act coaccag 74 O c catcct cag aaagaa.gaga aaaatgcgag togt catt C C cc aggcagc gaact taggg 8OO atggct catt gaacgatggt gigtaatatgg cqctaaaa.ca tacac cactgaaaacactac 86 O cattttct cott cacagttt ttcaacacat gtc.ctgg taa togaacaactt aatatagaaa 92 O atcc tt catt tacatcaa.cc cct atttgtg ggcagaaagc tict cattaca act cotcttic 98 O ataaggaaac aacticc caaa gatcaaaagg aaaatgtagg gtttagaaca cct act atta 2O4. O gaagat citat actggg tacc acaccaagaa citcct acticc ttittaagaat gcgcttgctg 21OO cticaggagaa aaaatatgga cct cittaaaa ttgtgtc.cca gcc acttgct ttcttggaag 216 O aagatatt.cg ggaagttitta aaagaagaala Ctggalacaga cct attic ct c aaagaggaag 222 O atgaac Ctgc titacaaaagc tigcaaacaag agaat accgc titctgggaag aaagt cagaa 228O aatcactagt Cittagata at tiggaaaaag aagaat Cagg CactCaactg ttgactgaag 234 O acattt caga catgcagt ca gaaaatagat titact acatc citt attaatg ataccattat 24 OO tggaaataca tdacaatagg togcaacttga titcctgaaaa acaagatata aattcaacca 246 O acaaaacata tacact tact aaaaagaaac caaac cctaa cact tccaaa gttgtcaaat 252O tggaaaagaa ticttcagt ca aattgttgaat gggaalacagt ggttt atggg alagacagaag 2580 acca actitat tatgactgaa caa.gcaagaa gat atctgag tacttacaca gctaccagta 264 O gtacttcaag agct ct cata citgitaattgt tattaaaatt gatgaaatgc cccactic cct 27 OO tactgcagtic tictact aaat taggttgcag tdaaatttitt citcaattagt tdtttittaaa 276 O gttgtaagat agc ccttitta atacagoat c titttittctat tctatatagt aggcagaaag 282O c tagtaagtic acttaagggg tagatagttt catagitttat tttittaa.gag atgagattitt 288O taaaaattgt ttittaaagaa caagatggga aaataataga atgttcatgg atttctaaaa 294 O gtaaattct catatatttitc titcacaagat atatgttgct act ct cittga tigctgcagtt 3 OOO ttgttataga taggtg tatgagtatatatg atttctgaaa ttagt citatg tatggaaagc 3 O 6 O acacatgatt titatgaagta Cttittgcc.ca tdtgctgatt tact taggct accatttaca 312 O aagaalacaca ttgaaaagga atttalaagga aggatagaaa gttgcactac taattittittg 318O tttitttittitt cagaagicagt aaaattaact acagtgttaa atgtattitat ttgagcatag 324 O tactgaaaac aaaaag catt caaaaaagag titttittctitt attagtaaat agtattittct 33 OO taatct caga ggagctgaga gttttgttga atgtattgta Cagtatgtag gag caggaga 3360 actttgtaaa ttggaaagaa gttctgtttitt ataatttatt tittatttitta aagcttaaat 342O gtagatattt atacgtatac agggtgcc ta gaa.gc.caatgttgtttcct g ttattacagc 3480 taacacagta aagaataatt ttgactittaa gtatgaaaca gtag taagtt atagotgcaa. 354 O agaatacaat atctatactg. tatgtcacat citacctaaat gttgcactat gcc ctittaaa 36OO

US 9,689,041 B2 61 62 - Continued aacaaaaagg actgaagaaa citctggalaca gagtaaaatg gacaagggac gaggatgata 54 O aattaaagaa gttggttgaa caa.catggaa citgatgattig gactictaatt gctagt catc 6OO ttcaaaatcg citctgattitt cagtgc.ca.gc atcgatggca gaaagttitta aatcc tigaat 660 tgataaaggg to Cttggact aaagaagaag atcagagggit tattgaatta gttcagaaat 72 O atgggc.calaa aagatggit ct ttaattgcaa alacatttaala aggaagaata ggcaa.gcagt 78O gtagagaaag atggcataat Catctgaatc Ctgaggtaaa gaaatct tcc tigacagaag 84 O aggaggacag gat Catct at galagcacata agcggttggg aaatcgttgg gCagaaattg 9 OO c caaac tact tccaggaagg actgataatt citat caaaaa to attggaat t c tactatoc 96.O gaagaaaagt ggalacaggag ggctatttac aagatggaat aaaat Cagaa Catctt cat O2O ctaaact tca acacaaacct tdtgcagota tdgat catat gcaaacccag aat cagttitt O8O acatacctgt toagat coct gggitat cagt atgtgtcacc tdaaggcaat td tatagaac 14 O atgttcagcc tacttctgcc tittatt cago aaccott cat tdatgaagat cotgataagg 2OO aaaagaaaat aaaggaactt gagatgct tc titatgtcagc tigagaatgala gttagaagaa 26 O agcgaatt CC at Cacagcct ggaagtttitt Ctagctggtc tigtagttt C ct catggatg 32O ataa catgtc. taatacticta aatagccttg acgagcacac tagtgagttt tacag tatgg 38O atgaaaatca gcctgtgtct gct cagoaga attcacccac aaagttcct g gcc.gtggagg 44 O caaacgctgt gttatcct ct ttgcagacca toccagaatt togcagagact citagaactta SOO ttgaatctga t cotgtagca toggagtgacg ttaccagttt tdatatttct gatgctgctg 560 Cttctic ct at caaatccacc C cagttaa at taatgagaat t cagcacaat galaggagc.ca 62O tggaatgc.ca atttalacgt.c agt cttgtac ttgaagggala aaaaaac act totaatggtg 68O gcaa.ca.gtga agctgttcct ttaa.catc cc caaatatago caagtttagc act coaccag 74 O c catcct cag aaagaa.gaga aaaatgcgag togt catt C C cc aggcagc gaact taggg 8OO atggct catt gaacgatggt gigtaatatgg cqctaaaa.ca tacac cactgaaaacactac 86 O cattttct cott cacagttt ttcaacacat gtc.ctgg taa togaacaactt aatatagaaa 92 O atcc tt catt tacatcaa.cc cct atttgtg ggcagaaagc tict cattaca act cotcttic 98 O ataaggaaac aacticc caaa gatcaaaagg aaaatgtagg gtttagaaca cct act atta 2O4. O gaagat citat actggg tacc acaccaagaa citcct acticc ttittaagaat gcgcttgctg 21OO cticaggagaa aaaatatgga cct cittaaaa ttgtgtc.cca gcc acttgct ttcttggaag 216 O aagatatt.cg ggaagttitta aaagaagaala Ctggalacaga cct attic ct c aaagaggaag 222 O atgaac Ctgc titacaaaagc tigcaaacaag agaat accgc titctgggaag aaagt cagaa 228O aatcactagt Cittagata at tgaaaaag aagaat Cagg cacticaactg ttgactgaag 234 O acattt Caga catgcagt ca aattgttgaat gggaalacagt ggttt atggg alagacagaag 24 OO acca actitat tatgactgaa caa.gcaagaa gat atctgag tacttacaca gctaccagta 246 O gtacttcaag agct ct cata citgitaattgt tattaaaatt gatgaaatgc cccactic cct 252O tactgcagtic tictact aaat taggttgcag tdaaatttitt citcaattagt tdtttittaaa 2580 gttgtaagat agc ccttitta atacagoat c titttittctat tctatatagt aggcagaaag 264 O c tagtaagtic acttaagggg tagatagttt catagitttat tttittaa.gag atgagattitt 27 OO taaaaattgt ttittaaagaa caagatggga aaataataga atgttcatgg atttctaaaa 276 O gtaaattct catatatttitc titcacaagat atatgttgct act ct cittga tigctgcagtt 282O ttgttataga taggtg tatgagtatatatg atttctgaaa ttagt citatg tatggaaagc 288O US 9,689,041 B2 63 64 - Continued acacatgatt titatgaagta Cttittgcc.ca tdtgctgatt tact taggct accatttaca 294 O aagaalacaca ttgaaaagga atttalaagga aggatagaaa gttgcactac taattittittg 3 OOO tttitttittitt cagaagicagt aaaattaact acagtgttaa atgtattitat ttgagcatag 3 O 6 O tactgaaaac aaaaag catt caaaaaagag titttittctitt attagtaaat agtattittct 312 O taatct caga ggagctgaga gttttgttga atgtattgta Cagtatgtag gag caggaga 318O actttgtaaa ttggaaagaa gttctgtttitt ataatttatt tittatttitta aagcttaaat 324 O gtagatattt atacgtatac agggtgcc ta gaa.gc.caatgttgtttcct g ttattacagc 33 OO taacacagta aagaataatt ttgactittaa gtatgaaaca gtag taagtt atagotgcaa. 3360 agaatacaat atctatactg. tatgtcacat citacctaaat gttgcactat gcc ctittaaa 342O t catgctggit tataaagtag ttctaaaaat gtact aaata ataatttaat attitt cittitt 3480 taaattatat cqggggtggit catatacatt aatctggtga tttgtatatg tdtttgaaat 354 O ttittgcattt tdtttaaaaa ataatatggit accttggtcc ctaaaaa.ca.g. tctgcactta 36OO gaagtttata t t tact cagt gtttcagaag toggagaacat tat cittittat ttataaaaat 366 O attttgtc.ct tttittaaatgttttgttgttt citctacaggit tacaa.cagtt gottcagttg 372 O cctgttittag gtgtttgcac ttattittatt tottcttgaaagaatttitta tittgcttittg 378 O tggtagagat tatatgtaat ttitttitt cag toatataatg gtgtgctgtc. aacttaalaca 384 O ctgacaggta aatagaattig tacactgtag tittgaattat ttataattga cacact citct 3900 c cct ct coac toctdaagta togctgctata gaaaatagoa gaatcggctt gctgctacga 396 O gaga aggaaa gagcgaccac cacttgcact gtgttgaaaag ataaaaaa.ca aatgatggca 4 O2O agttct caag ttaactaaat ggaat caacc attaccaggc aaattcttgc aaataccaaa 4 O8O atac tact at gcc titataaa acaaaatgaa agcaggittaa gattittctgc tictdtttgta 414 O tgttaataga aatggaaata ctaagtattt taatgct tag ct cittgaac a gtag acctaa 42OO aagggittitta agctatttaa atc tacttgc tagtttittgc at attittata tatatatata 426 O tittatatata tatatagtga gaagtgaaga aaatgitatgg tactaagatt atgcct tatt 432O gataaataga taalaccaatt tdaatcct ct tag catgttt aag tatgttg attgctttct 438 O aattaatgaa cittct cacag aaattt cact tagtgaaacc aatgattgta gcaaact cat 4 44 O actggat cat titcagttacc titgaactaat agcacataat gigtttitttgt tdttgttgtt 4500 tittaatgtag cccttacctg gatatacata gtctgcaatc accaaagtat aatat cittgt 456 O aaggctatat tttittaaag.c at atttitt to ttgagcatta aattatccta aatgg taata 462O tattgtggat aagtctgggc titattggaca taata catat ttgggttggit actggttgaa 468O t cct tcagtt aactgctttgttgctttittg caagatttitt tat cittaaac atgtcaggca 474. O t cittaagt ca cctittatact gttttgttcc tictdagtttc titt cagtatgttatacaaat 48OO gccaga cata acatgtagca gccatacttg catggaaact gactacacat acataatact 486 O gcattt tatt gtaaggttitt cacattaata cagcaattac cct gactaaa ttgagttittg 492 O tgatatatgg aaaact tcat td taa.gagaa tottgcatac aatgttgaca tattalacatc 498O caaaataaag catctgttgta caa.gctga 5 OO8

<210s, SEQ ID NO 9 &211s LENGTH: 2632 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

US 9,689,041 B2 73 74 The invention claimed is: one binding partner specific for an expression product 1. A method comprising: expressed from a target gene selected from the group measuring an amount of at least one expression product consisting of the FAM198B, MYBL1, PDZK11P1, and expressed from at least one target gene selected from VSIG10 genes, wherein the binding partner is attached the group consisting of the FAM198B, MYBL1, to a detectable label or solid substrate. PDZK11P1, and VSIG 10 genes, 16. The kit of claim 15, further comprising instructions for wherein the amount of the expression product is measured performing in vitro colorectal cancer testing using a periph using a peripheral blood sample obtained for in vitro eral blood sample obtained from an individual. colorectal cancer testing from an individual. 17. The kit of claim 15, comprising a plurality of binding 2. The method of claim 1, wherein the amount of the 10 partners specific for a plurality of expression products expression product is measured by an assay that comprises expressed from a plurality of target genes selected from the contacting the expression product with at least one binding group consisting of the FAM198B, MYBL1, PDZK11P1, partner specific for the expression product. and VSIG10 genes. 3. The method of claim 1, comprising measuring the 18. The kit of claim 15, comprising a combination of amounts of a plurality of expression products expressed 15 binding partners specific for the expression products from a plurality of target genes selected from the group expressed from the NEAT1, FAM198B, ITGAM, MYBL1, consisting of the FAM198B, MYBL1, PDZK11P1, and SIAH2, PDZK11P1, and VSIG 10 genes. VSIG10 genes, wherein the amounts of the expression 19. The kit of claim 15, wherein the one or more binding products are measured by an assay that comprises contacting partners include at least one hybridization probe. the expression products with binding partners specific for 20. The kit of claim 15, wherein the one or more binding the expression products. partners include at least one hybridization probe and at least 4. The method of claim 1, wherein the amounts of fewer one primer. than 50 expression products are measured. 21. The kit of claim 15, wherein the one or more binding 5. The method of claim 1, comprising measuring the partners include at least one hybridization probe and a pair amounts of expression products expressed from the NEAT1, 25 of primers. FAM198B, ITGAM, MYBL1, SIAH2, PDZK11P1, and 22. The kit of claim 15, wherein the one or more binding VSIG 10 genes. partners include at least one ligand. 6. The method of claim 1, wherein the expression product 23. The kit of claim 15, wherein the one or more binding is an RNA transcript or a polypeptide. partners include at least one antibody or affinity protein. 7. The method of claim 1, wherein the amount of the 30 24. The kit of claim 15, wherein the one or more binding expression product is measured using at least one of hybrid partners include at least two ligands specific for the same ization, amplification, or sequencing. expression product. 8. The method of claim 1, wherein the expression product 25. The method of claim 1, further comprising measuring is mRNA that is hybridized with at least one probe, at least an amount of at least one expression product expressed from one primer, or a combination thereof. 35 at least one of the NEAT1, SIAH2, and ITGAM target genes. 9. The method of claim 1, wherein cDNA of the expres 26. A method comprising: sion product is hybridized with at least one probe, at least measuring amounts of a plurality of expression products one primer, or a combination thereof. expressed from (i) at least one of the NEAT 1 and 10. The method of claim 1, wherein cFNA of the expres ITGAM target genes, and (ii) at least one target gene sion product is hybridized with at least one probe, at least 40 selected from the group consisting of the FAM198B, one primer, or a combination thereof. MYBL1, SIAH2, PDZK11P1, and VSIG 10 genes, 11. The method of claim 1, wherein the expression wherein the amounts of the expression products are product is a polypeptide that is bound by at least one ligand measured using a peripheral blood sample obtained for specific for the polypeptide. in vitro colorectal cancer testing from an individual. 12. The method of claim 11, wherein the ligand is an 45 27. A method comprising: antibody. measuring amounts of a plurality of expression products 13. The method of claim 11, wherein the ligand is an expressed from a plurality of target genes selected from affinity protein. the group consisting of the FAM198B, MYBL1, 14. The method of claim 11, wherein the polypeptide is SIAH2, PDZK11P1, and VSIG10 genes, bound by at least two ligands specific for the polypeptide. 50 wherein the amounts of the expression products are 15. A kit for in vitro colorectal cancer testing, comprising: measured using a peripheral blood sample obtained for one or more binding partners specific for from 1 to no in vitro colorectal cancer testing from an individual. more than 50 expression products that include at least