«2 United States Patent Van Laar 'US008999648B2 (10) Patent No.: US 8,999,648 B2 (45) Date of Patent: Apr. 7, 2015 SYSTEM AND METHOD FOR (CLASSIFICATION OF PATIENTS oa 03) 03) Inveator: Ryan Van Laar, New York, NY (US) Assignee: Signal Geneties, Ine, Carlsbad, CA ws) (*) Notice: Subject to any disclaimer, the term of this Patent is extended or adused under 35 USC. 154) by 0 days 2) 2) 66) Appl.Nos — 13/498,965 PCT Filed: Sep.30, 2010 PCT No, §371 (6), (2). (4) Date: PCTIAU2010001286 Oct. 10,2012 (87) PCT Pub. Nos W02011/038461 PCT Pub, Date: Ape. 7, 2011 Prior Publication Data US 2013002343441 Jan, 24,2013 6s) [Related US. Application Data Provisional epplication No, 61/247,802, filed on Oct 1.2000, (60) Gb Ineek 129 168 Gok 1924 GO6R 19720 us.cl. cee (2006.01) (2011.01), (011.01) (32) GOGF 19/24 2013.01): GOSF 19°20 (201301) (58) Fleld of Classification Search None See application ile for complete search history. (36) References Cited USS. PSTENT DOCUMENTS 20080058840 AL 32008 Dai etal FOREIGN PATENT DOCUMENTS. WO Wo 2006007240 421-2006 Wo Woo AL 42012 OTHER PUBLICATIONS Miley, Let al, “An expression signatue fr pS3 satus in human ‘sat cancer predicts mation sa, transerptional fst, and ati survival Proceedings ofthe Naianal Academy of Sconces, {US4, vo, 102 No, 38, pp. 13850-13888 2005), Interaional Search Report for Lternaional Application No. PCT! ‘AU2010'001286, Date of Mailing Nov. 18,2010. Notification Concerning Tansmital of Internationa Preliminary Report oa Pacnabty and Writes Opinion of the International Seuiching Aubonity fr leteational Application No. CT AU2010! ‘001285 Date of Mailing Apr 12,2012 Alfonso, M, cal, Breas!PRS isa gene expression assay tht states intermediates Oneotype DX paint into high of fw Fk for disease ecirence” frau! Cancer Research and Tearment, 13809) 708-715 2015) Desinal Ce "Siron Time Dependence of the 76Ciene Prog: rosie Sianiure for Node-Negaive Breast Cancer Patients inthe {TRANSBG Multicenter Independent Validation Series™ Cleal Concer Resear 13 82073214 2009) Ishin, A.V, eal, “Genetic Relasifeation of Histologic Grade Doligates New ‘Cinical Suluypes of Breast Cancer! Cancer eer 6 1292-10301 200. sen, Re No al, "Mtasasis- Associated Gene Expression anges re Poor Outcomes n Paes ws Dukes Sage Bad Colorectal Cances” Clinic Cancer Research, 15: TO2TOSL (Goo, Pavitan, ofa, “Gene expression profing spares early breast cancer pacts rom adjuvant therapy dived and validated in tw population-based cohorts” Breas! Cancer Research, 7 ROSS-ROG4 0085) Schmit. Metal, “The Humoral Immune System asa Key Prog: ‘nose Impact in Node-Negaive Breast Cancer” Cancer Reseaeh, 5405 5 ‘Shedden, Ket al, "Gene Expeston-Bated Survival Prion ia Tang Adcnocarinoma: A. Mul-Site, Blinds! Validation Sly ‘Nate Medicine, 1 823-827 (2008). ‘an Tas 1 “Design and MullstesVliation ofa Web-Based Gene Pxpesion Assy for Predicting Breast Cancer Recutenceand Pate Survival” De Journel of Molecular Diagnostics, 13) 297- 304011). ‘ha, C-Q, eal “Prognostic and Predictive Gene Signature for ‘Adjivat Chenery a Rect Now-Seall-CllLungCance” ournal of Cintcel Oncology. 229}: 4417-4424 2010), “Avnetitc HG-ULS3A.& HG-ULS8D Pra Soquence, published ‘Aug. 20,2008 [Retrieved om Inenet Oee 9, 2013]-“URL- Bip: ‘wwatlymetx com Auth analysis dowload data HG-UIS38 probe Estazip “URL ‘pw allt com Auth tal ‘Se downlads data HG-U/L3B pre fasta rp> Tnernatonl Search Report rom nieralional Apicton No. PCT! ‘AUR 001250 tied ne Marker Se ant Mtoe for Class Seaton of Cancer Paints” mailed on Nov. 24,20 International Preliminary Report on Patentability fon of Aprliation No PCT AL2011 01250,titledCene Marker Sts ant ‘Methods for Classification of Cancer Pala” male oa Ape 2018, Writen Opin: Primary Examiner — Sioa Ketor (74) Attornes, Agent, oF Firm Reynolds, PC on ABSTRACT A system (100) for classifying a biological test sample, ‘including a database (112) populated with reference expres sion data, The reference expression data inludes expression Ikvels of plurality of molecules (polynucleotides oF polypeptides), including a set of marker molecules in plu- rity of reference samples. Bach reference sample has 3 pre-assigned valve foreach of one or more eincally signifi cant variables. The system includes at least one processor (110) and at least one storage medium containing program Jnstmctions for execution hy said processor (110). The pro- ram insiuetions cause the processor to accept (122) input expression daa including atest vector of expression levels oF the marker molecules in the biological test sample; and pass the input expression data fo one or more analysis programs (130g, 130,38). The analysis programs include at least one Statistical classification rogram (138) for assigning a value fat eastone of sid clinically signilican! variables to the test sample Hamilton, Brook, Smith & 14 Claims, & Drawing SheetsU.S. Patent Apr. 7, 2015 Sheet 1 of § US 8,999,648 B2 Relational Bee ica Eo cee FIG1U.S. Patent Apr. 7, 2015 Sheet 2 of 8 US 8,999,648 B2 4.Pationt with 2 Cirician ‘metastatic —P takes biopsy cancer oftumor 220 | 3. RNA extracted & hybridized to Gene Chip 230 i 4. GeneChip 5, Data for 5k _— CEL fle ————» probe sets = uploaded to extracted & Quality Chip0X 240 normalized 59 ‘edule un | 20 7..ChipOx Tumor Origin Htdute in / | 210 a. Results compile and presented to clinician 200 FIG2U.S. Patent Apr. 7, 2015 Sheet 3 of 8 US 8,999,648 B2 FIG3US 8,999,648 B2 Sheet 4 of 8 Apr. 7, 2015 US. Patent FIG 4U.S. Patent Apr. 7, 2015 Sheet 5 of 8 US 8,999,648 B2 ’ipDX Tumor Origin Database: ont Sippcrune oregoaae: [aces Level 1 Anatomical system. => classes a. Gastonia! Sytem Level 2: Specific : 40 classes. tissue. S Calon Level 3: Tissue and 2 subtype wp 295 classes a. Gobnsigrt eng eat FIG6U.S. Patent Apr. 7, 2015 Sheet 6 of US 8,999,648 B2 g oo eoreeccocceecaee T T 7 T 0 500 tooo 1800-2000. 6 so 1000 100 2000 FIG7U.S. Patent Apr. 7, 2015 Sheet 7 of 8 Percent survival Percent survival 0 5 10 15 Survival time 100 : 80 60 40 20 0 2 4 6 8 10 Survival time FIG8 US 8,999,648 B2 “ Henwins DING Apa02 ° Al68384 Haseerd CARS Nvcaosiss esters CAPS 2U3871_s_at NM apenas Hs642660 Clee 6 206903 NM 02968 HR3S787 SALLEUS 8,999,648 B2 37 38 TABLE 4-comtinsed sie be itm Aceionsmber Unies _ Gem ot Sa a a TAL as eat wi isin Mase? = ian Absa fees anes Sete featies ExrDs Mame ftom Sar Scone hasnt bake SAC fieteps Saas a Somer este reo Mine Frca08 Bettas Seer arto cue dae Sons ficarws MEAs Sema Shee fons Kirt Scone fesinno Sinn 3349s A080 e380 FOXLUS 8,999,648 B2 39 40 TABLE 4-comtinsed sie be itm Aceionsmber Unies _ Gem ot awe Sono inns Gana Sut te Sse foo Fe draw Soon fbooe Guan pe fone Gan assume feacars Tae Sear thane mest toms SeSiteh Ee Sins fuse’ SM Mies fase Spy Seach festsos Toc bet fiestas Garas Sens tebe Ror isn fares Laws Scone titer foc avseno fhosnt TwEwec avieris ~US 8,999,648 B2 4 a TABLE 4-comtinsed = a 214309 NMOL He19722 CAMK2NI nse NM_ai2 Hs64173ATPOVIBI AMS His 644865 N06 Hetsi2s4 KLUX? AN659225 Hsavsond CASK 221577 x at AFIO3934 ‘Hs.616962 GDPISUS 8,999,648 B2 43 44 TABLE 4-comtinsed monn epee Acne AKIDMI27 . Aono fees ant diem thane 3 SiC ots een Ake ieee Saas fete Mow ie Soci fiaowe ee 2087850 NM_002217 Here —TTIKS Sania Stow fedinss Aten ioc Sone fine’ ne Boo Sao this Sane iene Sects faa iste Sines fenes Also incon Scomoes feats ror Scones fans roe 208367 at NMLO10 76 His.6S4301 CYP3AGUS 8,999,648 B2 45 46 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt Tana AnrOT Teste? TF 2am NM otsas. Hs103640 HAOT Bis toa NMA estes Te 2usts2— Apes Hss3t74SLOSAL 2 aa RAC 076 ersoan? OGr3BIs sds NXcavaar? Reoasl PAK Beransey eton? CrHRS waias7 Hs149282 CPSL Bawsis, eaens INHBE Uns eisoan? Garabrs ras sat Nvcavnaos Hessen PRL. hows Besr2300 Heaoos PoLyRe2 dnsors—» asa) elosys MITT NMC 00771 Ha2a2004 Baiwsons elo SETRC ee fea nee ane AL35S392_ He6SSS1_— C200ef114 a ARRAS H331879 RADIUS 8,999,648 B2 47 48 TABLE 4-comtinsed mei Fb Cenk Asinimber___UsipaeID_ One bol Sora rrr Tesn069 ADEs Sot arenes ficnoste Corto oa Bacto Hanae) CaeNapn immtca Soe tieaoas foua iit fedaao Aegrcl im Noses tietoaa Gets Sosa arama traps fowvsat eed tana iota sions fan Aonrm2 asses taose ND Cison Heaton AkRiC? rei teaoas ea Ano sass990 oxy Sates fisneeALDIOAL asians fesoaah bts BESSISI6_ e6sstns ALEK?US 8,999,648 B2 49 50 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt Bape Amos Tein? WON 184875 aa “ARETAAAG Hs202733 NRINE pana Ania Hesoraa) Loess ssa at Beus9is Presties NM. oso tie3s1306 LCI 20628 ANTON? HRISS64 PAX? Sito ‘Tyson 223610 Bco27I6 Hs210870 SEMASB. iw ‘sss Heasnnas COLBAL Sn aoe ftomes Doce Sonn Sex fetcens cow Scones fasion bre se fiaeas EAP ara e Scone tie” stom anes e202 GNAQUS 8,999,648 B2 51 52 TABLE 4-comtinsed ‘ime tbe niu Aceionsmber ___Uimae D_Gem pb ean Tisones Tease ORS Sores wae tosses 8k Sobas iksoos BSP din thiseor sper inte thoses Phe Rear 3s Str iors Sine fiteote cab iota tis fdas Fake Soon Moore feoses Ch iota Sasi fee? Faun Sa Sena these’ MN s aes fist MAPK x 1BF222895 He269782 GNAQ x Sine feiss Mncar 2 “ AUILS8O62 Hs.369007 coe teas Seca fea restUS 8,999,648 B2 53 34 TABLE 4-comtinsed mei Fb Cenk Asinimber___UsipaeID_ One bol ines aaneie asia) KINDY sia sian teaser BRL Seas roast fete Dex aa Sees tea bee ios eat Bowne fiammos Maras Sines fibers Faxes Miiomee fcan Marae Arsene fewor ‘Tea Sates fawn Mot Scns ticsors Tews Aiton Heseren NoCD Stoo fiegocsn Marke Avie fatima Sont bwsea, fis FEN Siar tesa igen 209800 at NMCoLay2 esis Daszae55 US 8,999,648 B2 TABLE 4-comtinsed 56 gst SAL oon areas? Ricoaies NM 008203 prowess Rae vars Nxcansaoe Uusr Ft inno Sows Naczaor “ALS Nvcainsio ALOT Racoon Tene ease keansis Heaps eases ere ease esas? ease ero weesoanoe ese Hs.201776 soos Heats earren ca ‘esa ‘osm ‘rosa tesa KUKe Tosa bist ‘Tosa Mer oes Cotten: rica cow hex tooo crUS 8,999,648 B2 37 58 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt ier sw Aisa Tiealsios SERINE! 2088825 NM_onsi2t HsAasT136 GETS. howe aot Umar eshon Nearer z Nowe Hessias NALEN Ne Hies14626.— NEURDOY NXcaoisie estos Gee Svcs Beavis cUzpt uses NM Hattie VNNI ions ana ean? REGIA splat 03 Hesonsse COLiMAL 2011085 AVRO6TS Haen6 TABS biicoat Noise Heart AKAPIS 2210095 Awa Harigen0 Locse7I6s avnwis HsaBoss4 FAMIBAS iegseionceLP 1sszm aa NM172037 Headend ROHUS 8,999,648 B2 59 60 TABLE 4-comtinsed sie be itm Aceionsmber Unies _ Gem ot saw Tora Terim0 oan tau Senn fives Poets hina Sacreae simon Xt mses at BOORSIST Hs385034 SPQ. stat Sonat faut Rete Mises titer ars Sidon teed Cae ar tkiows ae Seine fpr Get Metieet fee exon Miesise feo Marka ATS2874 . Sees femes6 anes Aon fess Age oats fesonet FaNea now iat felis Klas Alg2s74? Hs99913—ADRBLUS 8,999,648 B2 61 62 TABLE 4-comtinsed ‘ime tbe niu Aceionsmber ___Uimae D_Gem pb ioe Sirs Teaco ARonre 2707 at AWI38I43 in sins 2380 T6208) Aine wapsrycecxs Monee Hsa3SS12 PPPICA rvs hoo? Leese 2 ‘BESS2383, - Sines teams runs Mconsess iu Tne NM_024005 Hsetons | CPLXS Sos fais Socks NM_029087 Hisa79™}— FLI2SUT- Mier : Toone senses tenses casi 2277385 at AAS59, His298713 100199 isso Sacsas wosUS 8,999,648 B2 63 64 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt Apo Taesoa099 _ NKDI AROS? Hs27243 CCLIS Rate 204612 NM a0425 Hsa33700 PRIA his eat Bessey eapeas NOLL isan ae Uososs evosist TAX Boiss at pete Hieaanaed — COLbA2 2 NMC OLT786 390738 GOLSYN Fi Nvcaue esiosia TRIDe Fi Nvcaigan Heaeiyot FAMDEB 2 NMC oteois. Hs274267 — CXorS7 x Nvcate2s? eapeess NOLL ANS Hetosist TAX? Besenree Hegsasia TIMP ‘ose Hiestwo2 | FORLB ABSI421 Hs279709 TRIMGS.US 8,999,648 B2 65 66 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt rr Woo Tear CDI 213003 5 Aloto86 Hisave0s = MucL imei RAC noes eteeon? | MITE 3 wosetlt Heaieons PRACT ewaess eset Oxi Ona Bene BAX besser Henenie STscaus Nassar Bowes eamas Tri Browsiot Heater EVs Racoaess Hetbeos UCL Nvcanazes eaveate Moser NMot233 Hs16829—TSPANI2 NMC oom115 EDNRB Nome Haas MYosA Nice Hie1se6s2 TashUS 8,999,648 B2 67 68 TABLE 4-comtinsed sins Pb Gedunk Acomionsanier Unig D_ Gene al ose ew SC Ternog —xPere 228829 “AATIDEH Hsa77370 ABI Hua ASomna0 tiers me indo See as Hetesee Thou inset at Scns teense Pine : St tases AnCboL Nan trains flows Setso33 tease Maar Sines fishin AaDae avin tiavea SUdg2 ir a Bicinsoyr fetta MZ dows Rear tae x ‘ Abie feauesy rane x Sicinin tose aN x Seton fisniae Fx SOL sat arises Beasised Cidoes nei? essai? NCOUS 8,999,648 B2 69 70 TABLE 4-comtinsed sea cohut cease theo x Bras Hs371720 SYK AnS6412 Heomist LYN anh ER enw 291s Aras Hs12986 TAXIBPS Bint ies Ms AWAOTSST Hs37120 SYK ADSI Hia4i73 | AHNAK? A053 HeaSit0 —CNRE Soar Hews IY Bie” oe goes) 26a bos set3120 TGF Ara Belsiria C1QINETUS 8,999,648 B2 n nD TABLE 4-comtinsed roy Tisaoeoad FAP Axsinon eisvea TROAL vav0stt Heia30n0 — HOXDIO asso eaosies PHL Osan Heabeseh SMa: awison Kemas Duar ansiee Hearinis RMR Ues79 370284 RAD. ais Heasecte CASPs Bass eso? CSNRIAT Nvcabsaos eases PHeL DBetwasoe estate Gest Uesi39 His6ss5RADSD ene JUN! Apoisr9 Beans TTGAY ABIEORIS est ARNTL,US 8,999,648 B2 3 4 TABLE 4-comtinsed Afimetia Probe 1D Gesbank Acesion umber Unigene 1D _ Gone bol ay Mt oer este Lice SE AW292168 - inst nasser Heaneni2 NTRK? 2068875 at AC sown? Heasiais DIOL 2199 a NM O26512 Hes7345 ERC? NAC As629 Heséoae SLCSAS sco ‘xgostes Hesi2002 KIAAISOS ‘aanni99 Heapenn2 NRK? Nasei2 Heso40s) RASAT Bou20si9 He.386664 DUOXAL ANI 62 He6se778 RABIIN asst ‘nosass4 eavoonn HRATL aorsets at NAC 001367 5% TRAKI A380 Hea023s4 lo? see MBH Heide MYKSUS 8,999,648 B2 15 16 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt Tome SAL coat Tienes Or 230389 NM ote, Hs7s002 ARPP-21 hie Broo esos LMODS 233049 5 Alie02s2 Heaigi22 | MYHTB Sian eat Rtoase eabeia? COLA 23402 os Hsaoeia? — COLZA1 down ovis . b BeAsate Henna PEGIO ALI IIR Rxconssas Hieasoa7 gyro NXcansaio Heamays TNESFIO Nvcansoee Rented Sixt NM_onaas2 HsrLis6 SLCVARRE NNcauwors eases Sor Nv 014332. Hsaeas:— SMPX 2983 st AnIvS4 eas KLCSUS 8,999,648 B2 7 TABLE 4-comtinsed mes Pb cesbaa Accninsnter___Unieee ID _Gane nt ame Dae THealsor0 MBL TABLES 0 TABLE 8 S-continved 200 eas nonunion with cll vache to prett "cn cncer een are eae ening he Inptbess ecard prev of ‘a gene wet conjunton wth ln wile to pre et camer sence ik wae Pre teing he Inet fie exes dat prev of a say nada sat ed a Atom ‘pocin BRCTB™ —cwenmiat__ Gat ttre» PTD" cect nterCreD Sieos foto ks it at crkitis Mtn) kt ome tks ars ‘s Choeis3 ARATIGE Sans 1vt6 sat HDGERPS AW207488 0810 Sch Rs Bak IRS Ry SH iach Gems CAE SG Shae’ RS us nae ALU AFIS7689 aD Sx_atIPNAR? NM_0o04 3435 sw tam 3 re oe re 5 cho Man & ot in Aste as y oe on a "Eten An =m Biv ds Reluome Barta Era’ Bn! a Brees em cums anata Ki enous = fie" at Mite Son's" Eun Gan = 210564 at CFLAR ARIS x71 at LLGLE NM 08528 3003 Somcct Gitta Brows Bike, konto COROIB A134 (208633_x at MACFI Wo10s2 2409 corer. Sect Mia Naan te cis ete Beck ama Seon Se excel umas0 at _ NM_028051 pact Nwcaveass meee xCON NL oss40 me kia Simca Se Satan EEFIAL BEDRG672 68 Doses at NRICE AMB24US 8,999,648 B2 79 TABLE S-continaed 80 TABLE 5-comtinved Stil wav di ie SSS es ra NACo “a espe SME ous act ssn aon zaib1 ison 20 isea, —ANaion mea AMC anmi = hows Mato Pore, Ncain a ns NwLamer Fovber = Micaissee =m) Svcs Feet Natoma ess a Bicone 0 The invention claimed is FeKcp NAL ners Sy 1" s'gystem for perbming a method of determining the Pan Siansta EE isk of breast anor ecurence ina beast cancer pation, FN Brea Sst comprising pinky Rotor al cat e proces and Pp a aco “PMTGSP™? —atteost one sorage medium containing program instve- x Eicoan —Avissae bin > tons for execution by said processor, said program Bigelow: Siti Bt instetions causing said procestor to execute steps Hee RAG xsrnr ss comprising: pepe bey tie ce Scceping input datain the form of expression levels ofa x za Sitctntoe ‘oie et of nucleic aid marker molecules in a Bclogteal 2 me waste Se wy sample fiom the subjet, wherein the nucleic aed z BRE ANOS markers comprise nucleic acids ited in Table 5: x Roop Nicolae comparing the expression levels eferenee vale: and F meus) Misa ‘Ssigning clinical annotation wo the biological sample RNIN Rucanose ‘on the basso the comparison, on RI Saco 4s wherein the clinical annotation i isk of reas cancer Eivar Nat wes tse 2. A‘system for classifying a biological sample from a som Noe ‘So! cancer patient comprising Seis? Stoo tbe at lest one processor and F eee UNE 4 at east one storage medium containing program inte x Slee? Sato ma tions for exeuton by said processor said program 2 sagea — aubiets on insinctions easing sid processor to execute steps > Sit Nitoims sent compris fi Bore ons ea scceping input datain he form ofexpresion eves ofa x Sari Niners fees Seofmuelic uid marker molecule ia the Biological x Sox Boseno0 ss Sample, wherein the mcleie acid marker molecules, 2 eo stoop aa ren combination of 100 or more genes in Table 4 a eS ed an comparing the expresion evelsof the setof melee aid x Seay Mtoisus ears marker molecules inthe biologie sale to expres 2 ioc As NMG sionlevelso thesetof muceie ae marker molecules 2 a te, ina setof reference samples cach member of te set 3 Tamnwe sacoien Ser ‘of reference samples having presssigned value for x Teese NACL? Saas ach of one or more clinically significant varibles x 0 mise ne Selected fram the group consining of diease sta z ora use? TS ss isease prognosis, and teatment response, fi a cere ian the comparing of expression levels in the biologics! x ars Seas Sate sample to the expression levels in the reference x ance SNOT ss Samples comprising one or more analysis methods, 2 uns Nwcongs7 ais the analysis methods comprising atleast one statist z Trea Bewiee Iie: 6 al classification program trained to dstinguish, x Gettin me ‘mong suid pre-assigned valves onthe bass ofthat F peo Aono we parol thereferenee data conesponding wo expression 2 ee ae levels ofthe uci aid marker molecules; and F oom a signing a pre-assigned valve ofthe one oe mor chin E mx 250265 cally ificant variables the Biological sample on 3 Sows ot thehass of thecomparson of the expestionlevelsoF the set of mueleie seid marker molecules in the bio=US 8,999,648 B2 81 logical sample to expression levels of the set of nucleic acid marker molecules in the set of reference ‘samples using the statistical classification program, ‘wherein the one or more clinically significant variables is selected from the group consisting of disease sate, disease prognosis, and treatment response 3. The system elaim2, wherein the patient has metastatic 4. The system of claim 2, wherein thet leastone statistical a program i selected from the group consisting sarest neighbors (KN), linear discriminant analysis, prinejpal components analysis, nearest centroid elasifica- ‘ion, and support vector machines. ', The system of elaim 2, wherein the one oF mare efini= cally significant variables are organized according to hicr- ‘arch, wherein the levels of the hieraechy are selected from the group consisting of anatomical system, tissue type, and rumour subtype 6. The system of claim 2, wherein the patient has breast 7. The system of claim 2, wherein the expression level is zenerated ising a platform selcte rom the group eonsisting of cDNA micrournys, oligonucleotide | microarray mietoRNA (miRNA) arays, and high-liroughput quantitae tive polymerase chain reaction (qPCR), ‘8. Thesystem of claim 2, wherein the expression level is at least partly assessed according toa distribution across refer ‘ence samples of one or more statsties derived from reference ‘data, wherein the statistics are selected from the group con- sisting of background intensity, percentage of molecules hove detection threshold, ratio of 3 expression level to 5° ‘expression level, slope of RNA degradation curve, nommali- sation factor, and log (base 10) ratio of mean intensity tomean background intensity 82 9. The system of claim 2, further comprising normalizing the expression eves inthe sample to be comparable with the reference expression levels, 10, The system of claim 1, whercin the system further comprises testing additional elinical covariates forthe subject Selected from patient age, grade, nodes, mmour size or FR status, 11, Thesystemofelaim 1, futher comprising eaeulating a prognostic index acconding to Formula 1 = Yn 01301 ae etm 0.247) © 12. The system of claim 1, wherein the expression levels are generated using a platform selected from the group eon- sisting of cDNA microarrays, oligonucleotide microarrays :mieroRNA (miRNA) arrays, and high-throughput quantita- tive polymerase chain reaction (qPCR), 13. The system of claim I, wherein the expression levels area least parly assessed aovording to a disteibuion across reference samples of one oF more statistics derived from reference dala, wherein the statistics are selected from the group consisting of background intensity, percentage of mol- ecules above detection threshold, ratio of 3' expression evel {0 expression level, slope of RNA degradation curve, nor valization factor and log (hase 10) ratio of mean intensity to ‘mean background intensity 14, The system of claim 13, further comprising normalz- ing the distribution of the expression levels tobe comparable ‘with te distribution of reference expression levels