WO2006015312A2 - Prognosis of breast cancer patients - Google Patents
Prognosis of breast cancer patients Download PDFInfo
- Publication number
- WO2006015312A2 WO2006015312A2 PCT/US2005/027243 US2005027243W WO2006015312A2 WO 2006015312 A2 WO2006015312 A2 WO 2006015312A2 US 2005027243 W US2005027243 W US 2005027243W WO 2006015312 A2 WO2006015312 A2 WO 2006015312A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- genes
- expression
- prognosis
- gene
- good prognosis
- Prior art date
Links
- 238000004393 prognosis Methods 0.000 title claims abstract description 298
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 217
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 215
- 230000014509 gene expression Effects 0.000 claims abstract description 408
- 239000000523 sample Substances 0.000 claims abstract description 283
- 238000000034 method Methods 0.000 claims abstract description 214
- 238000010837 poor prognosis Methods 0.000 claims abstract description 144
- 238000002493 microarray Methods 0.000 claims abstract description 110
- 206010027476 Metastases Diseases 0.000 claims abstract description 72
- 230000009401 metastasis Effects 0.000 claims abstract description 63
- 238000003745 diagnosis Methods 0.000 claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 claims description 489
- 239000003550 marker Substances 0.000 claims description 156
- 206010028980 Neoplasm Diseases 0.000 claims description 137
- 102000040430 polynucleotide Human genes 0.000 claims description 83
- 108091033319 polynucleotide Proteins 0.000 claims description 83
- 239000002157 polynucleotide Substances 0.000 claims description 83
- 150000007523 nucleic acids Chemical class 0.000 claims description 75
- 102000039446 nucleic acids Human genes 0.000 claims description 74
- 108020004707 nucleic acids Proteins 0.000 claims description 74
- 102000015694 estrogen receptors Human genes 0.000 claims description 72
- 108010038795 estrogen receptors Proteins 0.000 claims description 72
- 238000009396 hybridization Methods 0.000 claims description 69
- 238000004590 computer program Methods 0.000 claims description 26
- 230000000295 complement effect Effects 0.000 claims description 24
- 238000005259 measurement Methods 0.000 claims description 16
- 230000007246 mechanism Effects 0.000 claims description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 11
- 238000003860 storage Methods 0.000 claims description 10
- 238000009826 distribution Methods 0.000 claims description 6
- 210000000481 breast Anatomy 0.000 claims description 5
- 201000010700 sporadic breast cancer Diseases 0.000 claims description 2
- 108700026220 vif Genes Proteins 0.000 claims 1
- 230000002596 correlated effect Effects 0.000 abstract description 14
- 230000002068 genetic effect Effects 0.000 abstract description 7
- 238000007405 data analysis Methods 0.000 abstract description 4
- 238000007619 statistical method Methods 0.000 abstract description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 55
- 210000004027 cell Anatomy 0.000 description 55
- 108020004414 DNA Proteins 0.000 description 31
- 238000004422 calculation algorithm Methods 0.000 description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 26
- 102000004169 proteins and genes Human genes 0.000 description 26
- 230000000875 corresponding effect Effects 0.000 description 24
- 201000010099 disease Diseases 0.000 description 24
- 108020004999 messenger RNA Proteins 0.000 description 24
- 238000012549 training Methods 0.000 description 24
- 239000002299 complementary DNA Substances 0.000 description 21
- 238000003491 array Methods 0.000 description 20
- 201000011510 cancer Diseases 0.000 description 19
- 239000002773 nucleotide Substances 0.000 description 19
- 125000003729 nucleotide group Chemical group 0.000 description 19
- 238000003752 polymerase chain reaction Methods 0.000 description 17
- 230000006870 function Effects 0.000 description 15
- 210000001165 lymph node Anatomy 0.000 description 15
- 108091034117 Oligonucleotide Proteins 0.000 description 14
- 238000011285 therapeutic regimen Methods 0.000 description 14
- 230000001413 cellular effect Effects 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 239000000470 constituent Substances 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- 101150094765 70 gene Proteins 0.000 description 10
- 238000002372 labelling Methods 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 9
- 239000013615 primer Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 230000003321 amplification Effects 0.000 description 8
- 239000000975 dye Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 238000012545 processing Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 7
- 239000013614 RNA sample Substances 0.000 description 7
- 238000011226 adjuvant chemotherapy Methods 0.000 description 7
- 238000013459 approach Methods 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 108091000080 Phosphotransferase Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 230000011278 mitosis Effects 0.000 description 6
- 238000010369 molecular cloning Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 238000010606 normalization Methods 0.000 description 6
- 239000013610 patient sample Substances 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 238000003757 reverse transcription PCR Methods 0.000 description 6
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 6
- 102100031780 Endonuclease Human genes 0.000 description 5
- 230000032823 cell division Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000010195 expression analysis Methods 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 102000020233 phosphotransferase Human genes 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108700039887 Essential Genes Proteins 0.000 description 4
- 108091034057 RNA (poly(A)) Proteins 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 101710137500 T7 RNA polymerase Proteins 0.000 description 4
- 108010006785 Taq Polymerase Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 102000052609 BRCA2 Human genes 0.000 description 3
- 108700020462 BRCA2 Proteins 0.000 description 3
- 101150008921 Brca2 gene Proteins 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 239000007987 MES buffer Substances 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 238000012341 Quantitative reverse-transcriptase PCR Methods 0.000 description 3
- 108010077895 Sarcosine Proteins 0.000 description 3
- PGAVKCOVUIYSFO-XVFCMESISA-N UTP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-XVFCMESISA-N 0.000 description 3
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 3
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 3
- 238000009098 adjuvant therapy Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000010804 cDNA synthesis Methods 0.000 description 3
- 231100000504 carcinogenesis Toxicity 0.000 description 3
- 108091092328 cellular RNA Proteins 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 239000013068 control sample Substances 0.000 description 3
- 238000002790 cross-validation Methods 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- -1 e.g. Substances 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 238000011223 gene expression profiling Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000013074 reference sample Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 229940043230 sarcosine Drugs 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000011269 treatment regimen Methods 0.000 description 3
- PGAVKCOVUIYSFO-UHFFFAOYSA-N uridine-triphosphate Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 102000016736 Cyclin Human genes 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 2
- 108010007005 Estrogen Receptor alpha Proteins 0.000 description 2
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000011446 adjuvant hormonal therapy Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000001325 log-rank test Methods 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000010208 microarray analysis Methods 0.000 description 2
- 238000012775 microarray technology Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 238000002966 oligonucleotide array Methods 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 150000008300 phosphoramidites Chemical class 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 238000011176 pooling Methods 0.000 description 2
- 238000007639 printing Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 230000022983 regulation of cell cycle Effects 0.000 description 2
- 108020004418 ribosomal RNA Proteins 0.000 description 2
- 238000011524 similarity measure Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 101150088993 62 gene Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091007914 CDKs Proteins 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 108010060387 Cyclin B2 Proteins 0.000 description 1
- 102000003903 Cyclin-dependent kinases Human genes 0.000 description 1
- 108090000266 Cyclin-dependent kinases Proteins 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101150044894 ER gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102100037854 G1/S-specific cyclin-E2 Human genes 0.000 description 1
- 102100033201 G2/mitotic-specific cyclin-B2 Human genes 0.000 description 1
- 206010056740 Genital discharge Diseases 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 208000033640 Hereditary breast cancer Diseases 0.000 description 1
- 101000738575 Homo sapiens G1/S-specific cyclin-E2 Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 238000000342 Monte Carlo simulation Methods 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108010020713 Tth polymerase Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000011256 aggressive treatment Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000000315 cryotherapy Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000025581 hereditary breast carcinoma Diseases 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000002134 immunopathologic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007641 inkjet printing Methods 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000011551 log transformation method Methods 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000010801 machine learning Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000009607 mammography Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 101150024228 mdm2 gene Proteins 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 235000020004 porter Nutrition 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- RUOJZAUFBMNUDX-UHFFFAOYSA-N propylene carbonate Chemical compound CC1COC(=O)O1 RUOJZAUFBMNUDX-UHFFFAOYSA-N 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000009121 systemic therapy Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/20—Supervised data analysis
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/30—Unsupervised data analysis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00351—Means for dispensing and evacuation of reagents
- B01J2219/00378—Piezoelectric or ink jet dispensers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00351—Means for dispensing and evacuation of reagents
- B01J2219/00385—Printing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/00527—Sheets
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/0054—Means for coding or tagging the apparatus or the reagents
- B01J2219/00572—Chemical means
- B01J2219/00576—Chemical means fluorophore
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00585—Parallel processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00596—Solid-phase processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/0061—The surface being organic
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00623—Immobilisation or binding
- B01J2219/00626—Covalent
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
- B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00675—In-situ synthesis on the substrate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00693—Means for quality control
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00709—Type of synthesis
- B01J2219/00711—Light-directed synthesis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
- B01J2219/00722—Nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
Definitions
- the present invention relates to the identification of marker genes useful in the diagnosis and prognosis of breast cancer. More particularly, the invention relates to the identification of sets of marker genes able to distinguish individuals having breast cancer with good clinical prognosis from individuals with poor clinical prognosis. The invention further relates to methods of distinguishing breast cancer-related conditions using the identified sets of markers. The invention further provides methods for determining the course of treatment of a patient with breast cancer.
- breast cancer is the most common cancer in women and the second most common cause of cancer death in the United States, hi 1997, it was estimated that 181,000 new cases were reported in the U.S., and that 44,000 people would die of breast cancer (Parker et al, CA Cancer J. Clin. 47:5-27 (1997); Chu et al, J. Nat. Cancer Inst. 88:1571-1579 (1996)).
- the cumulative lifetime risk of breast cancer for women who carry the mutant BRCAl is predicted to be approximately 92%, while the cumulative lifetime risk for the non-carrier majority is estimated to be approximately 10%.
- BRCAl is a tumor suppressor gene that is involved in DNA repair and cell cycle control, which are both important for the maintenance of genomic stability. More than 90% of all mutations reported so far result in a premature truncation of the protein product with abnormal or abolished function.
- the histology of breast cancer in BRCAl mutation carriers differs from that in sporadic cases, but mutation analysis is the only way to find the carrier.
- BRCA2 is involved in the development of breast cancer, and like BRCAl plays a role in DNA repair. However, unlike BRCAl, it is not involved in ovarian cancer.
- a marker-based approach to tumor identification and characterization promises improved diagnostic and prognostic reliability.
- diagnosis of breast cancer requires histopathological proof of the presence of the tumor, hi addition to diagnosis, histopathological examinations also provide information about prognosis and selection of treatment regimens. Prognosis may also be established based upon clinical parameters such as tumor size, tumor grade, the age of the patient, and lymph node metastasis.
- Diagnosis and/or prognosis may be determined to varying degrees of effectiveness by direct examination of the outside of the breast, or through mammography or other X-ray imaging methods (Jatoi, Am. J. Surg. 177:518-524 (1999)).
- mammography or other X-ray imaging methods Jatoi, Am. J. Surg. 177:518-524 (1999)
- the latter approach is not without considerable cost, however. Every time a mammogram is taken, the patient incurs a small risk of having a breast tumor induced by the ionizing properties of the radiation used during the test.
- the process is expensive and the subjective interpretations of a technician can lead to imprecision. For example, one study showed major clinical disagreements for about one-third of a set of mammograms that were interpreted individually by a surveyed group of radiologists.
- Adjuvant systemic therapy has been shown to substantially improve the disease-free and overall survival in both premenopausal and postmenopausal women up to age 70 with lymph node negative and lymph node positive breast cancer. See Early Breast Cancer Trialists' Collaborative Group, Lancet 352(9132):930-942 (1998); Early Breast Cancer Trialists' Collaborative Group, Lancet 351(9114):1451-1467 (1998).
- the absolute benefit from adjuvant treatment is larger for patients with poor prognostic features and this has resulted in the policy to select only these so-called 'high-risk' patients for adjuvant chemotherapy.
- the present invention provides a method for classifying an individual with breast cancer as having a good prognosis or a poor prognosis, wherein said individual is 55 years of age or older, comprising detecting a difference in the expression of a first plurality of genes in a cell sample taken from the individual relative to a control, said first plurality of genes comprising 10 of the genes corresponding to the different markers listed in any of Tables 1-8, wherein "good prognosis" is a desired outcome and "poor prognosis” is an undesired outcome.
- said plurality comprises 20 of the genes corresponding to the different markers listed in any of Tables 1-8.
- said plurality comprises 50 of the genes corresponding to the different markers listed in any of Tables 1-8. In another specific embodiment, said plurality comprises each of the genes corresponding to the markers listed in Table 1. In another specific embodiment, said plurality comprises each of the genes corresponding to the markers listed in Table 3. In another specific embodiment, said individual is identified as ER+, and said plurality comprises 10 of the genes corresponding to the markers listed in Table 5. In another specific embodiment, said individual is identified as ER+, and said plurality comprises 50 of the genes corresponding to the markers listed in Table 5. In another specific embodiment, said individual is identified as ER+, and said plurality comprises each of the genes corresponding to the markers listed in Table 5.
- said individual is identified as ER+, and said plurality comprises 10 of the genes corresponding to the markers listed in Table 7. In another embodiment, said individual is identified as ER+, and said plurality comprises 50 of the genes corresponding to the markers listed in Table 7. In another specific embodiment, said individual is identified as ER+, and said plurality comprises each of the genes corresponding to the markers listed in Table 7.
- said control comprises nucleic acids derived from a pool of tumors from individual sporadic patients.
- said good prognosis is the non reoccurrence or metastasis within five years of initial diagnosis
- said poor prognosis is the reoccurrence or metastasis within five years of initial diagnosis.
- said detecting comprises the steps of: (a) generating a good prognosis template by hybridization of nucleic acids derived from a plurality of good prognosis patients against nucleic acids derived from a pool of tumors from individual patients; (b) generating a poor prognosis template by hybridization of nucleic acids derived from a plurality of poor prognosis patients against nucleic acids derived from said pool of tumors from said plurality of individual patients; (c) hybridizing an nucleic acids derived from and individual sample against said pool; and (d) determining the similarity of marker gene expression in the individual sample to the good prognosis template and the poor prognosis template, wherein if said expression is more similar to the good prognosis template, the sample is classified as having a good prognosis, and if said expression is more similar to the poor prognosis template, the sample is classified as having a poor prognosis.
- the invention further provides a method for classifying a sample as derived from an individual having a good prognosis or derived from an individual having a poor prognosis, wherein said individual is 55 years of age or older, by calculating the similarity between the expression of at least 10 of the different markers listed in any of Tables 1-8 in the sample to the expression of the same markers in a good prognosis nucleic acid pool and a poor prognosis nucleic acid pool, comprising the steps of: (a) labeling nucleic acids derived from a sample, with a first fluorophore to obtain a first pool of fluorophore-labeled nucleic acids; (b) labeling with a second fluorophore a first pool of nucleic acids derived from two or more samples from individuals having a good prognosis (good prognosis pool), and a second pool of nucleic acids derived from two or more samples from individuals having a poor prognosis (poor prognosis pool
- said similarity is calculated by determining a first sum of the differences of expression levels for each marker between said first fluorophore-labeled nucleic acid and said first pool of second fluorophore-labeled nucleic acid, and a second sum of the differences of expression levels for each marker between said first fluorophore-labeled nucleic acid and said second pool of second fluorophore-labeled nucleic acid, wherein if said first sum is greater than said second sum, the sample is classified as derived from an individual having a poor prognosis, and if said second sum is greater than said first sum, the sample is classified as derived from an individual having a good prognosis.
- the invention further provides a method for determining a set of marker genes whose expression is associated with a particular phenotype, comprising the steps of: (a) selecting a phenotype having two or more phenotype categories; (b) identifying a first plurality of genes, wherein the expression of said genes in a first plurality of samples is correlated or anticorrelated with one of the phenotype categories; (c) predicting the phenotype category of each sample in said plurality of samples based on the expression level of each of said plurality of genes across all other samples in said plurality of samples; (d) selecting those samples for which the phenotype category is correctly predicted, to form a second plurality of samples; (e) identifying a second plurality of genes, wherein the expression of said genes in said second plurality of samples is correlated or anticorrelated with one of the phenotype categories; wherein said second plurality of genes is a set of marker genes whose expression is associated with a particular phenotype.
- said phenotype is breast cancer, and said phenotype categories are good prognosis and poor prognosis.
- said second plurality of marker genes is validated by: (a) using a statistical method to randomize the association between said second plurality of marker genes and said phenotype category, thereby creating a control correlation coefficient for each marker gene; (b) repeating step (a) one hundred or more times to develop a frequency distribution of said control correlation coefficients for each marker gene; (c) determining the number of marker genes having a control correlation coefficient above a preselected threshold, thereby creating a control marker gene set; and (d) comparing the number of control marker genes so identified to the number of marker genes, wherein if the difference between the number of marker genes and the number of control genes is statistically significant, said set of marker genes is validated.
- said second plurality of marker genes is optimized by the method comprising: (a) rank-ordering the genes by amplitude of correlation or by significance of the correlation coefficients to create a rank-ordered list, and (b) selecting an arbitrary number n of marker genes from the top of the rank-ordered list.
- said set of marker genes is further optimized by the method comprising: (a) calculating an error rate for said arbitrary number n of marker genes; (b) increasing by 1 the number of genes selected from the top of the rank-ordered list; (c) calculating an error rate for said number of genes selected from the top of the rank-ordered list; (d) repeating steps (b) and (c) until said number of genes selected from the top of the rank-ordered list includes all genes included in said rank ordered list, and (e) identifying said number of genes selected from the top of the rank-ordered list for which the error rate is smallest, wherein said set of marker genes is optimized when the error rate is the smallest.
- the invention also provides a method for assigning a person to one of a plurality of categories in a clinical trial, comprising determining for each said person the level of expression of at least 10 of the different prognosis markers listed in any of Tables 1- 8, determining therefrom whether the person has an expression pattern that correlates with a good prognosis or a poor prognosis, and assigning said person to one category in a clinical trial if said person is determined to have a good prognosis, and a different category if that person is determined to have a poor prognosis.
- the invention further provides a microarray comprising a plurality of probes complementary and hybridizable to at least 10 different genes for which markers are listed in any one of Tables 1-8, wherein said plurality of probes is at least 50% of probes on said microarray.
- said plurality of probes is at least 50% of probes on said microarray.
- said plurality of probes is at least 70% of probes on said microarray.
- said plurality of probes is at least 90% of probes on said microarray.
- said plurality of probes is at least 95% of probes on said microarray.
- the invention also provides a microarray for distinguishing a cell sample from an individual having a good prognosis from a cell sample from an individual having a poor prognosis, wherein said individual is 55 years of age or older, comprising a positionally-addressable array of polynucleotide probes bound to a support, said polynucleotide probes comprising a plurality of polynucleotide probes of different nucleotide sequences, each of said different nucleotide sequences comprising a sequence complementary and hybridizable to a different gene, said plurality consisting of at least 10 of the different genes corresponding to the markers listed in any of Tables 1-8, wherein said plurality of polynucleotide probes is at least 50% of probes on said microarray.
- the invention further provides a kit for determining whether a sample is derived from a patient having a good prognosis or a poor prognosis, wherein said patient is 55 years of age or older, comprising at least one microarray comprising probes to at least 10 of the different genes corresponding to the markers listed in any of Tables 1-8, and a computer readable medium having recorded thereon one or more programs for determining the similarity of the level of nucleic acid derived from the markers listed in Table 1-8 in a sample to that in a pool of samples derived from individuals having a good prognosis and a pool of samples derived from individuals having a good prognosis, wherein the one or more programs cause a computer to perform a method comprising computing the aggregate differences in expression of each marker between the sample and the good prognosis pool and the aggregate differences in expression of each marker between the sample and the poor prognosis pool, or a method comprising determining the correlation of expression of the markers in the sample to the expression in the good prognosis
- the invention also provides a method for classifying a breast cancer patient according to prognosis, wherein said patient is 55 years of age or older, comprising: (a) comparing the respective levels of expression of at least 10 different genes for which markers are listed in any of Tables 1-8 in a cell sample taken from said breast cancer patient to respective control levels of expression of said at least 10 genes; and (b) classifying said breast cancer patient according to prognosis based on the similarity between said levels of expression in said cell sample and said control levels.
- step (b) comprises determining whether said similarity exceeds one or more predetermined threshold values of similarity.
- the method further comprises determining prior to step (a) said level of expression of said at least five genes.
- said control levels are the mean levels of expression of each of said at least five genes in a pool of tumor samples obtained from a plurality of breast cancer patients who have no distant metastasis within five years of initial diagnosis.
- said control levels comprise the expression levels of said genes in breast cancer patients who have had no distant metastasis within five years of initial diagnosis.
- said control levels comprise, for each of said at least five genes, mean log intensity values stored on a computer.
- the invention further provides a computer program product for classifying a breast cancer patient according to prognosis, said patient being 55 years of age or older, the computer program product for use in conjunction with a computer having a memory and a processor, the computer program product comprising a computer readable storage medium having a computer program encoded thereon, wherein said computer program product can be loaded into the one or more memory units of a computer and causes the one or more processor units of the computer to execute the steps of: (a) receiving a first data structure comprising the respective levels of expression of each of at least 10 different genes for which markers are listed in any of Tables 1-8 in a cell sample taken from said patient; (b) determining the similarity of the level of expression of each of said at least ten genes to respective control levels of expression of said at least five genes to obtain a patient similarity value; (c) comparing said patient similarity value to a selected threshold value of similarity of said respective levels of expression of each of said at least 10 genes to said respective control levels of expression of said at least 10 genes;
- said threshold value of similarity is a value stored in said computer.
- said control levels of expression of said at least 10 genes are stored in said computer.
- said computer program when loaded into memory, further causes said one or more processor units of the computer to execute the steps of receiving a data structure comprising clinical data specific to said breast cancer patient.
- said respective control levels of expression of said at least 10 genes comprises a set of single-channel mean hybridization intensity values for each of said at least 10 genes, stored on said computer readable storage medium.
- said single-channel mean hybridization intensity values are log transformed.
- said computer program product causes said processing unit to perform said comparing step (c) by calculating the difference between the level of expression of each of said at least five genes in said cell sample taken from said breast cancer patient and said respective control levels of expression of said at least five genes.
- said computer program product causes said processing unit to perform said comparing step (c) by calculating the mean log level of expression of each of said at least 10 genes in said control to obtain a control mean log expression level for each gene, calculating the log expression level for each of said at least 10 genes in a breast cancer sample from said patient to obtain a patient log expression level, and calculating the difference between the patient log expression level and the control mean log expression for each of said at least 10 genes.
- said computer program product causes said processing unit to perform said comparing step (c) by calculating similarity between the level of expression of each of said at least 10 genes in said cell sample taken from said patient and said respective control levels of expression of said at least 10 genes, wherein said similarity is expressed as a similarity value, hi a more specific embodiment, said similarity value is a correlation coefficient.
- FIG. 1 Overview of gene expression data for a sample group of 153 breast cancer tumors from patients of age > 55 years over approximately 10,000 significant genes. Each row displays a tumor profile, and each column displays the data for a gene. White indicates the most overexpression relative to the reference pool, black indicates the most underexpression relative to the reference pool, and medium gray indicates no change.
- FIG. 2 The predictive power of the 70 marker classifier ⁇ see Van't Veer et al, Nature 415(6871):530-536 (2002)) for 153 tumors from this study.
- the overall odds ratio is 2.5 [26 38 19 70], and 5 year odds ratio is 5.2 [21 30 8 59].
- the overall error rate is 0.387 and 5 year error rate is 0.306. Error rates for prediction of outcome for good outcome samples and poor outcome samples were calculated based upon the selected threshold (X- axis). Circles: Error rate for good prognosis samples. Stars: Error rates for poor prognosis samples. Line: average of good prognosis and poor prognosis error rates.
- FIG. 4 Procedures used in identifying the optimal set of discriminating genes for the purpose of prognosis (the "homogeneous method”, also called “iterative algorithm”).
- FIG. 5 The classification error rate for type 1 and type 2 together as a function of the number of discriminating genes used in the classifier. The combined optimal error rate is reached by approximately 200 discriminating marker genes.
- the classifier was constructed by using the same method used in out previous study (see Van't Veer et al., Nature 415(6871):530-536 (2002)) ("the Nature method") and as described herein. Circles: error for a particular number of markers used in the classifier.
- X-axis number of reporters.
- Y-axis error rate.
- FIG. 7 The classification error rate for type 1 and type 2 together as a function of the number of discriminating genes used in the classifier. The combined optimal error rate is reached at approximately 100 discriminating marker genes.
- the classifier is modeled by using the new method discussed in the text ("the homogenous method"). Circles: error for a particular number of markers used in the classifier. X-axis: number of reporters. Y-axis: error rate.
- FIGS. 8 A, 8B FIGS. 8 A, 8B.
- FIG. 8 A Scattering plots between the correlation of tumor profiles to "poor outcome group” and the correlation of tumor profiles to "good outcome group” based on the new optimal classifier. Filled circles: good outcome patients. Squares: poor outcome patients.
- FIG. 8B Type 1 error rate, type 2 error rate, and average type 1 and type 2 error rate as a function of threshold.
- FIG. 9 Gene expression pattern of 100 genes identified by the "iterative algorithm” (see Example 3) that can be used to predict the disease outcome.
- FIGS. lOA-lOC Kaplan-Meier plots of the metastasis free probability as a function of time since initial diagnosis.
- Patients with breast cancer in the age group > 55 years are classified as either "poor prognosis" or "good prognosis” group based on a classifier with 70 genes derived from data of age group ⁇ 55 years in our previous study (FIG. 10A), a classifier with 200 genes built with the same method but based on this data set (FIG. 10B), and a classifier with 100 genes built with a new method that looks for homogenous patterns in each group based on this data set (FIG. 10C).
- FIGS. HA, HB Classification error rate for type 1 and type 2 together as a function of the number of discriminating genes used in the classifier for ER+ sample group, by the previously-published method (Van't Veer (2002); FIG. 1 IA); and by the iterative method (see Example 3; FIG. HB).
- FIGS. 14A-14C Kaplan-Meier plots of the metastasis free probability as a function of time since initial diagnosis.
- Patients with breast cancer in the age group > 55 years and ER+ (118 patients total) are classified into a "poor prognosis" group and a "good prognosis” group based on a classifier with 70 genes derived from data of age group ⁇ 55 years in our previous study (FIG. 14A); a classifier with 200 genes built with the same method but based on this data set (FIG. 14B); and a classifier with 100 genes build with an iterative method (Example 3) that looks for homogenous patterns in each group based on this data set (FIG. 14C).
- age 55+ individuals means individuals that are age 55 or older.
- BRCAl tumor means a tumor having cells containing a mutation of the BRCAl locus.
- the "absolute amplitude" of a correlation coefficient means the absolute value of the correlation coefficient, e.g., both correlation coefficients -0.35 and 0.35 have an absolute amplitude of 0.35.
- Good prognosis means a desired outcome.
- a good prognosis may be an expectation of no reoccurrences or metastasis within two, three, four, five years or more of initial diagnosis of breast cancer.
- Prognosis means an undesired outcome.
- a poor prognosis may be an expectation of a reoccurrence or metastasis within two, three, four, or five years of initial diagnosis of breast cancer.
- Marker means a gene or gene products, or an EST derived from that gene, the expression or level of which changes between certain conditions. Where the expression of the gene or gene products correlates with a certain condition, the gene or its products are a marker for that condition.
- Marker-derived polynucleotides means the RNA transcribed from a marker gene, any cDNA or cRNA produced therefrom, and any nucleic acid derived therefrom, such as synthetic nucleic acid having a sequence derived from the marker gene.
- prognosis informative means statistically significantly correlated. For example, the expression of a particular gene is prognosis-informative if its expression is significantly correlated with either a good prognosis or a poor prognosis.
- a “similarity value” is a number that represents the degree of similarity between two things being compared.
- a similarity value may be a number that indicates the overall similarity between a patient's expression profile using specific phenotype-related markers and a control specific to that phenotype (for instance, the similarity to a "good prognosis" template, where the phenotype is a good prognosis).
- the similarity value may be expressed as a similarity metric, such as a correlation coefficient, or may simply be expressed as the expression level difference, or the aggregate of the expression level differences, between a patient sample and a template.
- ER designates the estrogen receptor status of a breast cancer patient.
- ER + designates a high ER level, while ER " designates a low ER level.
- the ER status of a breast cancer patient can be evaluated by various means.
- the ER level is determined by measuring an expression level of a gene encoding the estrogen receptor in a patient.
- the gene encoding the estrogen receptor is the estrogen receptor ⁇ gene.
- the expression level of the estrogen receptor ⁇ gene in the patient relative to the expression level of the gene in a pool of breast tumor samples is used as a measure of the ER status, and the ER level is classified as ER + if the logl ⁇ (ratio) of the expression level is greater than -0.65, and the ER level is classified as ER " if the logl ⁇ (ratio) of the expression level is equal to or less than -0.65.
- the ER status is evaluated based on the expression profile of a set of marker genes as described in PCT Publication No. WO 02/103320.
- the invention provides sets of genetic markers whose expression is correlated with the prognosis of breast cancer. These markers are listed as SEQ ID NOS: 1- 387 herein. These markers are particularly useful in the prognosis of breast cancer in individuals of age 55 or older.
- the invention provides a set of 387 breast cancer prognosis-informative markers, i.e., markers that are significantly correlated with either a good or a poor outcome in breast cancer patients. These markers are listed in Tables 1, 3, 5 and 7 or in Tables 2, 4, 6 and 8. Tables 1 and 2 list the same markers; Tables 1, 3, 5 and 7 correlate particular markers with SEQ ID NOS for the 387 markers, and Tables 2, 4, 6 and 8 provide gene names and descriptions for each of the 387 markers.
- the invention also provides subsets of at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the different markers present in Tables 1, 3, 5 and 7 or in Tables 2, 4, 6 and 8, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals.
- the invention further provides subsets of no more than 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the different markers in Tables 1, 3, 5 and 7 or in Tables 2, 4, 6 and 8, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals.
- the invention further provides subsets of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the different markers listed in Tables 1, 3, 5 and 7 or in Tables 2, 4, 6 and 8.
- a subset comprises all 387 different markers listed in Tables 1, 3, 5 and 7 or in Tables 2, 4, 6 and 8.
- the invention provides a set of 200 breast cancer prognosis-informative markers, i.e., markers that are significantly correlated with either a good or a poor outcome in breast cancer patients. These markers were identified using an algorithm previously described (see International Application Publication No. WO 02/103320), and are listed in Tables 1 and 2. Tables 1 and 2 list the same markers; Table 1 correlates particular markers with SEQ ID NOS for the 200 markers, and Table 2 provides gene names and descriptions for each of the 200 markers.
- the invention also provides subsets of at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the markers present in Tables 1 or 2, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals.
- the invention further provides subsets of no more than 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the markers listed in Tables 1 or 2, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals.
- the invention further provides subsets of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the markers listed in Table 1 or 2.
- a subset comprises 100 of the markers, and even more preferably comprises all 200 markers listed in Table 1 or 2.
- the invention provides a set of 100 breast cancer prognosis-informative markers. These markers were identified by an iterative sample- exclusion method described elsewhere herein (see Example 3). These markers are listed in both Tables 3 and 4; Table 3 correlates particular markers with SEQ ID NOS for the 100 markers, and Table 4 provides gene names and descriptions for each of the 100 markers.
- the invention also provides subsets of at least 10, 15, 20, 25, 30, 40, 50 or 75 of the markers present in Table 3 or 4, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals.
- the invention further provides subsets of no more than 10, 15, 20, 25, 30, 40, 50 or 75 of the markers present in Table 3 or 4, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals.
- the invention further provides subsets of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the markers listed in Table 3 or 4.
- a subset comprises 50 of the markers, and even more preferably comprises all 100 markers listed in Table 3 or 4.
- the invention provides a set of 200 breast cancer prognosis-informative markers, i.e., markers that are significantly correlated with either a good or a poor prognosis. These markers were identified using an algorithm previously described (see International Application Publication No. WO 02/103320) applied to samples from individuals with ER+ tumors. These markers are listed in Table 5 and 6. Table 5 and 6 list the same markers; Table 5 correlates particular markers with SEQ ID NOS for the 200 markers, and Table 6 provides gene names and descriptions for each of the 200 markers.
- the invention also provides subsets of at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the markers present in Table 5 or 6, which are particularly useful for prognosis of breast cancer in individuals having breast cancer, including age 55+, ER+ individuals.
- the invention further provides subsets of no more than 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the markers present in Table 5 or 6, which are particularly useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals with ER+ tumors.
- the invention further provides subsets of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the markers listed in Table 5 or 6.
- a subset comprises 100 of the markers, and even more preferably comprises all 200 markers listed in Table 5 or 6.
- the invention provides a set of 100 breast cancer prognosis-informative markers. These markers were identified by an iterative sample- exclusion method described elsewhere herein (see Example 3) using ER+ tumor samples. These markers are listed in both Table 7 and 8; Table 7 correlates particular markers with SEQ ID NOS for the 100 markers, and Table 8 provides gene names and descriptions for each of the 100 markers.
- the invention also provides subsets of at least 10, 15, 20, 25, 30, 40, 50 or 75 of the markers present in Table 7 or 8, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals having ER+ tumors.
- the invention further provides subsets of no more than 10, 15, 20, 25, 30, 40, 50 or 75 of the markers present in Table 7 or 8, which are useful for prognosis of breast cancer in individuals having breast cancer, including age 55+ individuals having ER+ tumors.
- the invention further provides subsets of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the markers listed in Table 7 or 8.
- a subset comprises 50 of the markers, and even more preferably comprises all 100 markers listed in Table 7 or 8.
- the invention provides subsets of at least 10, 15, 20,
- the invention further provides subsets of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the markers listed in any one or more of Tables 1, 3, 5, and 7, or in any one or more of Tables 2, 4, 6, and 8; that is, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the sequences of SEQ ID NOS: 1-387.
- prognosis- informative markers may be selected from any one or more of Tables 1, 3, 5, and 7, or any one or more of Tables 2, 4, 6, and 8, and used in the methods of the invention.
- preferred prognosis-informative markers are those derived from genes that encode kinases or cell cycle control proteins.
- Table 3 100 prognosis markers identified by an iterative method.
- Table 7 100 prognosis markers identified by an iterative method in sporadic, ER+ individuals.
- the present invention provides sets of markers for the identification of conditions or indications associated with breast cancer.
- the marker sets were identified by determining which of ⁇ 25,000 human markers had expression patterns that correlate with the conditions or indications.
- the methods for identification of sets of markers make use of measured cellular constituent profiles, e.g., expression profiles of a plurality of genes (e.g., measurements of abundance levels of the corresponding gene products), in tumor samples from a plurality of patients whose prognosis outcomes are known.
- the prognosis outcomes can be the prognosis at a predetermined time after initial diagnosis.
- the predetermined time can be any appropriate time period, e.g., 2, 3, 4, or 5 years.
- Prognosis markers can be obtained by identifying genes whose expression levels correlate with prognosis outcome, e.g., genes whose expression levels in good prognosis patients group are significantly different from those in poor prognosis patients.
- the tumor samples from the plurality of patients are separated into a good prognosis group and a poor prognosis group for the predetermined time period.
- Genes whose expression levels exhibit differences between the good and poor prognosis groups to at least a predetermined level are selected as the genes whose expression levels correlate with patient prognosis.
- the expression profile is a differential expression profile.
- Each measurement in the profile is a differential expression level of a marker in a breast tumor sample versus that in a reference sample (also termed a standard or control sample)
- the reference sample comprises polynucleotide molecules, derived from one or more samples from a plurality of normal individuals.
- the normal individuals may be persons not having breast cancer.
- the standard or control may also comprise polynucleotide molecules, derived from one or more samples derived from individuals having a different form or stage of breast cancer; a different disease or different condition, or individuals exposed or subjected to a different condition, than the individual from which the sample of interest was obtained.
- the reference or control may be a sample, or set of samples, taken from the individual at an earlier time, for example, to assess the progression of a condition, or the response to a course of therapy.
- the standard or control is a pool of target polynucleotide molecules derived from a plurality of different individuals.
- the pool may be a pool of proteins or the relevant biomolecule.
- the pool comprises samples taken from a number of individuals having sporadic-type tumors.
- the pool comprises an artificially- generated population of nucleic acids designed to approximate the level of nucleic acid derived from each marker found in a pool of marker-derived nucleic acids derived from tumor samples
- the pool also called a "mathematical sample pool”
- the pool is represented by a set of expression values, rather than a set of physical polynucleotides; the level of expression of relevant markers in a sample from an individual with a condition, such as a disease, is compared to values representing control levels of expression for the same markers in the mathematical sample pool.
- a control may be a set of values stored on a computer.
- Such artificial or mathematical controls may be constructed for any condition of interest.
- the reference sample is derived from a normal breast cell line or a breast cancer cell line.
- expressed proteins are used as markers
- the proteins are obtained from the individual's sample, and the standard or control could be a pool of proteins from a number of normal individuals, or from a number of individuals having a particular state of a condition, such as a pool of samples from individuals having a particular prognosis of breast cancer.
- the method for identifying marker sets is as follows.
- the expression of all markers (genes) in a sample X is compared to the expression of all markers in a standard or control.
- the standard or control comprises target polynucleotide molecules derived from a sample from a normal individual (i.e., an individual not having breast cancer).
- the standard or control is a pool of target polynucleotide molecules. The pool may be derived from collected samples from a number of normal individuals. In a preferred embodiment, the pool comprises samples taken from a number of individuals having sporadic-type tumors.
- the pool comprises an artificially-generated population of nucleic acids designed to approximate the level of nucleic acid derived from each marker found in a pool of marker-derived nucleic acids derived from tumor samples.
- the pool is derived from normal or breast cancer cell lines or cell line samples.
- the comparison may be accomplished by any means known in the art. For example, expression levels of various markers may be assessed by separation of target polynucleotide molecules (e.g., RNA or cDNA) derived from the markers in agarose or polyacrylamide gels, followed by hybridization with marker-specific oligonucleotide probes. Alternatively, the comparison may be accomplished by the labeling of target polynucleotide molecules followed by separation on a sequencing gel. Polynucleotide samples are placed on the gel such that patient and control or standard polynucleotides are in adjacent lanes. Comparison of expression levels is accomplished visually or by means of 27243
- the expression of all markers is assessed simultaneously by hybridization to a microarray.
- markers meeting certain criteria are identified as associated with breast cancer.
- genes are first screening genes based on significant variation in expression as compared to a standard or control sample in a set of breast cancer tumor samples. Genes may be screened, for example, by determining whether they show significant variation as compared to a standard or control sample in at least some samples among the set of samples. Genes that do not show significant variation in at least some samples in the set of samples are presumed not to be informative, and are discarded from further consideration. Genes showing significant variation in at least some samples in the sample set are retained as candidate informative genes. The degree of variation in expression of a gene may be estimated by determining a difference or ratio of the expression of the gene in a sample and a control.
- the difference or ratio of expression may be further transformed, e.g., by a linear or log transformation.
- Selection of candidate markers may be made based upon either significant up- or down-regulation of the gene in at least some samples in the set or based on the statistical significance (e.g., the p-value) of the variation in expression of the gene. Preferably, both selection criteria are used.
- genes showing both a more than two-fold change (increase or decrease) in expression as compared to a standard in at least three samples, and a p-value of variation in expression of the gene in the set of tumor samples as compared to the standard sample is no more than 0.01 (i.e., is statistically significant) are selected as candidate genes associated with prognosis of breast cancer.
- Expression profiles comprising a plurality of different genes in a plurality of n breast cancer tumor samples can be used to identify markers that correlate with, and therefore are useful for discriminating, different clinical categories.
- c represents the clinical parameters in the n tumor samples or categories and r represents the measured expression levels of a gene in the n tumor samples, e.g., each element in r can be the linear, logarithmic or any transform of the ratio of expression of the gene between a tumor sample and a control.
- Genes for which the coefficient of correlation exceeds a cutoff or threshold value are identified as breast cancer-related markers specific for a particular clinical type. Such a cutoff or threshold value may correspond to a certain significance of discriminating genes obtained by Monte Carlo simulations.
- markers are chosen if the correlation coefficient is greater than about 0.3 or less than about -0.3.
- the significance of the set of marker genes can be evaluated.
- the significance may be calculated by any appropriate statistical method.
- a Monte-Carlo technique is used to randomize the association between the expression profiles of the plurality of patients and the clinical categories to generate a set of randomized data.
- the same marker selection procedure as used to select the marker set is applied to the randomized data to obtain a control marker set.
- a plurality of such runs can be performed to generate a probability distribution of the number of genes in control marker sets, hi a preferred embodiment, 10,000 such runs are performed. From the probability distribution, the probability of finding a marker set consisting of a given number of markers when no correlation between the expression levels and phenotype is expected (i.e., based randomized data) can be determined.
- the significance of the marker set obtained from the real data can be evaluated based on the number of markers in the marker set by comparing to the probability of obtaining a control marker set consisting of the same number of markers using the randomized data. In one embodiment, if the probability of obtaining a control marker set consisting of the same number of markers using the randomized data is below a given probability threshold, the marker set is said to be significant.
- the markers may be rank-ordered in order of significance of discrimination.
- rank ordering is by the amplitude of correlation between the change in gene expression of the marker and the specific condition being discriminated.
- Another, preferred, means is to use a statistical metric, hi a specific embodiment, the metric is a Fisher-like statistic: t M-( ⁇ /V n k 2 ( «i - D + ⁇ 2 2 (n 2 - I)] ,Z(H 1 + /I 2 - I)I[IIn 1 + l/n 2 ) Equation (2)
- ( ⁇ 1 ) is the error-weighted average of the log ratio of transcript expression measurements within a first clinical group (e.g., good prognosis)
- (x 2 ) is the error- weighted average of log ratio within a second, related clinical group (e.g., poor prognosis)
- ⁇ x is the variance of the log ratio within the first clinical group (e.g.,
- the rank-ordered marker set may be used to optimize the number of markers in the set used for discrimination. This is accomplished generally in a "leave one out” method as follows, hi a first run, a subset, for example 5, of the markers from the top of the ranked list is used to generate a template, where out of X samples, X-I are used to generate the template, and the status of the remaining sample is predicted. This process is repeated for every sample until every one of the X samples is predicted once. In a second run, additional markers, for example 5, are added, so that a template is now generated from 10 markers, and the outcome of the remaining sample is predicted. This process is repeated until the entire set of markers is used to generate the template.
- type 1 error false negative
- type 2 errors false positive
- the optimal number of markers is that number where the type 1 error rate, or type 2 error rate, or preferably the total of type 1 and type 2 error rate is lowest.
- validation of the marker set may be accomplished by an additional statistic, a survival model.
- This statistic generates the probability of tumor distant metastasis as a function of time since initial diagnosis.
- a number of models may be used, including Weibull, normal, log-normal, log logistic, log-exponential, or log-Rayleigh (Chapter 12 "Life Testing", S-PLUS 2000 GUIDE TO STATISTICS, Vol. 2, p. 368 (2000)).
- the probability of distant metastasis P at time t is calculated as
- the above methods are not limited to the identification of markers associated with breast cancer, but may be used to identify set of marker genes associated with any phenotype.
- the phenotype can be the presence or absence of a disease such as cancer, or the presence or absence of any identifying clinical condition associated with that cancer, the phenotype may also be the response, or lack thereof, to a particular treatment regimen, for example, a Course of one or more anticancer drugs.
- the phenotype may be a prognosis such as a survival time, probability of distant metastasis of a disease condition, or likelihood of a particular response to a therapeutic or prophylactic regimen.
- the phenotype need not be cancer, or a disease; the phenotype may be a nominal characteristic associated with a healthy individual.
- the invention provides an "iterative" method for the identification of sets of genes associated with a particular phenotype.
- An important aspect of this method is that samples, within a set of samples used to construct a classifier for the phenotype, that are incorrectly predicted using classifier templates constructed using all samples in the set, are discarded, and samples the phenotype of which is accurately predicted are retained. The retained samples are then used to construct a second classifier, which is more likely to contain a set of genes that reflects the dominant underlying molecular mechanism for the particular phenotype.
- the invention provides a method for determining a set of marker genes whose expression is associated with a particular phenotype, comprising the steps of: (a) selecting phenotype having two or more phenotype categories; (b) identifying a first plurality of genes, wherein the expression of said genes in a first plurality of samples is correlated or anticorrelated with one of the phenotype categories; (c) predicting the phenotype category of each sample in said plurality of samples based on the expression level of each of said plurality of genes across all other samples in said plurality of samples; (d) selecting those samples for which the phonotype category is correctly predicted, to form a second plurality of samples; and (e) identifying a second plurality of genes, wherein the expression of said genes in said second plurality of samples is correlated or anticorrelated with one of the phenotype categories; wherein said second plurality of genes is a set of marker genes whose expression is associated with a particular phenotype.
- the phenotype is breast cancer.
- said phenotype categories are good prognosis and poor prognosis.
- said good prognosis means no reoccurrence or metastasis within five years of initial diagnosis of breast cancer
- poor prognosis means reoccurrence or metastasis within five years of initial diagnosis of breast cancer.
- said phenotype categories are response and non-response to a particular anticancer drag, or to a particular combination of anticancer drugs.
- This iterative method may be applied to any disease or condition for which two or more phenotype categories exist.
- the method may be applied to the original generation of sets of markers informative for a particular phenotype and phenotype category(ies), and may be used to improve existing sets of markers that were selected by less robust means.
- markers identified as being phenotype and/or phenotype category- informative may be considered likely targets for therapeutics for that phenotype.
- markers identified as breast cancer prognosis-informative represent genes, and/or their encoded proteins, that are targets for therapeutics against breast cancer.
- target polynucleotide molecules are extracted from a sample taken from an individual having breast cancer.
- the sample may be collected in any clinically acceptable manner, but must be collected such that marker-derived polynucleotides (i.e., RNA) are preserved.
- marker-derived polynucleotides i.e., RNA
- mRNA or nucleic acids derived therefrom i.e., cDNA or amplified DNA
- cDNA or amplified DNA are preferably labeled distinguishably from standard or control polynucleotide molecules, and both are simultaneously or independently hybridized to a microarray comprising some or all of the markers or marker sets or subsets described above.
- mRNA or nucleic acids derived therefrom may be labeled with the same label as the standard or control polynucleotide molecules, wherein the intensity of hybridization of each at a particular probe is compared.
- a sample may comprise any clinically relevant tissue sample, such as a tumor biopsy or fine needle aspirate, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascitic fluid, cystic fluid, urine or nipple exudate.
- the sample may be taken from a human, or, in a veterinary context, from non-human animals such as ruminants, horses, swine or sheep, or from domestic companion animals such as felines and canines.
- the sample may also be paraffin-embedded tissue sections (see, e.g., U.S. Patent Application Publication No. 2005/0048542A1, which is incorporated by reference herein in its entirety).
- the expression profiles of paraffin-embedded tissue samples are preferably obtained using quantitative reverse transcriptase polymerase chain reaction qRT-PCR (see Section 5.4.2.7., infra).
- RNA may be isolated from eukaryotic cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein.
- Cells of interest include wild-type cells (i.e., non-cancerous), drug-exposed wild-type cells, tumor- or tumor-derived cells, modified cells, normal or tumor cell line cells, and drug-exposed modified cells.
- RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (Chirgwin et al, Biochemistry 18:5294-5299 (1979)). PoIy(A)+ RNA is selected by selection with oligo-dT cellulose (see Sambrook et al., , MOLECULAR CLONING - A LABORATORY MANUAL (2ND ED.), VOIS. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).
- separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.
- RNAse inhibitors may be added to the lysis buffer.
- mRNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA).
- Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SephadexTM (see Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vol. 2, Current Protocols Publishing, New York (1994).
- poly(A)+ mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS.
- the sample of RNA can comprise a plurality of different mRNA molecules, each different mRNA molecule having a different nucleotide sequence.
- the mRNA molecules in the RNA sample comprise at least 100 different nucleotide sequences. More preferably, the mRNA molecules of the RNA sample comprise mRNA molecules corresponding to each of the marker genes.
- the RNA sample is a mammalian RNA sample.
- total RNA or mRNA from cells are used in the methods of the invention.
- the source of the RNA can be cells of a plant or animal, human, mammal, primate, non-human animal, dog, cat, mouse, rat, bird, yeast, eukaryote, prokaryote, etc.
- the method of the invention is used with a sample containing total mRNA or total RNA from 1 x 10 6 cells or less.
- proteins can be isolated from the foregoing sources, by methods known in the art, for use in expression analysis at the protein level.
- Probes to the homologs of the marker sequences disclosed herein can be employed preferably wherein non-human nucleic acid is being assayed.
- the present invention provides methods of using the marker sets to analyze a sample from an individual so as to determine the metastatic potential of an individual's tumor at a molecular level, i.e., to determine a prognosis for the individual from which the sample is obtained.
- the individual need not actually be having breast cancer.
- the expression of specific marker genes in the individual, or a sample taken therefrom is analyzed, e.g., compared to a standard or control, to determine if the pattern of expression indicates a good or a poor prognosis.
- the levels of expression of breast cancer prognostic markers for condition X in an individual can be compared to the respective levels of the marker-derived polynucleotides in a control, wherein the levels of expression in the control represent the levels of expression of the markers exhibited by samples having condition X.
- the expression of the markers in the individual's sample is substantially ⁇ i.e., statistically) similar to that of the control, then the individual is said to have condition X, whereas if the expression of the markers in the individual's sample is substantially (i.e., statistically) different from that of the control, then the individual does not have condition X.
- condition Y can be a good prognosis and a poor prognosis, respectively, as defined by the particular disease or condition, such as breast cancer, and the particular clinical status of the individual.
- the comparison to a control representing condition Y can also be performed.
- the expression of the markers in the individual's sample is substantially (i.e., statistically) similar to that of the control, then the individual is said to have condition Y.
- both are performed simultaneously, such that each control acts as both a positive and a negative control.
- the distinguishing result may thus either be a demonstrable difference from the expression levels (i.e., the amount of marker-derived RNA, or polynucleotides derived therefrom) represented by the control, or no significant difference.
- the method of determining a particular tumor- related status of an individual comprises the steps of (1) hybridizing labeled target polynucleotides from the individual to a microarray containing one of the above marker sets; (2) hybridizing standard or control polynucleotides molecules to the microarray, wherein the standard or control molecules are differentially labeled from the target molecules; and (3) determining the difference in transcript levels, or lack thereof, between the target and standard or control, wherein the difference, or lack thereof, determines the individual's tumor-related status.
- the standard or control molecules comprise marker-derived polynucleotides from a pool of samples from normal individuals, or a pool of tumor samples from individuals having sporadic-type tumors.
- the standard or control is an artificially-generated pool of marker- derived polynucleotides, which pool is designed to mimic the level of marker expression exhibited by clinical samples of normal or breast cancer tumor tissue having a particular clinical indication (i.e., good prognosis or poor prognosis; no reoccurrence or metastasis within five years of initial diagnosis or reoccurrence or metastasis within five years of initial diagnosis; etc.).
- the control molecules comprise a pool derived from normal or breast cancer cell lines.
- good prognosis from “poor prognosis” tumor types.
- good prognosis means no reoccurrence or metastasis, in the individual from which the sample was taken, within five years of initial diagnosis
- poor prognosis means reoccurrence or metastasis within five years of initial diagnosis.
- the level of polynucleotides (i.e., mRNA or polynucleotides derived therefrom) in a sample from an individual, expressed from the different markers provided in any of Tables 1-8 are compared to the level of expression of the same markers from a control, wherein the control comprises marker-related polynucleotides derived from samples obtained from individuals with no 5-year reoccurrence or metastasis, samples take from individuals having reoccurrence or metastasis within five years, or both.
- the comparison is to both, and preferably the comparison is to polynucleotide pools from a number of "good prognosis'" and "poor prognosis" samples, respectively.
- the individual's marker expression most closely resembles or correlates with the "good prognosis” control, and does not resemble or correlate with the "poor prognosis” control, the individual is classified as having a good prognosis.
- the pool is not pure "good prognosis” or "poor prognosis,” for example, a sporadic pool may be used.
- a set of experiments should be performed in which nucleic acids from individuals with known prognosis status are hybridized against the pool, in order to define the expression templates for the "good prognosis” and "poor prognosis” group. Nucleic acids from each individual with unknown prognosis status are hybridized against the same pool and the expression profile is compared to the template(s) to determine the individual's prognosis.
- control or standard may be presented in a number of different formats.
- control, or template, to which the expression of marker genes in a breast cancer tumor sample is compared may be the average absolute level of expression of each of the genes in a pool of marker-derived nucleic acids pooled from breast cancer tumor samples obtained from a plurality of breast cancer patients.
- the difference between the absolute level of expression of these genes in the control and in a sample from a breast cancer patient provides the degree of similarity or dissimilarity of the level of expression in the patient sample and the control.
- the absolute level of expression may be measured by the intensity of the hybridization of the nucleic acids to an array.
- the values for the expression levels of the markers in both the patient sample and control are transformed (see Section 5.4.3).
- the expression level value for the patient, and the average expression level value for the pool, for each of the marker genes selected may be transformed by taking the logarithm of the value.
- the expression level values may be normalized by, for example, dividing by the median hybridization intensity of all of the samples that make up the pool.
- the control may be derived from hybridization data obtained simultaneously with the patient sample expression data, or may constitute a set of numerical values stores on a computer, or on computer- readable medium.
- the invention provides for method of determining whether an individual having breast cancer will likely experience a relapse within five years of initial diagnosis (Le., whether an individual has a poor prognosis) comprising (1) comparing the level of expression of at least ten of the different markers listed in any of Tables 1-8 in a sample taken from the individual to the level of the same markers in a standard or control, where the standard or control levels represent those found in an individual with a poor prognosis; and (2) determining whether the level of the marker- related polynucleotides in the sample from the individual is significantly different than that of the control, wherein if no substantial difference is found, the patient has a poor prognosis, and if a substantial difference is found, the patient has a good prognosis.
- the markers associated with good prognosis can also be used as controls. In a more specific embodiment, both controls are run.
- the invention provides for a method of determining a course of treatment of a breast cancer patient, comprising determining whether the level of expression of at least 10 of the different markers listed in any of Tables 1-8, or one or more subsets thereof, correlates with the level of these markers in a sample representing a good prognosis expression pattern or a poor prognosis pattern; and determining a course of treatment, wherein if the expression correlates with the poor prognosis pattern, the tumor is treated as an aggressive tumor.
- any of the marker sets described in Section 5.1.2. can be used.
- the full set of markers may be used (Le., the complete set of different markers shown in any of Tables 1-8).
- all markers disclosed herein may be used, i.e., all 387 prognosis-informative markers.
- subsets of the markers may be used.
- the prognosis of an individual is determined using the markers listed in any of Tables 1-4 are used.
- the individual is identified as being ER+, and the prognosis of an individual is determined using the markers listed in any of Tables 5-8 are used.
- An individual may be identified as ER+ or ER- by an acceptable means (e.g., northern blot analysis, SDS-PAGE analysis, or microarray analysis).
- the level of expression of the ER gene alone may be determined, whereby, for example, if the level of expression is, or is nearly, zero, the individual is ER-, and higher levels of expression indicate that the individual is ER+.
- one may identify an sample as ER- or ER+ using gene expression levels, for example, those disclosed in International Application Publication No. WO 02/103320.
- the prognosis of an individual may be determined using one or more subsets of at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the different markers present in any one or more of Tables 1-8 (SEQ ID NOS: 1-387), up to the total number of markers 387.
- the prognosis of an individual is determined using only those markers listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
- the prognosis of an individual may be determined using one or more subsets of at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the different markers present in any of Tables 1-8, up to the total number of markers in a Table.
- the prognosis of an individual may be determined using one or more subsets of no more than 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 175 of the different markers present in any of Tables 1-8, up to the total number of markers in a Table.
- the different markers, or subsets of different markers, used are those listed in any of Tables 5-8.
- the invention provides a method for determining a prognosis of an individual having breast cancer, comprising classifying said individual as having a good prognosis or a poor prognosis based on an expression profile comprising measurements of expression levels of a plurality of genes in a cell sample taken from the individual, said plurality of genes comprising 10 different genes corresponding to the markers listed in any one or more of Tables 1, 3, 5 and 7 (SEQ ID NOS: 1-387), wherein a good prognosis predicts no reoccurrence or metastasis within a predetermined period after initial diagnosis, and wherein a poor prognosis predicts reoccurrence or metastasis within said predetermined period after initial diagnosis.
- the patient's cellular constituent profile comprising measurements of a set of markers, e.g., expression levels of marker genes, is evaluated to determine whether the profile indicates good prognosis or poor prognosis.
- the patient's prognosis is evaluated by comparing the cellular constituent profile to a predetermined cellular constituent template profile corresponding to a certain prognosis, e.g., a good prognosis template comprising measurements of the plurality of cellular constituents which are representative of levels of the cellular constituents in a plurality of good prognosis patients or a poor prognosis template comprising measurements of the plurality of cellular constituents which are representative of levels of the cellular constituents in a plurality of poor prognosis patients.
- a good prognosis patient is a patient who has no reoccurrence or metastasis within a period of time after initial diagnosis, e.g., a period of 1, 2, 3, 4, 5 or 10 years
- a poor prognosis patient is a patient who has reoccurrence or metastasis within a period of time after initial diagnosis, e.g., a period of 1, 2, 3, 4, 5 or 10 years.
- both periods are 5 years.
- the degree of similarity of the patient's cellular constituent profile to a template representing good or poor prognosis can be used to indicate whether the patient has good or poor prognosis.
- a patient is classified as having a good prognosis profile if the patient's cellular constituent profile has a high similarity to a good prognosis template, e.g., a similarity to a good prognosis template above a predetermined threshold value; and/or has a low similarity to a poor prognosis template, e.g., a similarity to a poor prognosis template no higher than a predetermined threshold value.
- a patient is classified as having a poor prognosis profile if the patient's cellular constituent profile has a low similarity to a good prognosis template and/or has a high similarity to a poor prognosis template.
- the similarity between the marker expression profile of an individual and that of a control or template can be assessed in a number of ways.
- the profiles can be compared visually in a printout of expression difference data.
- the similarity can be calculated mathematically.
- ⁇ x Associated with every value X 1 is error ⁇ x .
- the error-weighted arithmetic mean may be calculated using the following formula:
- templates are developed for sample comparison.
- the template can be defined as the error- weighted log ratio average of the expression difference for the group of marker genes able to differentiate the particular breast cancer- related condition. For example, templates are defined for "good prognosis” samples and for "poor prognosis” samples.
- a classifier parameter is calculated. This parameter may be calculated using either expression level differences between the sample and template, or by calculation of a correlation coefficient.
- the similarity is represented by a correlation coefficient between the patient's profile and the template.
- a correlation coefficient above a correlation threshold indicates high similarity, whereas a correlation coefficient below the threshold indicates low similarity.
- the correlation threshold is set as 0.3, 0.4, 0.5 or 0.6.
- similarity between a patient's profile and a template is represented by a distance between the patient's profile and the template.
- a distance below a given value indicates high similarity, whereas a distance equal to or greater than the given value indicates low similarity.
- Either one or both of the two classifier parameters can then be used to measure degrees of similarities between a patient's profile and the templates: P 1 measures the similarity between the patient' s profile 3; and the good prognosis template Z 1 , and P 2 measures the similarity between y and the poor prognosis template z 2 .
- y is classified as a good prognosis profile if P 1 is greater than a selected correlation threshold or if P 2 is equal to or less than a selected correlation threshold.
- y is classified as a poor prognosis profile if P 1 is less than a selected correlation threshold or if P 2 is above a selected correlation threshold. In still another embodiment, y is classified as a good prognosis profile if P 1 is greater than a first selected correlation threshold and y is classified as a poor prognosis profile if P 2 is greater than a second selected correlation threshold.
- the above method of determining a particular tumor-related status of an individual comprises the steps of (1) hybridizing labeled target polynucleotides from an individual to a microarray containing one of the above marker sets; (2) hybridizing standard or control polynucleotides molecules to the microarray, wherein the standard or control molecules are differentially labeled from the target molecules; and (3) determining the ratio (or difference) of transcript levels between two channels (individual and control), or simply the transcript levels of the individual; and (4) comparing the results from (3) to the predefined templates, wherein said determining is accomplished by means of the statistic of Equation 4 or Equation 6, and wherein the difference, or lack thereof, determines the individual's tumor-related status (for example, prognosis).
- the invention further provides a method for classifying a breast cancer patient according to prognosis, comprising comparing the levels of expression of at least 10 of the different genes for which markers are listed in any of Tables 1-8 in a cell sample taken from said breast cancer patient to control levels of expression of said at least five genes; and classifying said breast cancer patient according to prognosis of his or her breast cancer based on the similarity between said levels of expression in said cell sample and said control levels.
- the second step of this method comprises determining whether said similarity exceeds one or more predetermined threshold values of similarity.
- said control levels are the mean levels of expression of each of said at least ten genes in a pool of tumor samples obtained from a plurality of breast cancer patients having a good prognosis, e.g., who have no metastasis within five years of initial diagnosis.
- said control levels comprise the expression levels of said genes in breast cancer patients who have had no metastasis within five years of initial diagnosis.
- said control levels comprise, for each of said at least ten of the different genes for which markers are listed in any of Tables 1-8, mean log intensity values stored on a computer.
- said control levels comprise, for each of said at least ten of the genes for which markers are listed in any of Tables 1-8, mean log intensity values stored on a computer.
- the set of mean log intensity values listed in this table may be used as a "good prognosis" template for any of the prognostic methods described herein.
- the above method may also compare the level of expression of at least 10, 20, 30, 40, 50, 75, 100 or more different genes for which markers listed in any of Tables 1-8, or each of the genes for which markers are listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
- the present invention further provides a method of further classifying "good prognosis" patients into two groups: those having a "very good prognosis” and those having an “intermediate prognosis.” For each of the above classifications, the invention further provides recommended therapeutic regimens.
- the present invention also provides for the classification of a breast cancer patient into one of three prognostic categories comprising (a) determining the similarity between the level of expression of at least ten of the different genes for which markers are listed in any of Tables 1-8 to control levels of expression to obtain a patient similarity value; (b) providing a first threshold similarity value that differentiates persons having a good prognosis from those having a poor prognosis, and providing determining a second threshold similarity value, where said second threshold similarity value indicates a higher degree of similarity of the expression of said genes to said control than said first similarity value; and (c) classifying the breast cancer patient into a first prognostic category if the patient similarity value exceeds the first and second threshold similarity values, a second prognostic category if the patient similarity value equals or exceeds the first but not the second threshold similarity value, and a third prognostic category if the patient similarity value is less than the first threshold similarity value.
- the levels of expression of each of said at least five genes is determined first.
- the control comprises marker-related polynucleotides derived from breast cancer tumor samples taken from breast cancer patients clinically determined to have a good prognosis ("good prognosis” control), breast cancer patients clinically determined to have a poor prognosis “poor prognosis” control), or both.
- the control is a "good prognosis" control or template, i.e., a control or template comprising the mean levels of expression of said genes in breast cancer patients who have had no distant metastasis within five years of initial diagnosis.
- said control levels comprise a set of values, for example mean log intensity values, preferably normalized, stored on a computer.
- said determining in step (a) may be accomplished by a method comprising determining the difference between the absolute expression level of each of said genes and the average expression level of the same genes in a pool of tumor samples obtained from a plurality of breast cancer patients who have had no relapse of breast cancer within five years of initial diagnosis.
- said determining in step (a) may be accomplished by a method comprising determining the degree of similarity between the level of expression of each of said genes in a breast cancer tumor sample taken from a breast cancer patient and the level of expression of the same genes in a pool of tumor samples obtained from a plurality of breast cancer patients who have had no relapse of breast cancer within five years of initial diagnosis.
- said first threshold similarity value and said second threshold similarity values are selected by a method comprising (a) rank ordering in descending order said tumor samples that compose said pool of tumor samples by the degree of similarity between the level of expression of said genes in each of said tumor samples to the mean level of expression of the same genes of the remaining tumor samples that compose said pool to obtain a rank-ordered list, said degree of similarity being expressed as a similarity value; (b) determining an acceptable number of false negatives in said classifying, wherein said false negatives are breast cancer patients for whom the expression levels of said at least ten of the different genes for which markers are listed in any of Tables 1-8 in said cell sample predicts that said patient will have no distant metastasis within the first five years after initial diagnosis, but who has had a distant metastasis within the first five years after initial diagnosis; (c) determining a similarity value above which in said rank ordered list fewer than said acceptable number of tumor samples are false negatives; and (d) selecting said
- said second threshold similarity value is selected in step (e) by a method comprising determining which of said tumor samples, taken from patients having a distant metastasis within five years of initial diagnosis, in said rank ordered list has the greatest similarity value, and selecting said greatest similarity value as said second threshold similarity value.
- said first and second threshold similarity values are correlation coefficients, and said first threshold similarity value is 0.4 and said second threshold similarity value is greater than 0.4.
- said first similarity value is a similarity value above which at most 10% false negatives are predicted in a training set of tumors
- said second correlation coefficient is a coefficient above which at most 5% false negatives are predicted in said training set of tumors.
- said first correlation coefficient is a coefficient above which 10% false negatives are predicted in a training set of tumors
- said second correlation coefficient is a coefficient above which no false negatives are predicted in said training set of tumors.
- the first, second and third prognostic categories are characterized as "very good prognosis,” “intermediate prognosis,” and “poor prognosis,” respectively.
- Patients classified into the first prognostic category are likely not to have a distant metastasis within five years of initial diagnosis.
- Patients classified as having an "intermediate prognosis” are also unlikely to have a distant metastasis within five years of initial diagnosis, but may be recommended to undergo a different therapeutic regimen than patients having a "very good prognosis" marker gene expression profile ⁇ see below).
- Patients classified into the third prognostic category (“poor prognosis”) are likely to have a distant metastasis within five years of initial diagnosis.
- the similarity value is the degree of difference between the absolute ⁇ i.e., untransformed) level of expression of each of the genes in a tumor sample taken from a breast cancer patient and the mean absolute level of expression of the same genes in a control.
- the similarity value is calculated using expression level data that is transformed, hi another more specific embodiment, the similarity value is expressed as a similarity metric, such as a correlation coefficient, representing the similarity between the level of expression of the marker genes in the tumor sample and the mean level of expression of the same genes in a plurality of breast cancer tumor samples taken from breast cancer patients.
- said first and second similarity values are derived from control expression data obtained in the same hybridization experiment as that in which the patient expression level data is obtained.
- said first and second similarity values are derived from an existing set of expression data.
- said first and second correlation coefficients are derived from a mathematical sample pool. For example, comparison of the expression of marker genes in new tumor samples may be compared to the pre-existing template determined for these genes for patients in a previous study; the template, or average expression levels of each of the marker genes can be used as a reference or control for any tumor sample.
- the comparison is made to a template comprising the average expression level of at least ten of the different genes listed in any of Tables 1-8 for the 108 out of 153 patients ⁇ see Examples) clinically determined to have a good prognosis.
- the coefficient of correlation of the level of expression of these genes in the tumor sample to the "good prognosis" patient template is then determined to produce a tumor correlation coefficient.
- two similarity values may be derived: a first correlation coefficient that minimizes Type 1 and Type 2 error, and a second correlation coefficient that is higher than the first correlation coefficient.
- the second correlation coefficient is that of the actual poor prognosis sample in the rank-ordered list of samples having the highest correlation to the "good prognosis" template.
- the value of the second correlation coefficient will depend upon the set of samples selected for generation of the template. New breast cancer patients whose coefficients of correlation of the expression of these marker genes with the "good prognosis" template equal or exceed the second correlation coefficient are classified as having a "very good prognosis”; those having a coefficient of correlation of between the first and second correlation coefficients are classified as having an "intermediate prognosis”; and those having a correlation coefficient lower than the first correlation coefficient are classified as having a "poor prognosis.”
- the invention also provides a method of classifying a breast cancer patient according to prognosis, e.g., a breast cancer patient 55+ years of age or older, comprising the steps of (a) contacting first nucleic acids derived from a tumor sample taken from said breast cancer patient, and second nucleic acids derived from two or more tumor samples from breast cancer patients who have had no distant metastasis within five years of initial diagnosis, with an array under conditions such that hybridization can occur, detecting at each of a plurality of discrete loci on said array a first fluorescent emission signal from said first nucleic acids and a second fluorescent emission signal from said second nucleic acids that are bound to said array under said conditions, wherein said array comprises at least ten of the different genes for which markers are listed in any of Tables 1-4 and wherein at least 50% of the probes on said array are listed in Tables 1-8; (b) calculating the similar
- the patient's lymph node metastasis status (i.e., whether the patient is pN+ or pNO) is determined. Patients who are pNO and have a "very good prognosis” or “intermediate” expression profile may be treated without adjuvant chemotherapy. All other patients should be treated with adjuvant chemotherapy.
- the patient's estrogen receptor status is also identified ⁇ i.e., whether the patient is ER+ or ER-).
- patients classified as having an "intermediate prognosis” or “poor prognosis” who are ER+ are assigned a therapeutic regimen that additionally comprises adjuvant hormonal therapy.
- the invention provides for a method of assigning a therapeutic regimen to a breast cancer patient, e.g., a breast cancer patient 55+ years of age or older, comprising (a) classifying said patient as having a "poor prognosis,” “intermediate prognosis,” or “very good prognosis” on the basis of the levels of expression of at least ten of the different genes for which markers are listed in any of Tables 1-8; and (b) assigning said patient a therapeutic regimen, said therapeutic regimen comprising no adjuvant chemotherapy if the patient is lymph node negative and is classified as having a good prognosis or an intermediate prognosis, or comprising chemotherapy if said patient has any other combination of lymph node status and expression profile.
- the invention provides a method for assigning a therapeutic regimen for a breast cancer patient, comprising determining the lymph node status for said patient; determining the level of expression of at least ten of the different genes listed in any of Tables 1-8 in a tumor sample from said patient, thereby generating an expression profile; classifying said patient as having a "poor prognosis", “intermediate prognosis” or "very good prognosis” on the basis of said expression profile; and assigning the patient a therapeutic regimen, said therapeutic regimen comprising no adjuvant chemotherapy if the patient is lymph node negative and is classified as having a good prognosis or an intermediate prognosis, or a therapeutic regiment comprising chemotherapy if said patient has any other combination of lymph node status and expression profile.
- the ER status of the patient is additionally determined, and if the breast cancer patient is ER(+) and has an intermediate or poor prognosis, the therapeutic regimen additionally comprises hormonal therapy.
- the breast cancer patient is premenopausal.
- the breast cancer patient has stage I or stage II breast cancer.
- marker sets are not restricted to the prognosis of breast cancer- related conditions, and may be applied in a variety of phenotypes or conditions, clinical or experimental, in which gene expression plays a role.
- the marker set can be used to distinguish these phenotypes.
- the phenotypes may be the diagnosis and/or prognosis of clinical states or phenotypes associated with other cancers, other disease conditions, or other physiological conditions, wherein the expression level data is derived from a set of genes correlated with the particular physiological or disease condition.
- the expression of markers specific to other types of cancer may be used to differentiate patients or patient populations for those cancers for which different therapeutic regimens are indicated.
- control for example, the control can be the average level of expression of each of the markers, respectively, in a pool of individuals.
- the expression level values are preferably transformed in a number of ways.
- the expression level of each of the markers can be normalized by the average expression level of all markers the expression level of which is determined, or by the average expression level of a set of control genes.
- the markers are represented by probes on a microarray, and the expression level of each of the markers is normalized by the mean or median expression level across all of the genes represented on the microarray, including any non-marker genes.
- the normalization is carried out by dividing the median or mean level of expression of all of the genes on the microarray.
- the expression levels of the markers are normalized by the mean or median level of expression of a set of control markers.
- the control markers comprise a set of housekeeping genes.
- the normalization is accomplished by dividing by the median or mean expression level of the control genes.
- the sensitivity of a marker-based assay will also be increased if the expression levels of individual markers are compared to the expression of the same markers . in a pool of samples.
- the comparison is to the mean or median expression level of each the marker genes in the pool of samples.
- Such a comparison may be accomplished, for example, by dividing by the mean or median expression level of the pool for each of the markers from the expression level each of the markers in the sample. This has the effect of accentuating the relative differences in expression between markers in the sample and markers in the pool as a whole, making comparisons more sensitive and more likely to produce meaningful results that the use of absolute expression levels alone.
- the expression level data may be transformed in any convenient way; preferably, the expression level data for all is log transformed before means or medians are taken.
- the expression levels of the markers in the sample may be compared to the expression level of those markers in the pool, where nucleic acid derived from the sample and nucleic acid derived from the pool are hybridized during the course of a single experiment.
- Such an approach requires that new pool nucleic acid be generated for each comparison or limited numbers of comparisons, and is therefore limited by the amount of nucleic acid available.
- the expression levels in a pool are stored on a computer, or on computer-readable media, to be used in comparisons to the individual expression level data from the sample (i.e., single-channel data).
- the current invention provides the following method of classifying a first cell or organism as having one of at least two different phenotypes, where the different phenotypes comprise a first phenotype and a second phenotype.
- the level of expression of each of a plurality of genes in a first sample from the first cell or organism is compared to the level of expression of each of said genes, respectively, in a pooled sample from a plurality of cells or organisms, the plurality of cells or organisms comprising different cells or organisms exhibiting said at least two different phenotypes, respectively, to produce a first compared value.
- the first compared value is then compared to a second compared value, wherein said second compared value is the product of a method comprising comparing the level of expression of each of said genes in a sample from a cell or organism characterized as having said first phenotype to the level of expression of each of said genes, respectively, in the pooled sample.
- the first compared value is then compared to a third compared value, wherein said third compared value is the product of a method comprising comparing the level of expression of each of the genes in a sample from a cell or organism characterized as having the second phenotype to the level of expression of each of the genes, respectively, in the pooled sample.
- the first compared value can be compared to additional compared values, respectively, where each additional compared value is the product of a method comprising comparing the level of expression of each of said genes in a sample from a cell or organism characterized as having a phenotype different from said first and second phenotypes but included among the at least two different phenotypes, to the level of expression of each of said genes, respectively, in said pooled sample.
- a determination is made as to which of said second, third, and, if present, one or more additional compared values, said first compared value is most similar, wherein the first cell or organism is determined to have the phenotype of the cell or organism used to produce said compared value most similar to said first compared value.
- the compared values are each ratios of the levels of expression of each of said genes.
- each of the levels of expression of each of the genes in the pooled sample is normalized prior to any of the comparing steps.
- the normalization of the levels of expression is carried out by dividing by the median or mean level of the expression of each of the genes or dividing by the mean or median level of expression of one or more housekeeping genes in the pooled sample from said cell or organism.
- the normalized levels of expression are subjected to a log transform, and the comparing steps comprise subtracting the log transform from the log of the levels of expression of each of the genes in the sample.
- the two or more different phenotypes are different stages of a disease or disorder. In still another specific embodiment, the two or more different phenotypes are different prognoses of a disease or disorder. In yet another specific embodiment, the levels of expression of each of the genes, respectively, in the pooled sample or said levels of expression of each of said genes in a sample from the cell or organism characterized as having the first phenotype, second phenotype, or said phenotype different from said first and second phenotypes, respectively, are stored on a computer or on a computer-readable medium.
- the two phenotypes are good prognosis and poor prognosis. In a more specific embodiment, the two phenotypes are no metastasis within five years of initial diagnosis of breast cancer, and reoccurrence or metastasis within five years of initial diagnosis of breast cancer.
- the comparison is made between the expression of each of the genes in the sample and the expression of the same genes in a pool representing only one of two or more phenotypes.
- prognosis-correlated genes for example, one can compare the expression levels of prognosis-related genes in a sample to the average level of the expression of the same genes in a "good prognosis" pool of samples (as opposed to a pool of samples that include samples from patients having poor prognoses and good prognoses).
- a sample is classified as having a good prognosis if the level of expression of prognosis-correlated genes exceeds a chosen coefficient of correlation to the average "good prognosis" expression profile (i.e., the level of expression of prognosis-correlated genes in a pool of samples from patients having a "good prognosis.”
- Patients whose expression levels correlate more poorly with the "good prognosis" expression profile i.e., whose correlation coefficient fails to exceed the chosen coefficient
- the method can be applied to subdivisions of these prognostic classes.
- the phenotype is good prognosis and said determination comprises (1) determining the coefficient of correlation between the expression of said plurality of genes in the sample and of the same genes in said pooled sample; (2) selecting a first correlation coefficient value between 0.4 and +1 and a second correlation coefficient value between 0.4 and +1, wherein said second value is larger than said first value; and (3) classifying said sample as "very good prognosis” if said coefficient of correlation equals or is greater than said second correlation coefficient value, "intermediate prognosis” if said coefficient of correlation equals or exceeds said first correlation coefficient value, and is less than said second correlation coefficient value, or "poor prognosis” if said coefficient of correlation is less than said first correlation coefficient value.
- single-channel data may also be used without specific comparison to a mathematical sample pool.
- a sample may be classified as having a first or a second phenotype, wherein the first and second phenotypes are related, by calculating the similarity between the expression of at least 5 markers in the sample, where the markers are correlated with the first or second phenotype, to the expression of the same markers in a first phenotype template and a second phenotype template, by (a) labeling nucleic acids derived from a sample with a fluorophore to obtain a pool of fluorophore-labeled nucleic acids; (b) contacting said fluorophore-labeled nucleic acid with a microarray under conditions such that hybridization can occur, detecting at each of a plurality of discrete loci on the microarray a flourescent emission signal from said fluorophore-labeled nucleic acid that is bound to said microarray under said conditions; and (c
- the expression levels of the marker genes in a sample may be determined by any means known in the art.
- the expression level may be determined by isolating and determining the level (i.e., amount) of nucleic acid transcribed from each marker gene.
- the level of specific proteins translated from mRNA transcribed from a marker gene may be determined.
- the level of expression of specific marker genes can be accomplished by determining the amount of mRNA, or polynucleotides derived therefrom, present in a sample. Any method for determining RNA levels can be used. For example, RNA is isolated from a sample and separated on an agarose gel. The separated RNA is then transferred to a solid support, such as a filter. Nucleic acid probes representing one or more markers are then hybridized to the filter by northern hybridization, and the amount of marker-derived RNA is determined. Such determination can be visual, or machine-aided, for example, by use of a densitometer. Another method of determining RNA levels is by use of a dot-blot or a slot-blot.
- RNA, or nucleic acid derived therefrom, from a sample is labeled.
- the RNA or nucleic acid derived therefrom is then hybridized to a filter containing oligonucleotides derived from one or more marker genes, wherein the oligonucleotides are placed upon the filter at discrete, easily-identifiable locations.
- Hybridization, or lack thereof, of the labeled RNA to the filter-bound oligonucleotides is determined visually or by densitometer.
- Polynucleotides can be labeled using a radiolabel or a fluorescent (i.e., visible) label.
- the level of expression of particular marker genes may also be assessed by determining the level of the specific protein expressed from the marker genes. This can be accomplished, for example, by separation of proteins from a sample on a polyacrylamide gel, followed by identification of specific marker-derived proteins using antibodies in a western blot. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well-known in the art and typically involves isoelectric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension.
- marker-derived protein levels can be determined by constructing an antibody microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
- binding sites comprise immobilized, preferably monoclonal, antibodies specific to a plurality of protein species encoded by the cell genome.
- antibodies are present for a substantial fraction of the marker- derived proteins of interest.
- Methods for making monoclonal antibodies are well known ⁇ see, e.g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, New York, which is incorporated in its entirety for all purposes).
- monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell.
- proteins from the cell are contacted to the array, and their binding is assayed with assays known in the art.
- assays known in the art.
- the expression, and the level of expression, of proteins of diagnostic or prognostic interest can be detected through immunohistochemical staining of tissue slices or sections.
- tissue array Kononen et al, Nat. Med 4(7):844-7 (1998).
- tissue array multiple tissue samples are assessed on the same microarray. The arrays allow in situ detection of RNA and protein levels; consecutive sections allow the analysis of multiple samples simultaneously.
- polynucleotide microarrays are used to measure expression so that the expression status of each of the markers above is assessed simultaneously.
- the invention provides oligonucleotide or cDNA arrays comprising probes hybridizable to the genes corresponding to each of the marker sets described above ⁇ i.e., markers to distinguish patients 55 years and older with good prognosis versus patients with poor prognosis).
- the invention provides oligonucleotide arrays comprising probes having sequences identified by SEQ ID NOS: 388-774, corresponding respectively to markers identified by SEQ ID NOS: 1-387, or a subset or subsets of at least 10, 20, 30, 40, 50, 75, 100, 125, 150, 175 or 200 of these probes.
- the microarrays provided by the present invention may comprise probes hybridizable to the genes corresponding to markers able to distinguish the status of one, two, or all three of the clinical conditions noted above.
- the invention provides polynucleotide arrays comprising probes to a subset or subsets of at least 10, 20, 30, 40, 50, 75, 100, 125, 150, 175 or 200 of the different markers for which genes are listed in any of Tables 1-8.
- the invention provides polynucleotide arrays in which polynucleotide probes complementary and hybridizable to the breast cancer prognosis-related markers described herein are at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% of the probes on said array.
- the microarray of the invention comprises probes to at least 10 genes selected from Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
- the microarray of the convention comprises probes complementary and hybridizable to 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the genes listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7 or Table 8.
- Probes may be generated, of course, from the sequence of any of SEQ ID NOS: 1-387 for inclusion in a microarray of the invention.
- a microarray of the invention comprises probes to all 200 genes listed in Tables 1 or 2; all 100 genes listed in Tables 3 or 4; all 200 genes listed in Tables 5 or 6; and/or all 100 genes listed in Tables 7 or 8.
- the microarray of the invention comprises probes complementary and hybridizable to at least 10 of the genes listed in Tables 1-4, and probes complementary and hybridizable to at least 10 of the genes listed in Tables 5-8.
- the microarray may comprise probes complementary and hybridizable to 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the different markers listed in any of Tables 1-8; that is, may comprise probes complementary and hybridizable to 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the sequences of SEQ ID NOS: 1-387.
- microarrays that are used in the methods disclosed herein optionally comprise markers additional to at least some of the different markers listed in Tables 1-8.
- the microarray is a screening or scanning array as described in Altanner et ah, International Publication WO 02/18646, published March 7, 2002 and Scherer et ah, International Publication WO 02/16650, published February 28, 2002.
- the scanning and screening arrays comprise regularly-spaced, positionally-addressable probes derived from genomic nucleic acid sequence, both expressed and unexpressed.
- Such arrays may comprise probes corresponding to a subset of, or all of, the different markers listed in Tables 1-8, or a subset thereof as described above, and can be used to monitor marker expression in the same way as a microarray containing only markers listed in Tables 1-6.
- the microarray is a commercially- available cDNA microarray that comprises at least five of the different markers listed in Tables 1-8.
- a commercially-available cDNA microarray comprises all of the markers listed in Tables 1-8.
- such a microarray may comprise 5, 10, 15, 25, 50, 100, 150, 200, 250 or more of the different markers in any of Tables 1-8, up to the total number of markers listed in Tables 1-8.
- the different markers that are all or a portion of Tables 1-8 are at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the probes on the microarray.
- the microarray of the invention may additionally include sets of probes complementary and hybridizable to genes informative for related or unrelated conditions.
- a microarray comprising probes complementary and hybridizable to a plurality of the different prognosis-informative genes listed in any or all of Tables 1-8 may additionally comprise probes complementary and hybridizable to genes informative for ER tumor status, genes that may be used to distinguish sporadic from BRCA-I type tumors, or genes that are informative for any other clinical aspect of breast cancer, or any other related or unrelated condition.
- Microarrays are prepared by selecting probes which comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface.
- the probes may comprise DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA.
- the polynucleotide sequences of the probes may also comprise DNA and/or RNA analogues, or combinations thereof.
- the polynucleotide sequences of the probes may be full or partial fragments of genomic DNA.
- the polynucleotide sequences of the probes may also be synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
- the probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g., by PCR), or non-enzymatically in vitro.
- the probe or probes used in the methods of the invention are preferably immobilized to a solid support which may be either porous or non-porous.
- the probes of the invention may be polynucleotide sequences which are attached to a nitrocellulose or nylon membrane or filter covalently at either the 3 ' or the 5' end of the polynucleotide.
- hybridization probes are well known in the art (see, e.g., Sambrook et al., MOLECULAR CLONING - A LABORATORY MANUAL (2ND ED.), VOIS. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).
- the solid support or surface may be a glass or plastic surface.
- hybridization levels are measured to microarrays of probes consisting of a solid phase on the surface of which are immobilized a population of polynucleotides, such as a population of DNA or DNA mimics, or, alternatively, a population of RNA or RNA mimics.
- the solid phase may be a nonporous or, optionally, a porous material such as a gel.
- a microarray comprises a support or surface with an ordered array of binding (e.g., hybridization) sites or "probes" each representing one of the markers described herein.
- the microarrays are addressable arrays, and more preferably positionally addressable arrays.
- each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position in the array (Le., on the support or surface).
- each probe is covalently attached to the solid support at a single site.
- Microarrays can be made in a number of ways, of which several are described below. However produced, microarrays share certain characteristics. The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably, microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. The microarrays are preferably small, e.g., between 1 cm and 25 cm , between 12 cm and 13 cm , or 3 cm . However, larger arrays are also contemplated and may be preferable, e.g., for use in screening arrays.
- a given binding site or unique set of binding sites in the microarray will specifically bind (e.g., hybridize) to the product of a single gene in a cell (e.g., to a specific mRNA, or to a specific cDNA derived therefrom).
- a single gene in a cell e.g., to a specific mRNA, or to a specific cDNA derived therefrom.
- other related or similar sequences will cross hybridize to a given binding site.
- the microarrays of the present invention include one or more test probes, each of which has a polynucleotide sequence that is complementary to a subsequence of RNA or DNA to be detected.
- the position of each probe on the solid surface is known.
- the microarrays are preferably positionally addressable arrays.
- each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position on the array (i.e., on the support or surface).
- the microarray is an array (i.e., a matrix) in which each position represents one of the markers described herein.
- each position can contain a DNA or DNA analogue based on genomic DNA to which a particular RNA or cDNA transcribed from that genetic marker can specifically hybridize.
- the DNA or DNA analogue can be, e.g., a synthetic oligomer or a gene fragment.
- probes representing each of the markers is present on the array.
- the array comprises the 550 of the 2,460 RE-status markers, 70 of the 5/?CA2/sporadic markers, and all 231 of the prognosis markers.
- the "probe" to which a particular polynucleotide molecule specifically hybridizes according to the invention contains a complementary genomic polynucleotide sequence.
- the probes of the microarray preferably consist of nucleotide sequences of no more than 1,000 nucleotides. In some embodiments, the probes of the array consist of nucleotide sequences of 10 to 1,000 nucleotides.
- the nucleotide sequences of the probes are in the range of 10-200 nucleotides in length and are genomic sequences of a species of organism, such that a plurality of different probes is present, with sequences complementary and thus capable of hybridizing to the genome of such a species of organism, sequentially tiled across all or a portion of such genome.
- the probes are in the range of 10-30 nucleotides in length, in the range of 10-40 nucleotides in length, in the range of 20-50 nucleotides in length, in the range of 40-80 nucleotides in length, in the range of 50-150 nucleotides in length, in the range of 80-120 nucleotides in length, and most preferably are 60 nucleotides in length.
- the probes may comprise DNA or DNA "mimics" (e.g., derivatives and analogues) corresponding to a portion of an organism's genome.
- the probes of the microarray are complementary RNA or RNA mimics.
- DNA mimics are polymers composed of subunits capable of specific, Watson-Crick-like hybridization with DNA, or of specific hybridization with RNA.
- the nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.
- Exemplary DNA mimics include, e.g., phosphorothioates.
- DNA can be obtained, e.g. , by polymerase chain reaction (PCR) amplification of genomic DNA or cloned sequences.
- PCR primers are preferably chosen based on a known sequence of the genome that will result in amplification of specific fragments of genomic DNA.
- Computer programs that are well known in the art are useful in the design of primers with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences).
- each probe on the microarray will be between 10 bases and 50,000 bases, usually between 300 bases and 1,000 bases in length.
- PCR methods are well known in the art, and are described, for example, in Innis et al.
- An alternative, preferred means for generating the polynucleotide probes of the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (Froehler et al, Nucleic Acid Res. 14:5399- 5407 (1986); McBride et al, Tetrahedron Lett. 24:246-248 (1983)). Synthetic sequences are typically between about 10 and about 500 bases in length, more typically between about 20 and about 100 bases, and most preferably between about 40 and about 70 bases in length.
- synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine.
- nucleic acid analogues may be used as binding sites for hybridization.
- An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al, Nature 363:566-568 (1993); U.S. Patent No. 5,539,083).
- Probes are preferably selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure (see Friend et al., International Patent Publication WO 01/05935, published January 25, 2001; Hughes et al., Nat. Biotech. 19:342-7 (2001)).
- positive control probes e.g., probes known to be complementary and hybridizable to sequences in the target polynucleotide molecules
- negative control probes e.g., probes known to not be complementary and hybridizable to sequences in the target polynucleotide molecules, should be included on the array.
- positive controls are synthesized along the perimeter of the array. In another embodiment, positive controls are synthesized in diagonal stripes across the array. In still another embodiment, the reverse complement for each probe is synthesized next to the position of the probe to serve as a negative control, hi yet another embodiment, sequences from other species of organism are used as negative controls or as "spike-in" controls.
- the probes are attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material.
- a preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al, Science 270:467-470 (1995). This method is especially useful for preparing microarrays of cDNA (See also, DeRisi et al, Nature Genetics 14:457-460 (1996); Shalon et al, Genome Res. 5:639-645 (1996); and Schena et al, Proc. Natl. Acad. ScL U.S.A. 93:10539-11286 (1995)).
- a second preferred method for making microarrays is by making high- density oligonucleotide arrays.
- Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al, 1991, Science 251:767-773; Pease et al, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026; Lockhart et al, 1996, Nature Biotechnology 14:1675; U.S. Patent Nos.
- oligonucleotides e.g., 60-mers
- the array produced is redundant, with several oligonucleotide molecules per RNA.
- the arrays of the present invention are prepared by synthesizing polynucleotide probes on a support.
- polynucleotide probes are attached to the support covalently at either the 3 ' or the 5' end of the polynucleotide.
- microarrays of the invention are manufactured by means of an ink jet printing device for oligonucleotide synthesis, e.g., using the methods and systems described by Blanchard in U.S. Pat. No. 6,028,189; Blanchard et al, 1996, Biosensors and Bioelectronics 11:687-690; Blanchard, 1998, in SYNTHETIC DNA ARRAYS IN GENETIC ENGINEERING, Vol. 20, J.K. Setlow, Ed., Plenum Press, New York at pages 111-123.
- the oligonucleotide probes in such microarrays are preferably synthesized in arrays, e.g., on a glass slide, by serially depositing individual nucleotide bases in "microdroplets" of a high surface tension solvent such as propylene carbonate.
- the microdroplets have small volumes ⁇ e.g., 100 pL or less, more preferably 50 pL or less) and are separated from each other on the microarray (e.g., by hydrophobic domains) to form circular surface tension wells which define the locations of the array elements (i.e., the different probes).
- Microarrays manufactured by this ink-jet method are typically of high density, preferably having a density of at least about 2,500 different probes per 1 cm 2 .
- the polynucleotide probes are attached to the support covalently at either the 3 ' or the 5' end of the polynucleotide.
- the polynucleotide molecules which may be analyzed by the present invention may be from any clinically relevant source, but are expressed RNA or a nucleic acid derived therefrom (e.g., cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter), including naturally occurring nucleic acid molecules, as well as synthetic nucleic acid molecules.
- RNA or a nucleic acid derived therefrom e.g., cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter
- naturally occurring nucleic acid molecules as well as synthetic nucleic acid molecules.
- the target polynucleotide molecules comprise RNA, including, but by no means limited to, total cellular RNA, poly(A) + messenger RNA (mRNA) or fraction thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (Le., cRNA; see, e.g., Linsley & Schelter, U.S. Patent Application No. 09/411,074, filed October 4, 1999, or U.S. Patent Nos. 5,545,522, 5,891,636, or 5,716,785).
- RNA including, but by no means limited to, total cellular RNA, poly(A) + messenger RNA (mRNA) or fraction thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (Le., cRNA; see, e.g., Linsley & Schelter, U.S. Patent Application No. 09/411,074, filed October 4, 1999, or U.S. Patent Nos. 5,545,522, 5,891,636, or 5,716,785).
- RNA is extracted from cells of the various types of interest in this invention using guanidinium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al, 1979, Biochemistry 18:5294-5299).
- RNA is extracted using a silica gel-based column, commercially available examples of which include RNeasy (Qiagen, Valencia, California) and StrataPrep (Stratagene, La Jolla, California).
- RNA is extracted from cells using phenol and chloroform, as described in Ausubel et al , eds., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, VOI III, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York, at pp. 13.12.1-13.12.5).
- RNA can be selected, e.g., by selection with oligo-dT cellulose or, alternatively, by oligo-dT primed reverse transcription of total cellular RNA.
- RNA can be fragmented by methods known in the art, e.g. , by incubation with ZnCl 2 , to generate fragments of RNA.
- the polynucleotide molecules analyzed by the invention comprise cDNA, or PCR products of amplified RNA or cDNA.
- total RNA, mRNA, or nucleic acids derived therefrom is isolated from a sample taken from a person having breast cancer.
- Target polynucleotide molecules that are poorly expressed in particular cells may be enriched using normalization techniques (Bonaldo et al, 1996, Genome Res. 6:791-806).
- the target polynucleotides are detectably labeled at one or more nucleotides. Any method known in the art may be used to detectably label the target polynucleotides. Preferably, this labeling incorporates the label uniformly along the length of the RNA, and more preferably, the labeling is carried out at a high degree of efficiency.
- One embodiment for this labeling uses oligo-dT primed reverse transcription to incorporate the label; however, conventional methods of this method are biased toward generating 3' end fragments.
- random primers ⁇ e.g., 9- mers
- random primers may be used in conjunction with PCR methods or T7 promoter-based in vitro transcription methods in order to amplify the target polynucleotides.
- the detectable label is a luminescent label.
- fluorescent labels such as a fluorescein, a phosphor, a rhodamine, or a polymethine dye derivative.
- fluorescent labels examples include, for example, fluorescent phosphoramidites such as FluorePrime (Amersham Pharmacia, Piscataway, NJ.), Fluoredite (Millipore, Bedford, Mass.), FAM (ABI, Foster City, Calif.), and Cy3 or Cy5 (Amersham Pharmacia, Piscataway, N.J.).
- the detectable label is a radiolabeled nucleotide.
- target polynucleotide molecules from a patient sample are labeled differentially from target polynucleotide molecules of a standard.
- the standard can comprise target polynucleotide molecules from normal individuals (i.e., those not having breast cancer), hi a highly preferred embodiment, the standard comprises target polynucleotide molecules pooled from samples from normal individuals or tumor samples from individuals having sporadic-type breast tumors.
- the target polynucleotide molecules are derived from the same individual, but are taken at different time points, and thus indicate the efficacy of a treatment by a change in expression of the markers, or lack thereof, during and after the course of treatment (i.e., chemotherapy, radiation therapy or cryotherapy), wherein a change in the expression of the markers from a poor prognosis pattern to a good prognosis pattern indicates that the treatment is efficacious.
- different timepoints are differentially labeled.
- Nucleic acid hybridization and wash conditions are chosen so that the target polynucleotide molecules specifically bind or specifically hybridize to the complementary polynucleotide sequences of the array, preferably to a specific array site, wherein its complementary DNA is located.
- Arrays containing double-stranded probe DNA situated thereon are preferably subjected to denaturing conditions to render the DNA single-stranded prior to contacting with the target polynucleotide molecules.
- Arrays containing single-stranded probe DNA may need to be denatured prior to contacting with the target polynucleotide molecules, e.g., to remove hairpins or dimers which form due to self complementary sequences.
- Optimal hybridization conditions will depend on the length (e.g. , oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, or DNA) of probe and target nucleic acids.
- type e.g., RNA, or DNA
- oligonucleotides become shorter, it may become necessary to adjust their length to achieve a relatively uniform melting temperature for satisfactory hybridization results.
- General parameters for specific (i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook et al. , MOLECULAR CLONING - A LABORATORY MANUAL (2ND ED.), VOIS.
- Typical hybridization conditions for the cDNA microarrays of Schena et al. are hybridization in 5 X SSC plus 0.2% SDS at 65 °C for four hours, followed by washes at 25 0 C in low stringency wash buffer (1 X SSC plus 0.2% SDS), followed by 10 minutes at 25 0 C in higher stringency wash buffer (0.1 X SSC plus 0.2% SDS) (Schena et al, Proc. Natl. Acad. ScL U.S.A.
- Particularly preferred hybridization conditions include hybridization at a temperature at or near the mean melting temperature of the probes (e.g. , within 5°C, more preferably within 2°C) in 1 M NaCl, 50 mM MES buffer (pH 6.5), 0.5% sodium sarcosine and 30% formamide.
- the fluorescence emissions at each site of a microarray may be, preferably, detected by scanning confocal laser microscopy.
- a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
- a laser may be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al, 1996, "A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization," Genome Research 6:639-645, which is incorporated by reference in its entirety for all purposes).
- the arrays are scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective. Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultiplier tubes. Fluorescence laser scanning devices are described in Schena et al., Genome Res. 6:639-645 (1996), and in other references cited herein. Alternatively, the fiber-optic bundle described by Ferguson et al., Nature Biotech. 14:1681-1684 (1996), may be used to monitor mRNA abundance levels at a large number of sites simultaneously.
- Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 or 16 bit analog to digital board.
- the scanned image is despeckled using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site. If necessary, an experimentally determined correction for "cross talk" (or overlap) between the channels for the two fluors may be made.
- a ratio of the emission of the two fluorophores can be calculated. The ratio is independent of the absolute expression level of the cognate gene, but is useful for genes whose expression is significantly modulated in association with the different breast cancer-related condition.
- Quantitative reverse transcriptase PCR can also be used to determine the expression level of a marker gene.
- the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.
- the two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MLV-RT).
- AMV-RT avilo myeloblastosis virus reverse transcriptase
- MMV-RT Moloney murine leukemia virus reverse transcriptase
- the reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling.
- extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, Calif., USA), following the manufacturer's instructions.
- the derived cDNA can then be used as a template in the subsequent PCR reaction.
- the PCR step can use a variety of thermostable DNA-dependent
- DNA polymerases it typically employs the Taq DNA polymerase, which has a 5 '-3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity.
- TaqMan ® PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
- Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction.
- a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers.
- the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe.
- the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
- One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
- TaqMan ® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM. Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).
- the 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRISM 7700TM Sequence Detection SystemTM.
- the system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer.
- the system includes software for running the instrument and for analyzing the data.
- 5'-Nuclease assay data are initially expressed as Ct, or the threshold cycle.
- Fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction.
- the point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).
- RT-PCR is usually performed using an internal standard.
- the ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
- RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and ⁇ -actin.
- GPDH glyceraldehyde-3-phosphate-dehydrogenase
- ⁇ -actin glyceraldehyde-3-phosphate-dehydrogenase
- RT-PCR A more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan ® probe).
- Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
- quantitative competitive PCR where internal competitor for each target sequence is used for normalization
- quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
- kits comprising the marker sets above.
- the kit contains a microarray ready for hybridization to target polynucleotide molecules, plus software for the data analyses described above.
- a computer system comprises internal components linked to external components.
- the internal components of a typical computer system include a processor element interconnected with a main memory.
- the computer system can be an Intel 8086-, 80386-, 80486-, PentiumTM, or PentiumTM-based processor with preferably 32 MB or more of main memory.
- the computer system may also be a Macintosh or a Macintosh-based system, but may also be a minicomputer or mainframe.
- the external components may include mass storage.
- This mass storage can be one or more hard disks (which are typically packaged together with the processor and memory). Such hard disks are preferably of 1 GB or greater storage capacity.
- Other external components include a user interface device, which can be a monitor, together with an inputting device, which can be a "mouse", or other graphic input devices, and/or a keyboard.
- a printing device can also be attached to the computer.
- a computer system is also linked to network link, which can be part of an Ethernet link to other local computer systems, remote computer systems, or wide area communication networks, such as the Internet.
- This network link allows the computer system to share data and processing tasks with other computer systems.
- a software component comprises the operating system, which is responsible for managing computer system and its network interconnections.
- This operating system can be, for example, of the Microsoft Windows® family, such as Windows 3.1, Windows 95, Windows 98, Windows 2000, or Windows NT, or may be of the Macintosh OS family, or may be UNIX or an operating system specific to a minicomputer or mainframe.
- the software component represents common languages and functions conveniently present on this system to assist programs implementing the methods specific to this invention.
- the methods of this invention are programmed in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including some or all of the algorithms to be used, thereby freeing a user of the need to procedurally program individual equations or algorithms.
- Such packages include Mathlab from Mathworks (Natick, MA), Mathematica® from Wolfram Research (Champaign, IL), or S-Plus® from Math Soft (Cambridge, MA).
- the software component includes the analytic methods of the invention as programmed in a procedural language or symbolic package.
- the software to be included with the kit comprises the data analysis methods of the invention as disclosed herein.
- the software may include mathematical routines for marker discovery, including the calculation of similarity values between clinical categories (e.g., ER status) and marker expression.
- the software may also include mathematical routines for calculating the similarity between sample marker expression and control marker expression, using array-generated fluorescence data, to determine the clinical classification of a sample.
- the software may also include mathematical routines for determining the prognostic outcome, and recommended therapeutic regimen, for a particular breast cancer patient.
- Such software would include instructions for the computer system's processor to receive data structures that include the level of expression of ten or more of the different marker genes listed in any of Tables 1-8 in a breast cancer tumor sample obtained from the breast cancer patient; the mean level of expression of the same genes in a control or template; and the breast cancer patient's clinical information, for example including lymph node and ER status.
- the software may additionally include mathematical routines for transforming the hybridization data and for calculating the similarity between the expression levels for the marker genes in the patient's breast cancer tumor sample and the control or template.
- the software includes mathematical routines for calculating a similarity metric, such as a coefficient of correlation, representing the similarity between the expression levels for the marker genes in the patient's breast cancer tumor sample and the control or template, and expressing the similarity as that similarity metric.
- a similarity metric such as a coefficient of correlation
- the software may include decisional routines that integrate the patient's clinical and marker gene expression data, and recommend a course of therapy.
- the software causes the processor unit to receive expression data for the patient's tumor sample, calculate a metric of similarity of these expression values to the values for the same genes in a template or control, compare this similarity metric to a pre-selected similarity metric threshold or thresholds that differentiate prognostic groups, assign the patient to the prognostic group, and, on the basis of the prognostic group, assign a recommended therapeutic regimen.
- the software additionally causes the processor unit to receive data structures comprising clinical information about the breast cancer patient. In a more specific example, such clinical information includes the patient's age, stage of breast cancer, estrogen receptor status, and lymph node status.
- control is an expression template comprising expression values for marker genes within a group of breast cancer patients
- the control can comprise either hybridization data obtained at the same time (i.e., in the same hybridization experiment) as the patient's individual hybridization data, or can be a set of hybridization or marker expression values stores on a computer, or on computer-readable media. If the latter is used, new patient hybridization data for the selected marker genes, obtained from initial or follow-up tumor samples, or suspected tumor samples, can be compared to the stored values for the same genes without the need for additional control hybridizations.
- the software may additionally comprise routines for updating the control data set, i.e., to add information from additional breast cancer patients or to remove existing members of the control data set, and, consequently, for recalculating the average expression level values that comprise the template.
- said control comprises a set of single-channel mean hybridization intensity values for each of said at least ten of said genes, stored on a computer-readable medium.
- Clinical data relating to a breast cancer patient can be contained in a database of clinical data in which information on each patient is maintained in a separate record, which record may contain any information relevant to the patient, the patient's medical history, treatment, prognosis, or participation in a clinical trial or study, including expression profile data generated as part of an initial diagnosis or for tracking the progress of the breast cancer during treatment.
- one embodiment of the invention provides a computer program product for classifying a breast cancer patient according to prognosis, the computer program product for use in conjunction with a computer having a memory and a processor, the computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein said computer program product can be loaded into the one or more memory units of a computer and causes the one or more processor units of the computer to execute the steps of (a) receiving a first data structure comprising the level of expression of at least ten of the different genes for which markers are listed in any of Tables 1-8 in a cell sample taken from said breast cancer patient; (b) determining the similarity of the level of expression of said at least 10 genes to control levels of expression of said at least five genes to obtain a patient similarity value; (c) comparing said patient similarity value to selected first and second threshold values of similarity of said level of expression of said genes to said control levels of expression to obtain first and second similarity threshold values, respectively, wherein said second similarity threshold indicates greater
- said first threshold value of similarity and said second threshold value of similarity are values stored in said computer.
- said first prognosis is a "very good prognosis”
- said second prognosis is an “intermediate prognosis”
- said third prognosis is a "poor prognosis”
- said computer program mechanism may be loaded into the memory and further cause said one or more processor units of said computer to execute the step of assigning said breast cancer patient a therapeutic regimen comprising no adjuvant chemotherapy if the patient is lymph node negative and is classified as having a good prognosis or an intermediate prognosis, or comprising chemotherapy if said patient has any other combination of lymph node status and expression profile.
- said computer program mechanism may be loaded into the memory and further cause said one or more processor units of the computer to execute the steps of receiving a data structure comprising clinical data specific to said breast cancer patient.
- said clinical data includes the lymph node and estrogen receptor (ER) status of said breast cancer patient.
- said single-channel hybridization intensity values are log transformed.
- the computer implementation of the method may use any desired transformation method.
- the computer program product causes said processing unit to perform said comparing step (c) by calculating the difference between the level of expression of each of said genes in said cell sample taken from said breast cancer patient and the level of expression of the same genes in said control.
- the computer program product causes said processing unit to perform said comparing step (c) by calculating the mean log level of expression of each of said genes in said control to obtain a control mean log expression level for each gene, calculating the log expression level for each of said genes in a breast cancer sample from said breast cancer patient to obtain a patient log expression level, and calculating the difference between the patient log expression level and the control mean log expression for each of said genes.
- the computer program product causes said processing unit to perform said comparing step (c) by calculating similarity between the level of expression of each of said genes in said cell sample taken from said breast cancer patient and the level of expression of the same genes in said control, wherein said similarity is expressed as a similarity value.
- said similarity value is a correlation coefficient. The similarity value may, however, be expressed as any art-known similarity metric.
- a user first loads experimental data into the computer system. These data can be directly entered by the user from a monitor, keyboard, or from other computer systems linked by a network connection, or on removable storage media such as a CD-ROM, floppy disk (not illustrated), tape drive (not illustrated), ZIP® drive (not illustrated) or through the network. Next the user causes execution of expression profile analysis software which performs the methods of the present invention.
- a user first loads experimental data and/or databases into the computer system. This data is loaded into the memory from the storage media or from a remote computer, preferably from a dynamic geneset database system, through the network. Next the user causes execution of software that performs the steps of the present invention.
- the software and/or computer system comprises access controls or access control routines, such as encryption, password- controlled access, and the like.
- 153 tumor samples were collected from breast cancer patients, each of whom was at least 55 years of age. Of the 153 patients, 45 had metastasis and 108 had no metastasis. RNA samples from each patient were prepared, and each RNA sample was profiled using inkjet microarrays. Marker genes were then identified based on expression patterns, and classifiers were trained to use these marker genes to classify tumors into prognostic categories. These marker genes were then used to predict the prognostic outcome.
- An oligo-dT primer containing a T7 RNA polymerase promoter sequence was used to prime first strand cDNA synthesis, and random primers (pdN6) were used to prime second strand cDNA synthesis by MMLV Reverse Transcriptase. This reaction yielded a double-stranded cDNA that contained the T7 RNA polymerase (T7RNAP) promoter. The double-stranded cDNA was then transcribed into cRNA by T7RNAP.
- T7RNAP T7 RNA polymerase
- cRNA was labeled with Cy3 or Cy5 dyes using a two-step process.
- cRNA labeling a 3:1 mixture of 5-(3-Aminoallyl)uridine 5 '-triphosphate (Sigma) and UTP was substituted for UTP in the in vitro transcription (IVT) reaction. Allylamine-derivitized cRNA products were then reacted with N-hydroxy succinimide esters of Cy3 or Cy5 (CyDye, Amersham Pharmacia Biotech).
- Cy5 -labeled cRNA from one breast cancer patient were mixed with the same amount of Cy3-labeled product from the pool of equal amount of cRNA from each individual sporadic patient. Hybridizations were done in duplicate with fluor reversals. Before hybridization, labeled cRNAs were fragmented to an average size of approximately 50-100 nucleotides by heating at 60°C in the presence of 10 mM ZnCl 2 . Fragmented cRNAs were added to hybridization buffer containing 1 M NaCl, 0.5% sodium sarcosine and 50 mM MES, pH 6.5, which stringency was regulated by the addition of formamide to a final concentration of 30%.
- Hybridizations were carried out in a final volume of 3 ml at 40°C on a rotating platform in a hybridization oven (Robbins Scientific). After hybridization, slides were washed and scanned using a confocal laser scanner (Agilent Technologies). Fluorescence intensities on scanned images were quantified, normalized and corrected.
- the reference cRNA pool was formed by pooling equal amount of cRNAs from each individual patient.
- Hybridizations were carried out in duplicate, the second time after fluorescent dye reversals. Before hybridization, labeled cRNAs were fragmented to an average size of approximately 50-100 nucleotides by heating at 60°C in the presence of 10 mM ZnCl 2 . Fragmented cRNAs were added to hybridization buffer containing 1 M NaCl, 0.5% sodium sarcosine and 50 mM MES, pH 6.5, and hybridization stringency was regulated by the addition of formamide to a final concentration of 30%. Hybridizations were carried out in a final volume of 3 ml at 40°C on a rotating platform in a hybridization oven (Robbins Scientific).
- Hu25K microarrays represented the 24479 biological oligonucleotides plus 1281 control probes were used for this study. Sequences for microarrays were selected from RefSeq (a collection of non-redundant mRNA sequences, www.ncbi.nlm.nih.gov/LocusLink/refseq.html) and Phil Green EST contigs. Each mRNA or EST contig was represented on the Hu25K microarray by a single 60-mer oligonucleotide chosen by an oligo probe design program. After hybridization, slides were washed and scanned using a confocal laser scanner (Agilent Technologies).
- Fluorescence intensities on scanned images were quantified, normalized and corrected. Intensity ratios relative to the reference pool were calculated and the significance of the differential regulation was estimated by the error model developed for the transcript ratio measurements with two- color-labeled hybridization microarray system.
- the methodological invention consists of four parts.
- the first part is the overview of the gene expression patterns from all 153 tumors from patient group of > 55 year by two-dimensional unsupervised clustering to identify the dominant tumor types.
- the second part focuses on evaluating the 70-gene based classifier in the age group > 55 years to test whether there is a prognostic profile that are universally valid across age groups ( ⁇ 55 year and > 55 year) for breast cancer.
- a group of marker genes was also identified that can be used to classify sporadic breast cancer patients with age > 55 year into two different prognostic groups - poor prognosis group and good prognosis group.
- similar classifiers were identified for prognosis within patient groups with ER+.
- B Overall expression patterns of breast cancer tumors in patients with age > 55 year.
- correlation as similarity metric emphasizes the importance of co-regulation in clustering rather than the amplitude of expression.
- the set of -10,000 genes can also be clustered based on their similarities measured over the group of 153 tumor samples.
- the similarity measure between two genes is defined in the same way as in Equation (1) except that for each gene there are 153 components of log ratio measurements.
- the two-dimensional clustering results shown in FIG. 1 are genome- wide overview of data representation for the profiled 153 tumor samples.
- the overall pattern revealed by unsupervised clustering relates to the end-point of interest in this study, i.e., metastasis status. This indicates that the transcriptional profiles of RNA samples from breast tumors measured with microarray technology represent patient disease states of prognostic value, and therefore the use of supervised algorithms should allow identification of predictors and construction of classifiers to differentiate tumors by prognosis.
- the 95% confidence interval is 1.2-5.1 for the overall metastasis and 2.0-13.0 for the 5-year metastasis. These numbers were obtained at a fixed threshold that were defined in our previous study for the age group of ⁇ 55 years (see Van't Veer (2002)).
- FIG. 2 shows the total error rate (type 1 + type 2 errors) as a function of threshold for overall metastasis of all 153 tumor samples.
- FIG. 3 shows the gene expression pattern of the 70-reporters for 153 profiled tumor samples. Visually, there are expression patterns in the group of 70 genes that are indicative of disease outcome among the 153 tumors. These results indicate that the classifier based on data from patients with age ⁇ 55 years has predictive power in prognosis of breast tumors from patients with age > 55 years.
- no-metastasis group 45 samples exhibited metastasizes, among which 29 exhibited metastasis within 5 years of the initial diagnosis (collectively, the "metastasis group”).
- the goal was to identify a set of marker genes from this data set exhibiting certain expression patterns that allow differentiation of these two subgroups among "sporadic" patients in the age group of > 55 years.
- a "leave-one-out" cross-validation method was used to build and evaluate a classifier (See FIG. 4).
- this method one sample is reserved for cross validation each time the classifier is trained.
- the training of the classifier involves the following steps (1) - (3) for any reserved sample. Steps (1) - (3) are repeated N times for N samples so that each sample is reserved once. See van 't Veer et ah, Nature 415, 530-536 (2002).
- Non-informative genes in each group of patients were first filtered out. Only genes with
- a set of candidate discriminating genes was identified based on gene expression data of a subset of these 153 samples. The subset of samples used for feature selection were those from individuals having either a good outcome with a follow up time at least 5 years, or a poor outcome metastasized with in 5 years, and those that were not omitted.
- genes on the candidate list were rank-ordered based on the magnitude of correlation as calculated above. Classification based on marker series
- a subset of N genes (as specified by the classifier) from the top of this rank-ordered list was used as discriminating genes.
- a template was defined for "good prognosis” group (called z j ) by using the error- weighted log ratio average of the selected group of genes.
- a template was defined for "poor prognosis” group (called Z 2 ) by using the error- weighted log ratio average of the selected group of genes.
- Two classifier parameters ( P 1 and P 2 ) were defined based on either correlation or distance.
- P 1 measures the similarity between one sample y and the "good prognosis" template Z 1 over this selected group of genes.
- P 2 measures the similarity between one sample y and the "poor prognosis” template z 2 over this selected group of genes.
- the performance of a classifier may vary with the number of features used in the classifier. To find the optimal number of features, the above process was repeated by varying the number of features (Le., genes) starting from 10, and also in increments of 10, to several hundred genes. The error rate is quite stable for marker genes above 100 (see FIG. 7). A set of 200 genes was thus selected as the optimal set of marker genes to classify breast cancer tumors into "poor prognosis” group and "good prognosis” group (see Tables 1 and 2). The classification results made with this optimal set of 200 marker genes are shown in FIG. 6.
- Example 2 Iterative algorithm ("homogenous method) to build classifier for prognosis of breast tumors from patients with age > 55 years
- Another optimal prognosis classifier was constructed using a different algorithm than that described above.
- the basic algorithm for classification used here is similar to the method previously used, except as noted below.
- Non-informative genes were first filtered in each group of patients.
- the classifier features were selected according to correlation with outcome (i.e., good prognosis or poor prognosis). Because of the "iterative training sample selection,” the features selected from each step of the second loop of leave-one-out process were highly overlapping. The final “optimal” reporter genes were selected using all the “training samples” as the result of "re-substitution” because one classifier was needed for each group.
- a classifier-building method called "iterative training sample selection" was used, hi the first step of this method, only the samples of those patients who had metastasis shorter than 5 years or who were metastasis-free with more than 5 years of follow-up time were used as the training set. Based on these training samples, a complete LOOCV (including reselecting features) process was performed. During this step, the number of features was fixed at 50 genes. This number is chosen to provide a stable classifier by the algorithm.
- the error rate is the average error rate from two populations: the number of poor outcome samples mis-classified as good outcome, divided by the total number of poor samples; and the number of good outcome samples mis- classified as poor outcome, divided by the total number of good samples.
- Two odds ratios are reported for a given threshold for differentiating good-outcome samples form poor- outcome samples: (1) the overall odds ratio; and (2) the 5 year odds ratio.
- the 5 year odds ratio was calculated from samples from individuals who were metastasis free for more than five years, or from individuals that had metastasis within 5 years).
- the threshold was applied to corl - cor2, where "corl” stands for correlation to the "average good profile” in the training set, and “cor2” stands for the correlation to the "average poor profile” in the training set.
- the threshold in the final round of LOOCV was defined as follows. (1) For each of the N sample i left out for training, features were selected based on the training set. (2) Given a feature set, an incomplete LOOCV was performed using N-I samples; only the "average poor profile” and “average good profile” was varied depending on whether the left out sample was in the training set or not. (3) A threshold is then determined based on the minimum error rate from the N-I samples, and that threshold is assigned to sample i in step (1). This step was repeated for each sample i in the set of samples. (4) The mean threshold from all N samples was then calculated, and designated the final threshold. By this method, the threshold in the classifier did not necessarily correspond to the minimum error rate, hence avoiding overestimating the performance.
- the total error rate as a function of the number of discriminating genes is shown in FIG. 7.
- an optimal set of 100 genes was identified that was used to build a classifier to predict the prognosis (see Tables 3 and 4).
- the scattering plot between correlation to "poor prognosis" profile and the correlation to "good prognosis” profile is shown in FIG. 8A.
- the type 1 error rate, the type 2 error rate, and average error rate are all shown in FIG. 8B as a function of threshold.
- the heatmap of gene expression for these 100 genes in all 153 samples is shown in FIG. 9.
- Table 9 summarizes the results of odds ratio, 95% confidence interval, total error rate, and p-value of log rank comparison test of two survival curves on Kalpan-Meier plots (FIG. 10) for predictions based on leave-one-out procedure from the previously constructed 70-gene based classifier, the 200-gene based classifier constructed by the same method, and the 100-gene based classifier constructed by the new (iterative) method.
- Table 9 Comparison of three different classifier genesets in the prognosis of samples from individuals age 55+.
- the estrogen receptor (ER) level affects the expression of thousands genes. It hence makes sense to develop a prognosis classifier separately for the ER+ patients and for the ER- patients.
- All 153 patient samples were divided into two groups, ER+ and ER-. Measurements from a microarray for ESRl were used to determine the ER status. The threshold used was the same threshold established in the previous study (see Van't Veer (2002)). Samples with ESRl log(ratio) >-0.65 were called ER+ samples. Of the 153 patients, 118 were ER+ and 35 were ER-. Because of the limited number of samples in the ER- group, only results derived from the ER+ group are discussed herein. Both the old and new method described above were used to build two separate classifiers for disease outcome prediction within ER+ group.
- FIG. 11 shows the total error rate as a function of the number of discriminating genes for both methods. The error rates do not vary significantly with the number of genes in both cases. 200 reporter genes were therefore selected using the old algorithm (Tables 5 and 6), and 100 genes using the new algorithm (Tables 7 and 8). The discriminative patterns of these genes are shown in FIGS. 12 and 13, respectively.
- FIG. 14 compares the K-M plots for the 70 genes applied to the ER+ samples, the old algorithm and new algorithms.
- Table 10 Comparison of three different classifier genesets in the prognosis of samples from ER+ individuals age 55+.
- the gene-expression based classifiers for the purpose of prognosis suggests an application to clinical practices.
- the present classifier identifies a set of discriminating genes for the purposes of prognosis using gene expression profiles.
- the molecular classification of breast cancers on the basis of gene expression patterns can thus identify clinically significant subtype of cancers.
- the present study demonstrates that a global view of gene expression in breast cancer can bring clarity to previously difficult diagnostic categories. The precision of morphological diagnosis, even when assisted by immunohistochemstry for a few markers, was insufficient to identify diagnostic and prognostic subgroups.
- Example 5 Biological significance of diagnostic marker genes
- kinases are important regulators of intracellular signal transduction pathways mediating cell proliferation, differentiation and apoptosis. Their activity is normally tightly controlled and regulated. Overexpression of certain kinases is known to be involved in oncogenesis, such as vascular endothelial growth factor receptorl (VEGFRl or FLTl), a tyrosine kinase that is an indicator of poor prognosis, which plays a very important role in tumor angiogenesis.
- VEGF vascular endothelial growth factor
- VEGFR' s ligand is also an indicator of poor prognosis, which means both ligand and receptor are co-upregulated in poor prognostic patients by an unknown mechanism.
- Cancer is characterized by deregulated cell proliferation. On the simplest level, this requires division of the cell, or mitosis. By keyword searching, “cell division” or “mitosis” was found to be included in 7 genes respectively in the 72 annotated entries from 156 genes indicating poor prognosis, and in zero genes in the 28 annotated genes from 75 genes that are indicators of good prognosis.
- Cyclins the regulatory subunits of cyclin-dependent kinases, control cell division or mitosis through key check-points within the cell cycle. Dysregulated expression and function of cyclins can lead to loss of normal growth control and cause uncontrolled expansion and invasion. Cyclin B2 and E2 were found to be overexpressed in poor prognostic patients.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Medical Informatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Data Mining & Analysis (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Theoretical Computer Science (AREA)
- Evolutionary Biology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioethics (AREA)
- Public Health (AREA)
- Evolutionary Computation (AREA)
- Epidemiology (AREA)
- Databases & Information Systems (AREA)
- Software Systems (AREA)
- Organic Chemistry (AREA)
- Artificial Intelligence (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Hospice & Palliative Care (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Nanotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002575557A CA2575557A1 (en) | 2004-07-30 | 2005-08-01 | Prognosis of breast cancer patients |
EP05784452A EP1782315A4 (en) | 2004-07-30 | 2005-08-01 | Prognosis of breast cancer patients |
AU2005267756A AU2005267756A1 (en) | 2004-07-30 | 2005-08-01 | Prognosis of breast cancer patients |
US11/658,605 US20090239214A1 (en) | 2004-07-30 | 2005-08-01 | Prognosis of breast cancer patients |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US59285804P | 2004-07-30 | 2004-07-30 | |
US60/592,858 | 2004-07-30 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2006015312A2 true WO2006015312A2 (en) | 2006-02-09 |
WO2006015312A3 WO2006015312A3 (en) | 2007-01-18 |
Family
ID=35787901
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/027243 WO2006015312A2 (en) | 2004-07-30 | 2005-08-01 | Prognosis of breast cancer patients |
Country Status (5)
Country | Link |
---|---|
US (1) | US20090239214A1 (en) |
EP (1) | EP1782315A4 (en) |
AU (1) | AU2005267756A1 (en) |
CA (1) | CA2575557A1 (en) |
WO (1) | WO2006015312A2 (en) |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009032915A2 (en) * | 2007-09-06 | 2009-03-12 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Arrays, kits and cancer characterization methods |
US7514209B2 (en) | 2001-06-18 | 2009-04-07 | Rosetta Inpharmatics Llc | Diagnosis and prognosis of breast cancer patients |
EP2090665A2 (en) | 2006-10-20 | 2009-08-19 | Exiqon A/S | Novel human microRNAs associated with cancer |
EP2094862A1 (en) * | 2006-11-24 | 2009-09-02 | Licentia Ltd. | Method for predicting the response to a therapy |
WO2010063454A1 (en) * | 2008-12-01 | 2010-06-10 | University Of Ulster | Method of stratifying breast cancer patients based on gene expression |
WO2010118782A1 (en) * | 2009-04-17 | 2010-10-21 | Universite Libre De Bruxelles | Methods and tools for predicting the efficiency of anthracyclines in cancer |
US20110152113A1 (en) * | 2008-07-02 | 2011-06-23 | Escudero Ramonon Garcia | Genomic fingerprint of breast cancer |
US8119655B2 (en) | 2005-10-07 | 2012-02-21 | Takeda Pharmaceutical Company Limited | Kinase inhibitors |
US8188255B2 (en) | 2006-10-20 | 2012-05-29 | Exiqon A/S | Human microRNAs associated with cancer |
US8278450B2 (en) | 2007-04-18 | 2012-10-02 | Takeda Pharmaceutical Company Limited | Kinase inhibitors |
WO2012135845A1 (en) | 2011-04-01 | 2012-10-04 | Qiagen | Gene expression signature for wnt/b-catenin signaling pathway and use thereof |
WO2012152922A1 (en) * | 2011-05-12 | 2012-11-15 | Traslational Cancer Drugs Pharma S.L. | Kiaa1456 expression predicts survival in patients with colon cancer |
EP2839034A4 (en) * | 2012-04-20 | 2016-01-06 | Sloan Kettering Inst Cancer | Gene expression profiles associated with metastatic breast cancer |
EP2993473A1 (en) * | 2007-01-30 | 2016-03-09 | Pharmacyclics, Inc. | Methods for determining cancer resistance to histone deacetylase inhibitors |
US9492423B2 (en) | 2011-09-13 | 2016-11-15 | Pharmacyclics Llc | Formulations of histone deacetylase inhibitor in combination with bendamustine and uses thereof |
WO2018175970A1 (en) * | 2017-03-24 | 2018-09-27 | The Brigham And Women's Hospitla, Inc. | Systems and methods for automated treatment recommendation based on pathophenotype identification |
US10202605B2 (en) | 2007-06-28 | 2019-02-12 | The Trustees Of Princeton University | Methods of identifying and treating poor-prognosis cancers |
US10745701B2 (en) | 2007-06-28 | 2020-08-18 | The Trustees Of Princeton University | Methods of identifying and treating poor-prognosis cancers |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK2258872T3 (en) * | 2002-03-13 | 2013-11-18 | Genomic Health Inc | Gene expression profiling in tumor tissue biopsies |
US20080108091A1 (en) * | 2006-08-07 | 2008-05-08 | Hennessy Bryan T | Proteomic Patterns of Cancer Prognostic and Predictive Signatures |
ES2343996B1 (en) | 2008-12-11 | 2011-06-20 | Fundacion Para La Investigacion Biomedica Del Hospital Universitario La Paz | METHOD FOR SUBCLASSIFICATION OF TUMORS. |
US9771618B2 (en) * | 2009-08-19 | 2017-09-26 | Bioarray Genetics, Inc. | Methods for treating breast cancer |
DE102010043541B4 (en) | 2009-12-16 | 2012-01-26 | Technische Universität Dresden | Method and means for predicting survival in pancreatic carcinoma by analysis of biomarkers |
WO2011151321A1 (en) * | 2010-05-31 | 2011-12-08 | Institut Curie | Asf1b as a prognosis marker and therapeutic target in human cancer |
US20130236567A1 (en) * | 2010-06-04 | 2013-09-12 | Katherine J. MARTIN | Gene expression signature as a predictor of chemotherapeutic response in breast cancer |
KR101287600B1 (en) * | 2011-01-04 | 2013-07-18 | 주식회사 젠큐릭스 | Prognostic Genes for Early Breast Cancer and Prognostic Model for Early Breast Cancer Patients |
KR101548830B1 (en) * | 2013-12-30 | 2015-08-31 | 가천대학교 산학협력단 | Cmposition for predicting breast cancer prognosis using marker for breast cancer stem cell screened by culture method of stem cell |
US20170193165A1 (en) * | 2015-12-30 | 2017-07-06 | Sastry Subbaraya Mandalika | Method and system for managing patient healthcare prognosis |
WO2017193141A1 (en) * | 2016-05-06 | 2017-11-09 | Siyuan Zhang | Prognosis biomarkers and anti-tumor compositions of targeted therapeutic treatments for triple negative breast cancer |
CN117500942A (en) * | 2021-06-18 | 2024-02-02 | 江苏鹍远生物技术有限公司 | Substances and methods for assessing tumors |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002103320A2 (en) | 2001-06-18 | 2002-12-27 | Rosetta Inpharmatics, Inc. | Diagnosis and prognosis of breast cancer patients |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5143854A (en) * | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US5545522A (en) * | 1989-09-22 | 1996-08-13 | Van Gelder; Russell N. | Process for amplifying a target polynucleotide sequence using a single primer-promoter complex |
US5578832A (en) * | 1994-09-02 | 1996-11-26 | Affymetrix, Inc. | Method and apparatus for imaging a sample on a device |
US5539083A (en) * | 1994-02-23 | 1996-07-23 | Isis Pharmaceuticals, Inc. | Peptide nucleic acid combinatorial libraries and improved methods of synthesis |
US5556752A (en) * | 1994-10-24 | 1996-09-17 | Affymetrix, Inc. | Surface-bound, unimolecular, double-stranded DNA |
US6028189A (en) * | 1997-03-20 | 2000-02-22 | University Of Washington | Solvent for oligonucleotide synthesis and methods of use |
US6271002B1 (en) * | 1999-10-04 | 2001-08-07 | Rosetta Inpharmatics, Inc. | RNA amplification method |
US7171311B2 (en) * | 2001-06-18 | 2007-01-30 | Rosetta Inpharmatics Llc | Methods of assigning treatment to breast cancer patients |
EP1599977A2 (en) * | 2003-02-28 | 2005-11-30 | Motorola, Inc. | System and method for passing data frames in a wireless network |
AU2004256425A1 (en) * | 2003-06-09 | 2005-01-20 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
EP1651775A2 (en) * | 2003-06-18 | 2006-05-03 | Arcturus Bioscience, Inc. | Breast cancer survival and recurrence |
EP3170906B1 (en) * | 2003-06-24 | 2018-08-22 | Genomic Health, Inc. | Prediction of likelihood of cancer recurrence |
WO2005008213A2 (en) * | 2003-07-10 | 2005-01-27 | Genomic Health, Inc. | Expression profile algorithm and test for cancer prognosis |
-
2005
- 2005-08-01 CA CA002575557A patent/CA2575557A1/en not_active Abandoned
- 2005-08-01 WO PCT/US2005/027243 patent/WO2006015312A2/en active Application Filing
- 2005-08-01 EP EP05784452A patent/EP1782315A4/en not_active Withdrawn
- 2005-08-01 AU AU2005267756A patent/AU2005267756A1/en not_active Abandoned
- 2005-08-01 US US11/658,605 patent/US20090239214A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002103320A2 (en) | 2001-06-18 | 2002-12-27 | Rosetta Inpharmatics, Inc. | Diagnosis and prognosis of breast cancer patients |
Non-Patent Citations (9)
Title |
---|
AUSUBEL ET AL.: "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY", vol. 2, 1994, CURRENT PROTOCOLS PUBLISHING |
CHIRGWIN ET AL., BIOCHEMISTRY, vol. 18, 1979, pages 5294 - 5299 |
CHU ET AL., J. NAT CANCER INST., vol. 88, 1996, pages 1571 - 1579 |
HELD ET AL., GENOME RESEARCH, vol. 6, 1996, pages 986 - 994 |
MIKI ET AL., SCIENCE, vol. 266, 1994, pages 66 - 71 |
PARKER ET AL., CA CANCER J. CLIN., vol. 47, 1997, pages 5 - 27 |
SAMBROOK ET AL.: "MOLECULAR CLONING - A LABORATORY MANUAL", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY |
SAMBROOK: "MOLECULAR CLONING - A LABORATORY MANUAL", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY |
See also references of EP1782315A4 |
Cited By (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7863001B2 (en) | 2001-06-18 | 2011-01-04 | The Netherlands Cancer Institute | Diagnosis and prognosis of breast cancer patients |
US7514209B2 (en) | 2001-06-18 | 2009-04-07 | Rosetta Inpharmatics Llc | Diagnosis and prognosis of breast cancer patients |
US9909185B2 (en) | 2001-06-18 | 2018-03-06 | The Netherlands Cancer Institute | Diagnosis and prognosis of breast cancer patients |
US8119655B2 (en) | 2005-10-07 | 2012-02-21 | Takeda Pharmaceutical Company Limited | Kinase inhibitors |
EP2090665A2 (en) | 2006-10-20 | 2009-08-19 | Exiqon A/S | Novel human microRNAs associated with cancer |
US8188255B2 (en) | 2006-10-20 | 2012-05-29 | Exiqon A/S | Human microRNAs associated with cancer |
EP2094862A1 (en) * | 2006-11-24 | 2009-09-02 | Licentia Ltd. | Method for predicting the response to a therapy |
EP2094862A4 (en) * | 2006-11-24 | 2010-08-11 | Licentia Ltd | Method for predicting the response to a therapy |
EP2993473A1 (en) * | 2007-01-30 | 2016-03-09 | Pharmacyclics, Inc. | Methods for determining cancer resistance to histone deacetylase inhibitors |
US8278450B2 (en) | 2007-04-18 | 2012-10-02 | Takeda Pharmaceutical Company Limited | Kinase inhibitors |
US10745701B2 (en) | 2007-06-28 | 2020-08-18 | The Trustees Of Princeton University | Methods of identifying and treating poor-prognosis cancers |
US10202605B2 (en) | 2007-06-28 | 2019-02-12 | The Trustees Of Princeton University | Methods of identifying and treating poor-prognosis cancers |
WO2009032915A2 (en) * | 2007-09-06 | 2009-03-12 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Arrays, kits and cancer characterization methods |
WO2009032915A3 (en) * | 2007-09-06 | 2009-05-28 | Us Health | Arrays, kits and cancer characterization methods |
US20110152113A1 (en) * | 2008-07-02 | 2011-06-23 | Escudero Ramonon Garcia | Genomic fingerprint of breast cancer |
WO2010063454A1 (en) * | 2008-12-01 | 2010-06-10 | University Of Ulster | Method of stratifying breast cancer patients based on gene expression |
WO2010118782A1 (en) * | 2009-04-17 | 2010-10-21 | Universite Libre De Bruxelles | Methods and tools for predicting the efficiency of anthracyclines in cancer |
WO2012135845A1 (en) | 2011-04-01 | 2012-10-04 | Qiagen | Gene expression signature for wnt/b-catenin signaling pathway and use thereof |
WO2012152922A1 (en) * | 2011-05-12 | 2012-11-15 | Traslational Cancer Drugs Pharma S.L. | Kiaa1456 expression predicts survival in patients with colon cancer |
CN103649334A (en) * | 2011-05-12 | 2014-03-19 | 特拉斯雷神诺癌症医药有限公司 | Kiaa1456 expression predicts survival in patients with colon cancer |
JP2014513975A (en) * | 2011-05-12 | 2014-06-19 | トラスラショナル、キャンサー、ドラッグス、ファーマ、ソシエダッド、リミターダ | Predicting survival of patients with colon cancer by KIAA1456 expression |
US9492423B2 (en) | 2011-09-13 | 2016-11-15 | Pharmacyclics Llc | Formulations of histone deacetylase inhibitor in combination with bendamustine and uses thereof |
EP2839034A4 (en) * | 2012-04-20 | 2016-01-06 | Sloan Kettering Inst Cancer | Gene expression profiles associated with metastatic breast cancer |
WO2018175970A1 (en) * | 2017-03-24 | 2018-09-27 | The Brigham And Women's Hospitla, Inc. | Systems and methods for automated treatment recommendation based on pathophenotype identification |
Also Published As
Publication number | Publication date |
---|---|
AU2005267756A1 (en) | 2006-02-09 |
EP1782315A4 (en) | 2009-06-24 |
EP1782315A2 (en) | 2007-05-09 |
CA2575557A1 (en) | 2006-02-09 |
WO2006015312A3 (en) | 2007-01-18 |
US20090239214A1 (en) | 2009-09-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2006015312A2 (en) | Prognosis of breast cancer patients | |
JP7042717B2 (en) | How to Predict the Clinical Outcomes of Cancer | |
US20180305768A1 (en) | Diagnosis and prognosis of breast cancer patients | |
JP4619350B2 (en) | Diagnosis and prognosis of breast cancer patients | |
US8019552B2 (en) | Classification of breast cancer patients using a combination of clinical criteria and informative genesets | |
EP2304631A1 (en) | Algorithms for outcome prediction in patients with node-positive chemotherapy-treated breast cancer | |
US20060292623A1 (en) | Signature genes in chronic myelogenous leukemia | |
US8105777B1 (en) | Methods for diagnosis and/or prognosis of colon cancer | |
WO2009009662A1 (en) | Method of predicting non-response to first line chemotherapy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2575557 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005267756 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005784452 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2005267756 Country of ref document: AU Date of ref document: 20050801 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2005267756 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 2005784452 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 11658605 Country of ref document: US |