Tumor molecular profiling methods

Abstract

To provide methods of molecular profiling of a sample from an individual to identify treatments for an individual with a condition or disease, such as cancer, using a result of the molecular profiling.SOLUTION: A method of identifying a candidate treatment for a subject in need of the treatment comprises: performing, on a sample from the subject, an immunohistochemistry (IHC) analysis, a microarray analysis, a fluorescent in-situ hybridization (FISH) analysis, and DNA sequencing; and comparing the IHC expression profile, microarray expression profile, FISH mutation profile and sequencing mutation profile against a rules database comprising a mapping of treatments whose biological activity against cancer cells is known.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 61 / 151,758, filed February 11, 2009; U.S. Provisional Patent Application No. 61 / 170,565, filed April 17, 2009, and U.S. Provisional Patent Application No. 61 / 229,686, filed July 29, 2009, all of which are incorporated herein by reference in their entireties. [Background technology]

[0002] background Patients' pathologies are typically treated with treatment regimens or therapies that are selected based on clinical criteria; that is, based on the determination that the patient has been diagnosed with a particular disease (the diagnosis is made from classical diagnostic assays), the treatment regimen or therapies are selected for the patient.The molecular mechanisms behind various pathologies have been the subject of research for many years, but the specific application of the molecular profile of affected individuals in determining treatment regimens and therapies for the affected individuals is disease-specific and has not been widely promoted.

[0003] Some treatment plans are determined by combining molecular profiling with clinical characterization of the patient, such as physician findings (e.g., codes from the International Classification of Diseases and the dates on which such codes were determined), laboratory test results, x-rays, biopsy results, patient explanations, and any other medical information that physicians typically rely on to make a diagnosis in a particular disease. However, because some treatment plans, although associated with treating a particular type of disease condition, may also be effective against different disease conditions, using a combination of selection materials based on molecular profiling and clinical characterization to determine a treatment plan or therapy (such as a diagnosis of a particular type of cancer), carries the risk that a treatment plan that is effective for a particular individual may be overlooked.

[0004] Patients with refractory and metastatic cancers are of particular interest to treating physicians. The majority of patients with metastatic cancer eventually exhaust treatment options for their tumors. After standard first- and second-line (and sometimes third-line and beyond) treatments for their tumors have been pursued, these patients have very limited options. These patients can also participate in Phase I and II clinical trials of novel anticancer agents, but they must usually meet very strict eligibility criteria to participate. When patients participate in these types of trials, studies have shown that novel anticancer agents can result in response rates ranging from an average of 5%-10% in the Phase I setting to 12% in the Phase II setting. These patients also have the option to choose to receive the best supportive care to treat their symptoms.

[0005] In recent years, there has been a surge in interest in developing new anti-cancer drugs that target cell surface receptors or upregulated or amplified gene products more and more. This approach has had some success (e.g., trastuzumab against HER2 / neu in breast cancer cells, rituximab against CD20 in lymphoma cells, bevacizamab against VEGF, and cetuximab against EGFR). However, patients' tumors still eventually progress with these therapies. If more targets or molecular findings, such as molecular mechanisms, genes, gene expression proteins, and / or combinations thereof, are measured in patients' tumors, it may be possible to find additional targets or molecular findings that can be utilized by using specific therapeutic agents. Identifying multiple agents that can treat multiple targets or underlying mechanisms provides cancer patients with viable alternative treatments to existing treatment regimens.

[0006] Molecular profiling analysis identifies one or more individual profiles that often drive more informed and more effective individualized treatment options, which can lead to improved patient care and improved outcomes. The present invention provides methods and systems for identifying treatments for individuals by performing molecular profiling of samples from these individuals. Summary of the Invention

[0007] The present invention provides methods and systems for performing molecular profiling and using the results from the molecular profiling to identify treatments for an individual, in some embodiments, that were not originally identified as treatments for the disease.

[0008] In one aspect, the present invention provides a method of identifying a candidate treatment for a subject in need of such treatment, comprising the steps of: performing immunohistochemistry (IHC) analysis on a sample from a subject to determine an IHC expression profile for at least five proteins; performing microarray analysis on the sample to determine a microarray expression profile for at least ten genes; performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least one gene; performing DNA sequencing on the sample to determine a sequencing mutation profile for at least one gene; and comparing the IHC expression profile, the microarray expression profile, the FISH mutation profile, and the sequencing mutation profile to a regulatory database. The database of rules includes mappings for treatments with known biological activity against: i) cancer cells that over- or under-express one or more proteins included in the IHC expression profile; ii) cancer cells that over- or under-express one or more genes included in the microarray expression profile; iii) cancer cells that have no mutations or have one or more mutations in one or more genes included in the FISH mutation profile; and / or iv) cancer cells that have no mutations or have one or more mutations in one or more genes included in the sequencing mutation profile. A candidate treatment is identified if: i) the comparison step indicates that the treatment should have biological activity against the cancer; and ii) the comparison step indicates no contraindication to the treatment for treating the cancer.

[0009] In some embodiments, IHC expression profiling includes assaying one or more of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1.

[0010] In some embodiments, microarray expression profiling is performed on ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LC The method includes assaying one or more of K, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70.

[0011] In some embodiments, FISH mutation profiling comprises assaying EGFR and / or HER2.

[0012] In some embodiments, the sequencing mutation profiling comprises assaying one or more of KRAS, BRAF, c-KIT, and EGFR.

[0013] In another aspect, the present invention provides a method for the treatment of a cancer, comprising the steps of: performing immunohistochemistry (IHC) analysis on a sample from a subject to determine an IHC expression profile for at least five of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; performing microarray analysis on the sample to determine an IHC expression profile for at least five of ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, C-Kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; D33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1 , HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, The present invention provides a method for identifying a candidate treatment for a subject in need of such treatment, comprising determining a microarray expression profile for at least five of POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for EGFR and / or HER2; performing DNA sequencing on the sample to determine a sequencing mutation profile for at least one of KRAS, BRAF, c-KIT, and EGFR; and comparing the IHC expression profile, the microarray expression profile, the FISH mutation profile, and the sequencing mutation profile to a regulatory database.The rule database comprises mappings for treatments with known biological activity against i) cancer cells that overexpress or underexpress one or more proteins included in the IHC expression profile; ii) cancer cells that overexpress or underexpress one or more genes included in the microarray expression profile; iii) cancer cells that do not have a mutation or have one or more mutations in one or more genes included in the FISH mutation profile; and / or iv) cancer cells that do not have a mutation or have one or more mutations in one or more genes included in the sequencing mutation profile. A candidate treatment is identified when i) the comparison step indicates that the treatment should have biological activity against the cancer; and ii) the comparison step indicates that the treatment does not have a contraindication for treating the cancer. In some embodiments, IHC expression profiling is performed for at least 50%, 60%, 70%, 80%, or 90% of the biomarkers described. In some embodiments, microarray expression profiling is performed for at least 50%, 60%, 70%, 80%, or 90% of the biomarkers listed.

[0014] In a third aspect, the present invention provides a method for determining an IHC expression profile for at least a group of proteins consisting of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; and performing microarray analysis on the sample to determine an IHC expression profile for at least a group of proteins consisting of ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, C-Kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1. D33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HC K, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1 , PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least a group of genes consisting of EGFR and HER2; performing DNA sequencing on the sample to determine a sequencing mutation profile for at least a group of genes consisting of KRAS, BRAF, c-KIT, and EGFR; and comparing the IHC expression profile, the microarray expression profile, the FISH mutation profile, and the sequencing mutation profile against a regulatory database.The database of rules includes mappings for treatments with known biological activity against: i) cancer cells that over- or under-express one or more proteins included in the IHC expression profile; ii) cancer cells that over- or under-express one or more genes included in the microarray expression profile; iii) cancer cells that have no mutations or have one or more mutations in one or more genes included in the FISH mutation profile; and / or iv) cancer cells that have no mutations or have one or more mutations in one or more genes included in the sequencing mutation profile. A candidate treatment is identified if: i) the comparison step indicates that the treatment should have biological activity against the cancer; and ii) the comparison step indicates no contraindication to the treatment for treating the cancer.

[0015] In some embodiments of the method of the present invention, the sample comprises formalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen (FF) tissue, or tissue that is contained in a solution that preserves nucleic acid or protein molecules.In some embodiments, any one of microarray analysis, FISH mutation analysis, or sequencing mutation analysis is not performed.For example, the method cannot be performed unless the sample passes quality control test.In some embodiments, the quality control test comprises A260 / A280 ratio or Ct value of RT-PCR of RPL13a mRNA.For example, the quality control test may require A260 / A280 ratio lower than 1.5 or RPL13a Ct value higher than 30.

[0016] In some embodiments, microarray expression profiling is performed using a low-density microarray, an expression microarray, a comparative genomic hybridization (CGH) microarray, a single nucleotide polymorphism (SNP) microarray, a proteomics array, or an antibody array.

[0017] The method of the present invention may require assaying certain markers, including additional markers. In some embodiments, IHC expression profiling is performed on at least SPARC, TOP2A, and / or PTEN. Microarray expression profiling may be performed on at least CD52. IHC expression profiling further comprises assaying one or more of DCK, EGFR, BRCA1, CK 14, CK 17, CK 5 / 6, E-cadherin, p95, PARP-1, SPARC, and TLE3. In some embodiments, IHC expression profiling further comprises assaying Cox-2 and / or Ki-67. In some embodiments, microarray expression profiling further comprises assaying HSPCA. In some embodiments, FISH mutation profiling further comprises assaying c-Myc and / or TOP2A. Sequencing mutation profiling may include assaying PI3K.

[0018] According to the method of the present invention, several genes and gene products can be assayed.For example, the genes used for IHC expression profiling, microarray expression profiling, FISH mutation profiling, and sequencing mutation profiling include ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, androgen receptor, AR, AREG, ASNS, BCL2, BCRP, BDCA1, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDK2, CDW52, CES2, CK 14, CK 17, CK 5 / 6, c-KIT, c-Myc, COX-2, cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, E-cadherin, ECGF1, EGFR, EPHA2, epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, Her2 / Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IL 13RA1, IL2RA, KDR, KIT, K-RAS, LCK, LTB, lymphotoxin β receptor, LYN, MGMT, MLH1, MRP1, MS4A1, MSH2, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SPARC Independently including one or more of MC, SPARC PC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TOPO1, TOPO2B, topoisomerase II, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, and ZAP70.

[0019] In some embodiments, the microarray expression analysis comprises identifying whether a gene is up-regulated or down-regulated with statistical significance compared to reference.Statistical significance can be determined by a p-value of 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001 or less.The p-value can also be corrected for multiple comparisons.The correction for multiple comparisons can include Bonferroni correction or its improved method.

[0020] In some embodiments, the IHC analysis comprises determining whether 30% or more of the sample has a staining intensity of +2 or greater.

[0021] The rules contained within the rule database used by the methods of the present invention may be based on the effectiveness of various treatments specific to the target gene or gene product. The rule database may include the rules described in Table 1 and / or Table 2 herein.

[0022] In some embodiments of the methods of the present invention, a prioritized list of candidate therapies is identified. Prioritization can include ordering therapies from highest priority to lowest priority according to therapies based on microarray analysis and either IHC or FISH analysis; therapies based on IHC analysis but not microarray analysis; and therapies based on microarray analysis but not IHC analysis.

[0023] In some embodiments of the methods of the present invention, the candidate treatment comprises administering one or more candidate therapeutic agents. The one or more candidate therapeutic agents include 5-fluorouracil, abarelix, alemtuzumab, aminoglutethimide, anastrazole, aromatase inhibitors (anastrazole, letrozole), asparaginase, aspirin, ATRA, azacitidine, bevacizumab, bexarotene, bicalutamide, bortezomib, calcitriol, capecitabine, carboplatin, celecoxib, cetuximab, chemoendocrine therapy, cholecalciferol, cisplatin, carboplatin, cyclophosphamide, cyclophosphamide / vincristine, cytarabine, dasatinib, decitabine, doxorubicin, epirubicin, epirubicin, erlotinib, etoposide, exemestane, fluoropyrimidine, flutamide, It may be fulvestrant, gefitinib, gefitinib and trastuzumab, gemcitabine, gonadorelin, goserelin, hydroxyurea, imatinib, irinotecan, ixabepilone, lapatinib, letrozole, leuprolide, liposomal doxorubicin, medroxyprogesterone, megestrol, methotrexate, mitomycin, nab-paclitaxel, octreotide, oxaliplatin, paclitaxel, panitumumab, pegaspargase, pemetrexed, pentostatin, sorafenib, sunitinib, tamoxifen, tamoxifen-based therapy, temozolomide, topotecan, toremifene, trastuzumab, VBMCP / cyclophosphamide, vincristine, or any combination thereof.The one or more candidate therapeutic agents may also be selected from the group consisting of 5FU, bevacizumab, capecitabine, cetuximab, cetuximab plus gemcitabine, cetuximab plus irinotecan, cyclophosphamide, diethylstibesterol, doxorubicin, erlotinib, etoposide, exemestane, fluoropyrimidines, gemcitabine, gemcitabine plus etoposide, gemcitabine plus pemetrexed, irinotecan, irinotecan plus sorafenib, lapatinib, lapatinib plus tamoxifen, retrozole, ribozyme, ribozyme plus tamoxifen ... The treatment may be rituximab, letrozole plus capecitabine, mitomycin, nab-paclitaxel, nab-paclitaxel plus gemcitabine, nab-paclitaxel plus trastuzumab, oxaliplatin, oxaliplatin + 5FU + trastuzumab, panitumumab, pemetrexed, sorafenib, sunitinib, sunitinib, sunitinib + mitomycin, tamoxifen, temozolomide, temozolomide + bevacizumab, temozolomide + sorafenib, trastuzumab, vincristine, or any combination thereof.

[0024] In an embodiment of the method of the present invention, the sample comprises cancer cells. The cancer can be metastatic cancer. The cancer can be refractory to previous treatment. The previous treatment can be a standard of care for cancer. In some cases, the subject has been previously treated with one or more therapeutic agents for treating cancer. In some cases, the subject has not been previously treated with one or more candidate therapeutic agents identified.

[0025] In some embodiments, the cancer comprises prostate cancer, lung cancer, melanoma, small cell carcinoma (esophageal / retroperitoneal), cholangiocarcinoma, mesothelioma, head and neck cancer (SCC), pancreatic cancer, pancreatic neuroendocrine carcinoma, small cell carcinoma, gastric cancer, pseudomyxoma peritonei, anal canal carcinoma (SCC), vaginal carcinoma (SCC), cervical cancer, renal carcinoma, eccrine sweat gland adenocarcinoma, salivary gland adenocarcinoma, uterine soft tissue sarcoma (uterine), GIST (gastric), or anaplastic thyroid carcinoma. In some embodiments, the cancer comprises cancer of the accessory, sinuses, middle and inner ear, adrenal gland, appendix, hematopoietic system, bones and joints, spinal cord, breast, cerebellum, cervix, connective soft tissue, uterine body, esophagus, eye, nose, eyeball, fallopian tube, extrahepatic bile duct, oral cavity, intrahepatic bile duct, kidney, appendix-colon, larynx, lips, liver, lung and bronchus, lymph nodes, cerebrum, spinal cord, nasal cartilage, retina, eye, oropharynx, endocrine glands, female reproductive organs, ovaries, pancreas, penis and scrotum, pituitary gland, pleura, prostate, rectum renal pelvis, ureter, peritoneum, salivary gland, skin, small intestine, stomach, testis, thymus, thyroid, tongue, unknown, bladder, uterus, vagina, labia, and vulva. In some embodiments, the sample comprises cells selected from the group consisting of fat, adrenal cortex, adrenal gland, adrenal-medulla, appendix, bladder, blood, blood vessel, bone, osteochondral, brain, breast, cartilage, cervix, colon, sigmoid colon, dendritic cell, skeletal muscle, endometrium, esophagus, fallopian tube, fibroblast, gallbladder, kidney, larynx, liver, lung, lymph node, melanocyte, mesothelial lining, myoepithelial cell, osteoblast, ovary, pancreas, parotid gland, prostate, salivary gland, sinus tissue, skeletal muscle, skin, small intestine, smooth muscle, stomach, synovium, joint lining tissue, tendon, testis, thymus, thyroid, uterus, and uterine corpus. In some embodiments, the cancer comprises breast cancer, colorectal cancer, ovarian cancer, lung cancer, non-small cell lung cancer, cholangiocarcinoma, mesothelioma, sweat gland carcinoma, or GIST cancer.

[0026] The method of the present invention can be used to prolong the progression-free survival (PFS) or disease-free survival (DFS) of subjects.For example, PFS or DFS can be prolonged by at least about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 100% compared to previous treatment.In addition, the method of the present invention can be used to select candidate treatment to prolong the lifespan of patients. For example, the patient's life span may be extended by at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 2 years, 2 1 / 2 years, 3 years, 4 years, or at least 5 years. [The present invention 1001] 1. A method of identifying a candidate treatment for a subject in need of such treatment, comprising: (a) performing immunohistochemistry (IHC) analysis on a sample from the subject to determine an IHC expression profile for at least five proteins; (b) performing a microarray analysis on the sample to determine a microarray expression profile for at least 10 genes; (c) performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least one gene; (d) performing DNA sequencing on the sample to determine a sequencing mutation profile for at least one gene; and (e) i. cancer cells that over- or under-express one or more proteins contained in the IHC expression profile; ii. cancer cells that over- or under-express one or more genes contained in the microarray expression profile; iii. cancer cells that have no mutations or one or more mutations in one or more genes included in the FISH mutation profile, and / or iv. Cancer cells that have no mutations or one or more mutations in one or more genes included in the sequencing mutation profile. comparing said IHC expression profile, microarray expression profile, FISH mutation profile, and sequencing mutation profile to a rule database that includes mappings for treatments with known biological activity against; and (f) i. the comparison of step (e) indicates that the treatment should have biological activity against the cancer; and ii. identifying a candidate treatment if the comparison of step (e) does not indicate a contraindication to said treatment with respect to treating said cancer. [The present invention 1002] 1. A method of identifying a candidate treatment for a subject in need of such treatment, comprising: (a) performing immunohistochemistry (IHC) analysis on a sample from the subject to determine an IHC expression profile for at least five of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; (b) Microarray analysis was performed on the sample to detect the following: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, ML determining a microarray expression profile for at least five of H1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; (c) performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for EGFR and / or HER2; (d) performing DNA sequencing on the sample to determine a sequencing mutation profile for at least one of KRAS, BRAF, c-KIT, and EGFR; and (e) i. cancer cells that over- or under-express one or more proteins contained in the IHC expression profile; ii. cancer cells that over- or under-express one or more genes contained in the microarray expression profile; iii. cancer cells that have no mutations or one or more mutations in one or more genes included in the FISH mutation profile, and / or iv. Cancer cells that have no mutations or one or more mutations in one or more genes included in the sequencing mutation profile. comparing said IHC expression profile, microarray expression profile, FISH mutation profile, and sequencing mutation profile to a rule database that includes mappings for treatments with known biological activity against; and (f) i. the comparison of step (e) indicates that the treatment should have biological activity against the cancer; and ii. identifying a candidate treatment if the comparison of step (e) does not indicate a contraindication to said treatment with respect to treating said cancer. [The present invention 1003] 2. A method of identifying a candidate treatment for cancer in a subject in need of such treatment, comprising: (a) performing immunohistochemistry (IHC) analysis on a sample from the subject to determine an IHC expression profile for at least a group of proteins consisting of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; (b) Microarray analysis was performed on the sample to detect the following: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH determining a microarray expression profile for at least a group of genes consisting of: MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; (c) performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least a group of genes consisting of EGFR and HER2; (d) performing DNA sequencing on the sample to determine a sequencing mutation profile for at least a group of genes consisting of KRAS, BRAF, c-KIT, and EGFR; and (e) i. cancer cells that over- or under-express one or more proteins contained in the IHC expression profile; ii. cancer cells that over- or under-express one or more genes contained in the microarray expression profile; iii. cancer cells that have no mutations or one or more mutations in one or more genes included in the FISH mutation profile; and / or iv. Cancer cells that have no mutations or one or more mutations in one or more genes included in the sequencing mutation profile. comparing said IHC expression profile, microarray expression profile, FISH mutation profile, and sequencing mutation profile to a rule database that includes mappings for treatments with known biological activity against; and (f) i. the comparison of step (e) indicates that the treatment should have biological activity against the cancer; and ii. identifying a candidate treatment if the comparison of step (e) does not indicate a contraindication to said treatment with respect to treating said cancer. [The present invention 1004] The method of any one of claims 1001, 1002, or 1003, wherein the sample comprises formalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen (FF) tissue, or tissue contained in a solution that preserves nucleic acid or protein molecules. [The present invention 1005] The method of any one of 1001, 1002, or 1003, wherein any one of microarray analysis, FISH mutation analysis, or sequencing mutation analysis is not performed. [The present invention 1006] The method of any one of claims 1001, 1002, or 1003, wherein the sample passes quality control testing. [The present invention 1007] 1006. The method of claim 1006, wherein the quality control test comprises the A260 / A280 ratio or the Ct value of RT-PCR of RPL13a mRNA. [The present invention 1008] 1007. The method of claim 1007, wherein the quality control tests include an A260 / A280 ratio of less than 1.5 or an RPL13a Ct value of greater than 30. [The present invention 1009] The method of the present invention 1002, wherein IHC expression profiling is performed for at least 50%, 60%, 70%, 80%, or 90% of the biomarkers listed in step (a). [The present invention 1010] The method of claim 1002, wherein microarray expression profiling is performed for at least 50%, 60%, 70%, 80%, or 90% of the biomarkers listed in step (b). [The present invention 1011] The method of any one of claims 1001, 1002, or 1003, wherein the microarray expression profiling is performed using a low-density microarray, an expression microarray, a comparative genomic hybridization (CGH) microarray, a single nucleotide polymorphism (SNP) microarray, a proteomics array, or an antibody array. [The present invention 1012] The method of any one of claims 1001, 1002, or 1003, wherein IHC expression profiling is performed for at least SPARC, TOP2A, and / or PTEN. [The present invention 1013] The method of any one of claims 1001, 1002, or 1003, wherein microarray expression profiling is performed on at least CD52. [The present invention 1014] The method of claim 1002, wherein the IHC expression profiling further comprises assaying one or more of DCK, EGFR, BRCA1, CK 14, CK 17, CK 5 / 6, E-cadherin, p95, PARP-1, SPARC, and TLE3. [The present invention 1015] The method of claim 1002, wherein the IHC expression profiling further comprises assaying Cox-2 and / or Ki-67. [The present invention 1016] The method of claim 1002, wherein the microarray expression profiling further comprises assaying HSPCA. [The present invention 1017] The method of claim 1002, wherein the FISH mutation profiling further comprises assaying c-Myc and / or TOP2A. [The present invention 1018] The method of claim 1002, wherein the sequencing mutation profiling further comprises assaying for PI3K. [The present invention 1019] The genes used for IHC expression profiling, microarray expression profiling, FISH mutation profiling, and sequencing mutation profiling were ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, androgen receptor, AR, AREG, ASNS, BCL2, BCRP, BDCA1, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDK2, CDW52, CES2, CK14, CK17, and CK21. 5 / 6, c-KIT, c-Myc, COX-2, cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, E-cadherin, ECGF1, EGFR, EPHA2, epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, Her2 / Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IL 13RA1, IL2RA, KDR, KIT, K-RAS, LCK, LTB, lymphotoxin β receptor, LYN, MGMT, MLH1, MRP1, MS4A1, MSH2, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SPARC The method of the present invention 1001, wherein the method independently comprises one or more of MC, SPARC PC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TOPO1, TOPO2B, topoisomerase II, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, and ZAP70. [The present invention 1020] The method of the present invention 1001, wherein the IHC expression profiling comprises assaying one or more of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1. [The present invention 1021] Microarray expression profiling identified the following genes: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, M The method of the present invention 1001, comprising assaying one or more of LH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. [The present invention 1022] The method of any one of claims 1 to 5, wherein the FISH mutation profiling comprises assaying EGFR and / or HER2. [The present invention 1023] The method of claim 1001, wherein the sequencing mutation profiling comprises assaying one or more of KRAS, BRAF, c-KIT, and EGFR. [The present invention 1024] The method of any one of claims 1001, 1002, or 1003, wherein the microarray expression analysis comprises identifying genes that are up-regulated or down-regulated with statistical significance compared to a reference. [The present invention 1025] The method of the present invention 1024, wherein statistical significance is determined by a p-value of less than or equal to 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. [The present invention 1026] The method of the present invention 1025, wherein the p-value is corrected for multiple comparisons. [The present invention 1027] The method of the present invention, wherein the correction for multiple comparisons includes the Bonferroni correction or a modification thereof. [The present invention 1028] The method of any one of claims 1001, 1002, or 1003, wherein the IHC analysis comprises determining whether 30% or more of the sample has a staining intensity of +2 or greater. [The present invention 1029] The method of any one of claims 1001, 1002, or 1003, wherein the rules database comprises the rules set forth in Table 1 and / or Table 2. [The present invention 1030] The method of any one of claims 1001, 1002, or 1003, wherein the rules contained within the rule database are based on the effectiveness of various treatments specific to the target gene or gene product. [The present invention 1031] The method of any one of claims 1001, 1002, or 1003, wherein a prioritized list of candidate treatments is identified. [The present invention 1032] The method of the present invention 1031, wherein the prioritization includes ordering treatments from high priority to low priority according to: treatments based on microarray analysis and either IHC analysis or FISH analysis; treatments based on IHC analysis but not microarray analysis; and treatments based on microarray analysis but not IHC analysis. [The present invention 1033] The method of any one of claims 1001, 1002, or 1003, wherein the treatment comprises administration of one or more candidate therapeutic agents. [The present invention 1034] The one or more candidate therapeutic agents may be 5-fluorouracil, abarelix, alemtuzumab, aminoglutethimide, anastrazole, aromatase inhibitors (anastrazole, letrozole), asparaginase, aspirin, ATRA, azacitidine, bevacizumab, bexarotene, bicalutamide, bortezomib, calcitriol, capecitabine, carboplatin, celecoxib, cetuximab, chemoendocrine therapy, cholecalciferol, cisplatin, carboplatin, cyclophosphamide, cyclophosphamide / vincristine, cytarabine, dasatinib, decitabine, doxorubicin, epirubicin, erlotinib, etoposide, exemestane, fluoropyrimidine, flutamide, fulvestine, The method of the present invention 1033 includes the use of trastuzumab, gemcitabine, gonadorelin, goserelin, hydroxyurea, imatinib, irinotecan, ixabepilone, lapatinib, letrozole, leuprolide, liposomal doxorubicin, medroxyprogesterone, megestrol, methotrexate, mitomycin, nab-paclitaxel, octreotide, oxaliplatin, paclitaxel, panitumumab, pegaspargase, pemetrexed, pentostatin, sorafenib, sunitinib, tamoxifen, tamoxifen-based therapy, temozolomide, topotecan, toremifene, trastuzumab, VBMCP / cyclophosphamide, vincristine, or any combination thereof. [The present invention 1035] The one or more candidate therapeutic agents may be 5FU, bevacizumab, capecitabine, cetuximab, cetuximab + gemcitabine, cetuximab + irinotecan, cyclophosphamide, diethylstibesterol, doxorubicin, erlotinib, etoposide, exemestane, fluoropyrimidine, gemcitabine, gemcitabine + etoposide, gemcitabine + pemetrexed, irinotecan, irinotecan + sorafenib, lapatinib, lapatinib + tamoxifen, letrozole, or retroviral therapy. The method of the present invention 1033, comprising administering to a patient in need thereof a combination of temozolomide, temozolomide + capecitabine, mitomycin, nab-paclitaxel, nab-paclitaxel + gemcitabine, nab-paclitaxel + trastuzumab, oxaliplatin, oxaliplatin + 5FU + trastuzumab, panitumumab, pemetrexed, sorafenib, sunitinib, sunitinib, sunitinib + mitomycin, tamoxifen, temozolomide, temozolomide + bevacizumab, temozolomide + sorafenib, trastuzumab, vincristine, or any combination thereof. [The present invention 1036] The method of any one of claims 1001 to 1002, wherein the sample comprises cancer cells. [The present invention 1037] The method of any one of claims 1001, 1002, or 1003, wherein the subject has previously been treated with one or more therapeutic agents for treating cancer. [The present invention 1038] The method of any one of claims 1001, 1002, or 1003, wherein the subject has not been previously treated with one or more candidate therapeutic agents identified in step (f). [The present invention 1039] The method of any one of claims 1001, 1002, or 1003, wherein the cancer comprises a metastatic cancer. [The present invention 1040] The method of any one of claims 1001, 1002, or 1003, wherein the cancer is refractory to previous treatments. [The present invention 1041] The method of claim 1040, wherein the prior treatment comprises standard of care for the cancer. [The present invention 1042] The method of invention 1001, 1002, or 1003, wherein the cancer comprises prostate cancer, lung cancer, melanoma, small cell carcinoma (esophageal / retroperitoneal), cholangiocarcinoma, mesothelioma, head and neck cancer (SCC), pancreatic cancer, pancreatic neuroendocrine carcinoma, small cell carcinoma, gastric cancer, pseudomyxoma peritonei, anal canal carcinoma (SCC), vaginal carcinoma (SCC), cervical cancer, renal carcinoma, eccrine sweat gland adenocarcinoma, salivary gland adenocarcinoma, uterine soft tissue sarcoma (uterus), GIST (gastric), or anaplastic thyroid carcinoma. [The present invention 1043] The method of any one of claims 1001, 1002, or 1003, wherein the cancer comprises cancer of the accessory, sinuses, middle and inner ear, adrenal gland, appendix, hematopoietic system, bones and joints, spinal cord, breast, cerebellum, cervix, connective soft tissue, uterus, esophagus, eye, nose, eyeball, fallopian tube, extrahepatic bile duct, oral cavity, intrahepatic bile duct, kidney, appendix-colon, larynx, lips, liver, lung and bronchi, lymph nodes, cerebrum, spinal cord, nasal cartilage, retina, eye, oropharynx, endocrine glands, female reproductive organs, ovaries, pancreas, penis and scrotum, pituitary gland, pleura, prostate, rectum renal pelvis, ureter, peritoneum, salivary gland, skin, small intestine, stomach, testis, thymus, thyroid, tongue, unknown, bladder, uterus, vagina, labia, and vulva. [The present invention 1044] 1001, 1002, or 1003, wherein the sample comprises cells selected from the group consisting of adipose, adrenal cortex, adrenal gland, adrenal-medulla, appendix, bladder, blood, blood vessels, bone, osteochondral, brain, breast, cartilage, cervix, colon, sigmoid colon, dendritic cells, skeletal muscle, endometrium, esophagus, fallopian tube, fibroblasts, gallbladder, kidney, larynx, liver, lung, lymph node, melanocytes, mesothelial lining, myoepithelial cells, osteoblasts, ovary, pancreas, parotid gland, prostate, salivary gland, sinus tissue, skeletal muscle, skin, small intestine, smooth muscle, stomach, synovium, joint lining tissue, tendon, testis, thymus, thyroid, uterus, and uterine corpus. [The present invention 1045] The method of any one of claims 1001, 1002, or 1003, wherein the cancer comprises breast cancer, colorectal cancer, ovarian cancer, lung cancer, non-small cell lung cancer, cholangiocarcinoma, mesothelioma, sweat gland carcinoma, or GIST cancer. [The present invention 1046] The method of any one of claims 1001, 1002, or 1003, wherein the subject's progression free survival (PFS) or disease free survival (DFS) is extended. [The present invention 1047] The method of the present invention 1046, wherein PFS or DFS is extended by at least about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 100% compared to previous treatment. [The present invention 1048] The method of any one of claims 1001, 1002, or 1003, wherein the selection of the candidate treatment extends the lifespan of the subject. [The present invention 1049] The method of the present invention 1048, wherein the patient's life span is extended by at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 2 years, 2.5 years, 3 years, 4 years, or at least 5 years.

[0027] INCORPORATION BY REFERENCE All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0028] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Figure 1] FIG. 1 shows a block diagram of an exemplary embodiment of a system for determining personalized medical interventions for specific disease conditions utilizing non-disease specific molecular profiling of a patient's biological specimen. [Diagram 2] 1 is a flow chart of an exemplary embodiment of a method for determining personalized medical intervention for a particular pathology utilizing non-disease specific molecular profiling of a patient's biological specimen. [Figure 3A] 2. An exemplary patient profile report according to step 80 of FIG. [Figure 3B] 2. An exemplary patient profile report according to step 80 of FIG. [Figure 3C] 2. An exemplary patient profile report according to step 80 of FIG. [Figure 3D] 2. An exemplary patient profile report according to step 80 of FIG. [Figure 4] 1 is a flow chart of an exemplary embodiment of a method for identifying drug therapies / agents that may interact with a target. [Diagram 5] 1 is a flow chart and diagrams illustrating various portions of the information-based personalized medicine discovery system and method according to the present invention; [Figure 6] 1 is a flow chart and diagrams illustrating various portions of the information-based personalized medicine discovery system and method according to the present invention; [Figure 7] 1 is a flow chart and diagrams illustrating various portions of the information-based personalized medicine discovery system and method according to the present invention; [Figure 8] 1 is a flow chart and diagram illustrating various portions of the system and method for information-based personalized medicine discovery in accordance with the present invention; [Figure 9] 1 is a flow chart and diagrams illustrating various portions of the information-based personalized medicine discovery system and method according to the present invention; [Figure 10] 1 is a flow chart and diagrams illustrating various portions of the information-based personalized medicine discovery system and method according to the present invention; [Figure 11] 1 is a flow chart and diagrams illustrating various portions of the information-based personalized medicine discovery system and method according to the present invention; [Figure 12] 1 is a flow chart and diagram illustrating various portions of the system and method for information-based personalized medicine discovery in accordance with the present invention; [Figure 13] 1 is a flow chart and diagram illustrating various portions of the system and method for information-based personalized medicine discovery in accordance with the present invention; [Figure 14] 1 is a flow chart and diagram illustrating various portions of the system and method for information-based personalized medicine discovery in accordance with the present invention; [Figure 15] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 16] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 17] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 18] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 19] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 20] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 21] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 22] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Diagram 23] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 24] 5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Diagram 25]5-14 are computer screen printouts relating to various portions of the information-based personalized medicine discovery system and method shown in FIGS. [Figure 26A] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 26B] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 26C] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 26D] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 26E] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 26F] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 26G] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Fig. 26H] A table showing the frequency of significant changes in gene expression protein expression by tumor type is shown. [Figure 27A] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 27B] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 27C] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 27D] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 27E] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 27F] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 27G] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Fig. 27H] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 28A] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28B] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28C] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28D] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28E] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28F] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28G] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Fig. 28H] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28I] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28J] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28K] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28L] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28M] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28N] A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 28O]A table showing the frequency of significant changes in expression of specific gene expression proteins by tumor type is shown. [Figure 29] 29 is a table showing biomarkers (gene expressed proteins) tagged as targets in order of frequency based on FIG. 28. [Figure 30A] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30B] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30C] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30D] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30E] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30F] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30G] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30H] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30I] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30J] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30K] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30L] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30M] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30N] A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Figure 30O]A table showing the frequency of significant changes in expression of specific genes by tumor type is shown. [Diagram 31] 31 is a table showing genes tagged as targets in order of frequency based on FIG. 30. [Diagram 32] Progression-free survival (PFS) with therapy selected by molecular profiling (period B) and PFS with the most recent therapy at the time the patient started progressing (period A) are shown. Patients were defined as benefiting from therapy selected by molecular profiling if their PFS(B) / PFS(A) ratio was ≥ 1.3. [Diagram 33] FIG. 1 is a schematic diagram of a method for identifying treatments by molecular profiling once a target has been identified. [Diagram 34] 1 shows the distribution of patients in the study conducted in Example 1. [Diagram 35] Graph showing results of a study in which 18 / 66 (27%) patients had a PFS ratio >= 1.3. [Diagram 36] Waterfall plot for all patients for maximum % change in total diameter of target lesions relative to baseline diameter. [Figure 37] This shows the relationship between what clinicians chose to use to treat patients before knowing what the molecular profiling results suggested. No agreement was found for the 18 patients with a PFS ratio ≥ 1.3. [Figure 38] Schematic representation of overall survival for 18 patients with a PFS ratio ≥ 1.3 and for all 66 patients. [Figure 39] 1 shows microarray profiling results and an exemplary output of calls made using cutoff values. [Figure 40A] 1 shows an exemplary patient report based on molecular profiling. [Figure 40B] 1 shows an exemplary patient report based on molecular profiling. [Figure 40C] 1 shows an exemplary patient report based on molecular profiling. [Figure 40D]1 shows an exemplary patient report based on molecular profiling. [Figure 40E] 1 shows an exemplary patient report based on molecular profiling. [Diagram 40F] 1 shows an exemplary patient report based on molecular profiling. [Figure 40G] 1 shows an exemplary patient report based on molecular profiling. [Figure 40H] 1 shows an exemplary patient report based on molecular profiling. [Fig. 40I] 1 shows an exemplary patient report based on molecular profiling. [Figure 40J] 1 shows an exemplary patient report based on molecular profiling. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The present invention provides a method and system for identifying therapeutic targets by using molecular profiling. The molecular profiling approach provides a method for selecting candidate therapies for individuals that can favorably change the clinical course of individuals with conditions or diseases such as cancer. The molecular profiling approach can provide clinical benefits to individuals, such as longer progression-free survival (PFS), longer disease-free survival (DFS), longer overall survival (OS), or extended life span, when treated with the molecular profiling approach, compared to using traditional approaches to select treatment plans. Molecular profiling can suggest candidate therapies when a disease is refractory to current therapies, such as after cancer develops resistance to standard care.

[0030] Molecular profiling can be performed by any known means for detecting molecules in biological samples. Profiling can be performed in any applicable biological sample. The sample is typically from an individual with a suspected or known disease or disorder, including but not limited to a biopsy sample from a cancer patient. Molecular profiling of a sample can also be performed by any number of techniques that evaluate the amount or state of biological factors such as DNA sequences, mRNA sequences, or proteins. Such techniques include, but are not limited to, immunohistochemistry (IHC), in situ hybridization (ISH), fluorescent in situ hybridization (FISH), various types of microarrays (mRNA expression arrays, protein arrays, etc.), various types of sequencing (Sanger, pyrosequencing, etc.), comparative genomic hybridization (CGH), next generation sequencing, Northern blots, Southern blots, immunoassays, and any other suitable techniques under development for assaying the presence or amount of a biological molecule of interest. Any one or more of these methods can be used simultaneously or sequentially with each other.

[0031] Molecular profiling is used to select candidate treatments for disorders in subjects. For example, the candidate treatments may be treatments that are known to have an effect on cells that differentially express genes as identified by molecular profiling techniques. Differential expression may include both overexpression and underexpression of biological products, such as genes, mRNA, or proteins, compared to a control. The control may include cells that are similar to the sample but do not contain the disease. The control may be from the same patient, such as a normal adjacent part of the same organ as the diseased cells, or the control may be from healthy tissue from another patient. The control may be a control found in the same sample, such as a housekeeping gene or its product (e.g., mRNA or protein). For example, the control nucleic acid may be one that is known not to differ depending on the cancerous or non-cancerous state of the cells. The expression level of the control nucleic acid may be used to normalize the signal levels in the test and reference populations. Exemplary control genes include, but are not limited to, for example, β-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1. Multiple controls or types of controls can be used. The causes of differential expression can be different. For example, the number of gene copies can increase in cells, which leads to increased expression of the gene. Alternatively, the transcription of the gene can be altered, for example, by chromatin remodeling, differential methylation, differential expression or activity of transcription factors, etc. For example, translation can be altered by differential expression of factors that degrade mRNA, translate mRNA, or repress translation, for example, microRNA or siRNA. In some embodiments, differential expression includes differential activity. For example, a protein can carry a mutation that increases the activity of the protein, such as constitutive activation, thereby contributing to a diseased state. Molecular profiling that reveals changes in activity can be used to guide treatment selection.

[0032] In the case where molecular profiling reveals that multiple drug targets are differentially expressed, decision rules can be introduced to prioritize the selection of specific treatments. Treatments can be prioritized using any such rules that help prioritize treatments, such as direct results of molecular profiling, predicted efficacy, previous history with the same or other treatments, predicted side effects, availability, cost, drug-drug interactions, and other factors considered by the treating physician. The physician can ultimately decide the course of treatment. Thus, molecular profiling can select candidate treatments based on the individual characteristics of diseased cells, such as tumor cells, and other individual factors in the subject in need of treatment, as opposed to relying on the traditional one-size-fits-all approach taken to target treatments to certain symptoms. In some cases, the recommended treatment is one that is not typically used to treat the disease or disorder that the subject suffers from. In some cases, the recommended treatment is used after standard care no longer provides adequate efficacy.

[0033] Nucleic acid includes deoxyribonucleotides or ribonucleotides, and their polymers in single-stranded or double-stranded form, and their complements. Nucleic acid can include known nucleotide analogs or modified backbone residues or bonds, which are synthetic, natural, and non-natural, and have similar binding properties as reference nucleic acid, and are metabolized in a similar manner as reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, and polypeptide nucleic acids (PNAs). In addition to the sequences shown, nucleic acid sequences can also include conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell Probes 8:91-98 (1994)). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0034] A particular nucleic acid sequence may also implicitly encompass nucleic acid sequences that code for that particular sequence and "splice variants" and truncated forms. Similarly, a particular protein encoded by a nucleic acid may encompass any protein encoded by a splice variant or truncated form of that nucleic acid. A "splice variant" is, as the name suggests, a product of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced ​​such that different (alternative) nucleic acid splice products code for different polypeptides. The mechanisms for producing splice variants vary, but include alternative splicing of exons. Alternative polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splice reaction, including recombinant splice products, is included in this definition. Nucleic acids may be truncated at the 5' or 3' end. Polypeptides may be truncated at the N- or C-terminus. Truncated forms of nucleic acid or polypeptide sequences may occur naturally or may be produced recombinantly.

[0035] The terms "gene variant" and "nucleotide variant" are used interchangeably herein to refer to changes or alterations to a reference human gene or cDNA sequence at a particular locus, including but not limited to deletions, insertions, inversions, and substitutions of nucleotide bases in coding and non-coding regions. Deletions can be of a single nucleotide base, a portion or region of the nucleotide sequence of a gene, or the entire gene sequence. Insertions can be of one or more nucleotide bases. Gene variants or nucleotide variants can occur in transcriptional regulatory regions, untranslated regions of mRNA, exons, introns, exon / intron junctions, etc. Gene variants or nucleotide variants can potentially result in stop codons, frameshifts, deletions of amino acids, changes in gene transcript splice forms, or changes in amino acid sequence.

[0036] Alleles or gene alleles generally include naturally occurring genes having a reference sequence or genes that contain particular nucleotide variants.

[0037] A haplotype refers to a combination of genetic (nucleotide) variants in a region of mRNA or genomic DNA on a single chromosome found in an individual. Thus, a haplotype contains several genetically linked polymorphic variants that are typically inherited together as a unit.

[0038] The term "amino acid variant" as used herein refers to the amino acid change relative to the reference protein sequence resulting from a genetic or nucleotide variant relative to a reference human gene encoding a reference human protein. The term "amino acid variant" is intended to include not only single amino acid substitutions, but also amino acid deletions, insertions, and other significant changes in the amino acid sequence in the reference protein.

[0039] The term "genotype" as used herein refers to the nucleotide characteristics of a particular nucleotide variant marker (or locus) in one or both alleles of a gene (or a particular chromosomal region). For a particular nucleotide position of a gene of interest, the nucleotides or their equivalents at that locus in one or both alleles form the genotype of the gene at that locus. The genotype may be homozygous or heterozygous. Thus, "genotyping" refers to determining the genotype, i.e., the nucleotide at a particular locus. Genotyping can also be performed by determining the amino acid variant at a particular position of a protein, which can be used to infer the corresponding nucleotide variant.

[0040] The term "locus" refers to a specific position or site in a gene sequence or protein. Thus, one or more consecutive nucleotides may be present at a particular locus, or one or more amino acids may be present at a particular locus in a polypeptide. Additionally, a locus may refer to a specific position in a gene where one or more nucleotides have been deleted, inserted, or inverted.

[0041] As used herein, the terms "polypeptide", "protein" and "peptide" are used interchangeably to refer to an amino acid chain in which amino acid residues are linked by covalent peptide bonds. The amino acid chain can be of any length of at least two amino acids, including full-length proteins. Unless otherwise specified, polypeptides, proteins and peptides also encompass their various modified forms, including but not limited to glycosylated forms, phosphorylated forms, etc. A polypeptide, protein or peptide may also be referred to as a gene product.

[0042] Herein is provided a list of genes and gene products that can be assayed by molecular profiling techniques. The list of genes may be provided in conjunction with molecular profiling techniques that detect gene products (e.g., mRNA or protein). Those skilled in the art will understand that this refers to the detection of gene products of the described genes. Similarly, the list of gene products may be provided in conjunction with molecular profiling techniques that detect gene sequence or copy number. Those skilled in the art will understand that this refers to the detection of genes corresponding to the gene products, including, for example, DNA that codes for the gene products. As will be understood by those skilled in the art, "biomarker" or "marker" includes genes and / or gene products depending on the context.

[0043] The terms "label" and "detectable label" may refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or similar methods. Such labels include biotin for staining with labeled streptavidin conjugates, magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, etc.), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means for detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using light-sensitive film or scintillation counters, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. Labels can include, for example, ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can function as specific binding pair members for labeled ligands. General information on labels, labeling procedures, and detection of labels can be found in Polak and Van Noorden Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and Haugland Handbook of Fluorescent Probes and Research Chemicals, a combined handbook and catalogue Published by Molecular Probes, Inc. (1996).

[0044] Detectable labels include, but are not limited to, nucleotides (labeled or unlabeled), compomers, sugars, peptides, proteins, antibodies, chemical compounds, conductive polymers, binding moieties such as biotin, mass tags, colorimetric agents, luminescent agents, chemiluminescent agents, light scattering agents, fluorescent tags, radioactive tags, charge tags (electric or magnetic charge), volatile tags and hydrophobic tags, biomolecules (e.g., members of the binding pairs antibody / antigen, antibody / antibody, antibody / antibody fragment, antibody / antibody receptor, antibody / protein A or protein G, hapten / anti-hapten, biotin / avidin, biotin / streptavidin, folate / folate binding protein, vitamin B12 / intrinsic factor, chemically reactive groups / complementary chemically reactive groups (e.g., members of the sulfhydryl / maleimide, sulfhydryl / haloacetyl derivatives, amine / isotriocyanate, amine / succinimidyl ester, and amine / sulfonyl halide), and the like.

[0045] The term "antibody" as used herein includes natural and non-natural antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, and humanized antibodies, as well as antigen-binding fragments thereof (e.g., Fab', F(ab')2, Fab, Fv, and rIgG). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). See also, for example, Kuby, J., Immunology, 3.sup.rd Ed., WH Freeman & Co., New York (1998). Such non-natural antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, for example, as described in Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. For example, these and other methods of making chimeric antibodies, humanized antibodies, CDR-grafted antibodies, single-chain antibodies, and bifunctional antibodies are well known to those skilled in the art.See, for example, Winter and Harris, Immunol. Today 14:243-246 (1993);Ward et al., Nature 341:544-546 (1989);Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York, 1988;Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992);Borrebaeck, Antibody Engineering, 2d ed. (Oxford University Press 1995);each of which is incorporated herein by reference.

[0046] Unless otherwise specified, antibodies can include both polyclonal and monoclonal antibodies. Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described, for example, in Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al. (1993) Proc Natl Acad Sci USA. 90:6444, Gruber et al. (1994) J Immunol :5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1997) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[0047] Typically, an antibody has a heavy chain and a light chain. Each of the heavy and light chains contains a constant region and a variable region (these regions are also known as "domains"). The light and heavy chain variable regions contain four framework regions interrupted by three hypervariable regions, also called complementarity determining regions (CDRs). The extent of the framework regions and CDRs are defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to the antigen epitope. The CDRs of each chain are typically numbered sequentially from the N-terminus and designated CDR1, CDR2, and CDR3, and are typically identified by the chain in which that particular CDR is located. Thus, the V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found and is L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. H References to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. L References to the variable region of an immunoglobulin light chain refer to the light chain of an Fv, scFv, dsFv or Fab.

[0048] The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy and light chains of a conventional two-chain antibody are linked to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site. A "chimeric antibody" is an immunoglobulin molecule in which (a) the constant region or a portion thereof has been altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof has been altered, replaced, or exchanged with a variable region having a different or altered antigen specificity.

[0049] A "humanized antibody" is an immunoglobulin molecule that contains minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity determining region (CDR) of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity have been replaced with residues from the recipient's CDR. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions being those of a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Humanization can essentially be performed by following the method of Winter and coworkers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than an entire human variable domain has been substituted by the corresponding sequence from a non-human species.

[0050] The terms "epitope" and "antigenic determinant" refer to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or from non-contiguous amino acids arranged in close proximity by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically contains at least 3, more commonly at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0051] The terms "primer", "probe", and "oligonucleotide" are used interchangeably herein to refer to relatively short nucleic acid fragments or sequences. They may include DNA, RNA, or hybrids thereof, or chemically modified analogs or derivatives thereof. Typically, they are single-stranded. However, they may be double-stranded, with two complementary strands that can be separated by denaturation. Usually, primers, probes, and oligonucleotides have a length of about 8 nucleotides to about 200 nucleotides, preferably about 12 nucleotides to about 100 nucleotides, more preferably about 18 to about 50 nucleotides. For various molecular biology applications, they can be labeled with detectable markers or modified using conventional methods.

[0052] The term "isolated" when used in reference to a nucleic acid (e.g., genomic DNA, cDNA, mRNA, or fragments thereof) is intended to mean that the nucleic acid molecule is present in a form substantially separated from other naturally occurring nucleic acids that are normally associated with the molecule. Since a naturally occurring chromosome (or its viral equivalent) contains a long nucleic acid sequence, an isolated nucleic acid may be a nucleic acid molecule that has only a portion of the nucleic acid sequence in the chromosome, without one or more other portions that are present on the same chromosome. More specifically, an isolated nucleic acid may contain a naturally occurring nucleic acid sequence that is adjacent to the nucleic acid in the naturally occurring chromosome (or its viral equivalent). An isolated nucleic acid may be substantially separated from other naturally occurring nucleic acids on different chromosomes of the same organism. An isolated nucleic acid may also be a composition in which a particular nucleic acid molecule is significantly enriched, such that the particular nucleic acid molecule constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the total nucleic acid in the composition.

[0053] An isolated nucleic acid may be a hybrid nucleic acid in which a specific nucleic acid molecule is covalently linked to one or more nucleic acid molecules that are not adjacent to the specific nucleic acid in nature.For example, an isolated nucleic acid may be present in a vector.In addition, a specific nucleic acid may have a nucleotide sequence identical to that of a natural nucleic acid, or a modified or mutant form thereof with one or more mutations, such as nucleotide substitutions, deletions / insertions, inversions, etc.

[0054] An isolated nucleic acid can be prepared from a recombinant host cell (wherein the nucleic acid has been recombinantly amplified and / or expressed), or it can be a chemically synthesized nucleic acid having a naturally-occurring nucleotide sequence or an artificially modified version thereof.

[0055] The term "isolated polypeptide" as used herein is defined as a polypeptide molecule that exists in a form other than that found in nature. Thus, an isolated polypeptide may be a non-naturally occurring polypeptide. For example, an isolated polypeptide may be a "hybrid polypeptide". An isolated polypeptide may also be a polypeptide derived from a natural polypeptide by addition or deletion or substitution of amino acids. An isolated polypeptide may also be a "purified polypeptide", which is used herein to mean a composition or preparation in which a particular polypeptide molecule is significantly enriched such that it constitutes at least 10% of the total protein content in the composition. As will be apparent to one of skill in the art, a "purified polypeptide" may be obtained from natural or recombinant host cells by standard purification techniques, or may be obtained by chemical synthesis.

[0056] The terms "hybrid protein," "hybrid polypeptide," "hybrid peptide," "fusion protein," "fusion polypeptide," and "fusion peptide" are used interchangeably herein to refer to a non-naturally occurring or isolated polypeptide in which a particular polypeptide molecule is covalently linked to one or more other polypeptide molecules that are not naturally linked to the particular polypeptide. Thus, a "hybrid protein" may be two natural proteins or fragments thereof that are covalently linked together. A "hybrid protein" may also be a protein formed by covalently linking two artificial polypeptides together. Typically, but not necessarily, two or more polypeptide molecules are linked or "fused" together by peptide bonds that form a single, unbranched polypeptide chain.

[0057] The term "high stringency hybridization conditions" when used with respect to nucleic acid hybridization includes overnight hybridization at 42°C in a solution containing 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH 7.6, 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured and sheared salmon sperm DNA, and washing of the hybridization filter in 0.1x SSC at about 65°C. The term "moderately stringent conditions" when used in reference to nucleic acid hybridization includes hybridization overnight at 37° C. in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate, pH 7.6, 5×Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured and sheared salmon sperm DNA, and washing of the hybridization filter in 1×SSC at about 50° C. It should be noted that many other hybridization methods, solutions, and temperatures can be used to achieve comparable stringent hybridization conditions, as will be apparent to one of skill in the art.

[0058] For the purpose of comparing two different nucleic acid or polypeptide sequences, one sequence (test sequence) can be stated to be identical to the other sequence (comparison sequence) at a certain percentage. The identity percentage can be determined by the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which is incorporated into various BLAST programs. The identity percentage can be determined by the "BLAST 2 Sequences" tool available on the National Center for Biotechnology Information (NCBI) website. See Tatusova and Madden, FEMS Microbiol. Lett., 174(2):247-250 (1999). For pairwise comparison between DNA, the BLASTN program is used with default parameters (match: 1; mismatch: -2; open gap: 5 penalty; extension gap: 2 penalty; gap x_dropoff: 50; expectation: 10; and word size: 11, with filters). For pairwise comparison between protein sequences, the BLASTP program can be used with default parameters (matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15; expectation: 10.0; and word size: 3, with filter). The identity percentage of two sequences is calculated by aligning the test sequence with the comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence. When BLAST is used to compare two sequences, it aligns the sequences and gives the identity percentage over a defined alignment region. When two sequences are aligned over their entire length, the identity percentage obtained by BLAST is the identity percentage of the two sequences.If BLAST does not align two sequences over their entire length, the number of identical amino acids or nucleotides in the unaligned regions of the test and comparison sequences is assumed to be zero, and the percentage identity is calculated by adding the number of identical amino acids or nucleotides in the aligned regions and dividing that number by the length of the comparison sequences. Various versions of the BLAST program can be used to compare sequences, for example, BLAST 2.1.2 or BLAST+ 2.2.22.

[0059] The subject may be any animal that can benefit from the method of the present invention, including, for example, humans, and non-human mammals such as primates, rodents, horses, dogs, and cats.The subject includes, but is not limited to, eukaryotic organisms, most preferably primates, such as chimpanzees or humans, cows; dogs; cats; rodents, such as guinea pigs, rats, mice; mammals such as rabbits; or birds; reptiles; or fish.The subject specifically intended for treatment using the method described herein includes humans.The subject may be referred to as an individual or a patient.

[0060] Treatment of a disease or individual according to the present invention is an approach to obtain beneficial or desired medical results, including clinical results, but not necessarily cures. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the extent of disease, stable (i.e., not worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, amelioration or alleviation of pathology, and remission (whether partial or total). Treatment also includes prolonging survival compared to expected survival without treatment or with a different treatment. Treatment may include administration of a therapeutic agent, which may be an agent that exerts a cytotoxic, cytostatic, or immunomodulatory effect on diseased cells, such as cancer cells, or other cells that may promote a pathological condition. There is no limit to the therapeutic agent selected by the method of the present invention. Any therapeutic agent that may be found to have a correlation between molecular profiling and the potential efficacy of the agent may be selected. Therapeutic agents include, but are not limited to, small molecules, protein therapy, antibody therapy, viral therapy, gene therapy, and the like. Cancer treatments or therapies include apoptosis-mediated and non-apoptosis-mediated cancer therapies, including, but not limited to, chemotherapy, hormonal therapy, radiation therapy, immunotherapy, and combinations thereof. Chemotherapeutic agents include therapeutic agents and combinations of therapeutic agents that treat, e.g., kill, cancer cells. Examples of various types of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., nitrogen mustard derivatives, ethylenimines, alkyl sulfonates, hydrazines and triazines, nitrosoureas, and metal salts), plant alkaloids (e.g., vinca alkaloids, taxanes, podophyllotoxins, and camptothecan analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, etc.), antimetabolites (e.g., folate antagonists, pyrimidine antagonists, purine antagonists, and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors, and various antineoplastic agents (e.g., ribonucleotide reductase inhibitors, corticosteroid inhibitors, enzymes, antimicrotubule agents, and retinoids).

[0061] As used herein, sample includes any relevant sample that can be used for molecular profiling, such as, for example, a biopsy sample or a section of tissue, such as tissue taken during surgery or other treatment, an autopsy sample, and a frozen section taken for histological purposes. Such samples include blood and blood fractions or blood products (e.g., serum, buffy coat, plasma, platelets, red blood cells, etc.), sputum, buccal tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, other biological or bodily fluids (e.g., prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, etc.), and the like. Samples may be processed according to techniques understood by those skilled in the art. Samples may be fresh, frozen or fixed, without limitation. In some embodiments, samples include formalin-fixed paraffin-embedded (FFPE) tissue or fresh frozen (FF) tissue. Samples may include cultured cells, including primary or immortalized cell lines derived from a subject sample. Samples may also refer to extracts from a sample from a subject. For example, the sample may include DNA, RNA, or protein extracted from tissue or body fluid. Many techniques and commercial kits are available for such purposes. Fresh samples from individuals can be treated with agents to preserve RNA before further processing, such as cell lysis and extraction. Samples may include frozen samples taken for other purposes. Samples may be accompanied by relevant information such as the subject's age, sex, and clinical condition; the source of the sample; and the method of sample collection and storage. Samples are typically taken from subjects.

[0062] Biopsy includes the process of taking a tissue sample for diagnostic or prognostic evaluation, and the tissue specimen itself. Any biopsy technique known in the art can be applied to the molecular profiling method of the present invention. The biopsy technique applied may depend on the type of tissue to be evaluated (e.g., colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cells, lung, breast, etc.), the size and type of tumor (e.g., solid or suspended, blood or ascites), among other factors. Exemplary biopsy techniques include, but are not limited to, excision biopsy, incision biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An "excision biopsy" refers to the taking of an entire tumor mass with a small margin of surrounding normal tissue. An "incision biopsy" refers to the taking of a wedge of tissue that includes the cross-sectional diameter of the tumor. Molecular profiling can use a "core needle biopsy" of the tumor mass, or a "fine needle aspiration biopsy" that generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70 and throughout Part V.

[0063] Standard molecular biology techniques known in the art and not specifically described are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), and Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and Watson et al., Recombinant DNA, Scientific American Books, New York, and Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998), and in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659, and 5,272,057, which are hereby incorporated by reference. Polymerase chain reaction (PCR) can be generally performed as described in PCR Protocol: A Guide To Methods And Application, Academic Press, San Diego, Calif. (1990).

[0064] Gene expression profiling In some aspects of the present invention, biomarkers are evaluated by gene expression profiling. Methods of gene expression profiling include methods based on polynucleotide hybridization analysis and methods based on polynucleotide sequencing. Commonly used methods known in the art for quantifying mRNA expression in a sample include Northern blotting and in situ hybridization (Parker & Barnes (1999) Methods in Molecular Biology 106:247-283); RNAse protection assay (Hod (1992) Biotechniques 13:852-854); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al. (1992) Trends in Genetics 8:263-264). Alternatively, antibodies can be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes, or DNA-protein duplexes. Representative methods of sequencing-based gene expression analysis include serial analysis of gene expression (SAGE) and expression analysis by massively parallel signature sequencing (MPSS).

[0065] Reverse transcription PCR (RT-PCR) RT-PCR can be used to measure the RNA levels, such as mRNA or miRNA levels, of the biomarkers of the present invention.RT-PCR can be used to compare such RNA levels of the biomarkers of the present invention in different sample populations, with or without drug treatment, in normal and tumor tissues, to characterize patterns of gene expression, to distinguish closely related RNAs, and to analyze RNA structure.

[0066] The first step is the isolation of RNA, for example mRNA, from a sample.The starting material can be total RNA isolated from human tumor or tumor cell line and corresponding normal tissue or cell line.Thus, RNA can be isolated from a sample, for example tumor cell or tumor cell line, and compared with pooled DNA from healthy donors.When the source of mRNA is a primary tumor, mRNA can be extracted, for example, from frozen tissue sample, or from archival tissue sample that is paraffin-embedded and fixed (for example, formalin-fixed).

[0067] General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al. (1997) Current Protocols of Molecular Biology, John Wiley and Sons. Methods for extracting RNA from paraffin-embedded tissues are disclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, following the manufacturer's instructions (QIAGEN Inc., Valencia, CA). For example, total RNA can be isolated from cultured cells using Qiagen RNeasy mini columns. Many RNA isolation kits are commercially available and can be used in the methods of the present invention.

[0068] Alternatively, the first step is the isolation of miRNA from target sample.The starting material is typically the total RNA isolated from human tumor or tumor cell line and corresponding normal tissue or cell line.Thus, RNA can be isolated from various primary tumors or tumor cell lines, together with pooled DNA from healthy donors.When the source of miRNA is primary tumor, miRNA can be extracted, for example, from frozen tissue sample, or from archival tissue sample that is paraffin-embedded and fixed (e.g., formalin-fixed).

[0069] General methods for miRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al. (1997) Current Protocols of Molecular Biology, John Wiley and Sons. Methods for extracting RNA from paraffin-embedded tissues are disclosed, for example, in Rupp & Locker (1987) Lab Invest. 56:A67, and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, following the manufacturer's instructions. For example, total RNA can be isolated from cultured cells using Qiagen's RNeasy mini-columns. Many RNA isolation kits are commercially available and can be used in the method of the present invention.

[0070] Whether the RNA comprises mRNA, miRNA, or other RNA types, gene profiling by RT-PCR may involve reverse transcription of the RNA template into cDNA followed by amplification in a PCR reaction. Commonly used reverse transcriptases include, but are not limited to, avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed with specific primers, random hexamers, or oligo-dT primers, depending on the context and purpose of expression profiling. For example, extracted RNA can be reverse transcribed using a Gene Amp RNA PCR kit (Perkin Elmer, Calif., USA) according to the manufacturer's instructions. The derived cDNA can then be used as a template in a subsequent PCR reaction.

[0071] While a variety of thermostable DNA-dependent DNA polymerases can be used in the PCR step, Taq DNA polymerase is typically used, which has 5'-3' nuclease activity but lacks 3'-5' proofreading endonuclease activity. TaqMan PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe that is bound to its target amplicon, although any enzyme with comparable 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, the probe, is designed to detect the nucleotide sequence located between the two PCR primers. This probe cannot be extended by Taq DNA polymerase enzyme and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. When the two dyes are in close proximity to each other on the probe, any laser-induced emission from the reporter dye is quenched by the quencher dye. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resulting probe fragments dissociate in solution, and the signal from the released reporter dye is freed from the quenching effect of the second fluorophore. Because one molecule of reporter dye is liberated for each new molecule synthesized, detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0072] TaqMan™ RT-PCR can be performed using commercially available equipment, such as the ABI PRISM 7700™ Sequence Detection System™ (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or the Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In one particular embodiment, the 5' nuclease procedure is performed on a real-time quantitative PCR device, such as the ABI PRISM 7700™ Sequence Detection System™. The system consists of a thermocycler, a laser, a charge-coupled device (CCD), a camera, and a computer. The system amplifies samples in a 96-well format on the thermocycler. During amplification, the laser-induced fluorescent signal is collected in real time for all 96 wells via fiber optic cables and detected by the CCD. The system includes software for running the instrument and for analyzing the data.

[0073] TaqMan data is first expressed as Ct, or threshold cycle. As above, fluorescence values ​​are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. The point at which the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

[0074] To minimize errors and the effects of sample-to-sample variation, RT-PCR is usually performed with an internal standard. An ideal internal standard is expressed at a constant level among different tissues and is not affected by the experimental treatment. The RNAs most frequently used to normalize patterns of gene expression are the mRNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin.

[0075] Real-time quantitative PCR (also quantitative real-time polymerase chain reaction, QRT-PCR, or Q-PCR) is a more recent variation of the RT-PCR technique. Q-PCR can measure the accumulation of PCR products through a dual-labeled fluorogenic probe (i.e., TaqMan™ probe). Real-time PCR is compatible with both quantitative competitive PCR, in which an internal competitor for each target sequence is used for normalization, and quantitative comparative PCR, which uses a normalization gene or a housekeeping gene for RT-PCR that is contained in the sample. See, for example, Held et al. (1996) Genome Research 6:986-994.

[0076] Immunohistochemistry (IHC) IHC is the process of locating an antigen (e.g., a protein) within the cells of a tissue that binds to an antibody specific for that antigen in the tissue. The antigen-binding antibody can be bound or fused to a tag that allows its detection, for example, by visualization. In some embodiments, the tag is an enzyme that can catalyze a color reaction, such as alkaline phosphatase or horseradish peroxidase. The enzyme can be fused to the antibody or non-covalently bound, for example, using the biotin-avidin system. Alternatively, the antibody can be tagged with a fluorophore, such as fluorescein, rhodamine, DyLight Fluor, or Alexa Fluor. The antigen-binding antibody can be directly tagged, or the antigen-binding antibody itself can be recognized by a detection antibody that carries a tag. IHC can be used to detect one or more proteins. The expression of a gene product can be related to its staining intensity compared to a control level. In some embodiments, a gene product is considered to be differentially expressed if its staining changes by at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9, or 10 fold in a sample relative to a control.

[0077] Microarrays The biomarkers of the present invention can also be identified, confirmed, and / or measured using microarray technology. Thus, the expression profile biomarkers can be measured in either fresh or paraffin-embedded tumor tissues using microarray technology. In this method, the polynucleotide sequences of interest are plated or arrayed on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. The source of mRNA can be total RNA isolated from samples such as human tumors or tumor cell lines and corresponding normal tissues or cell lines. In this way, RNA can be isolated from various primary tumors or tumor cell lines. When the source of mRNA is a primary tumor, for example, mRNA can be extracted from frozen tissue samples that are routinely prepared and stored in daily clinical practice, or from archival tissue samples that are paraffin-embedded and fixed (e.g., formalin-fixed).

[0078] Microarray technology can be used to measure the expression profile of biomarkers in either fresh or paraffin-embedded tumor tissue or body fluid.In this method, the polynucleotide sequence of interest is plated or arrayed on a microchip substrate.The arrayed sequence is then hybridized with specific DNA probes from cells or tissues of interest.Similar to RT-PCR, the source of miRNA is typically the total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines, including body fluids such as serum, urine, tears and exosomes.In this way, RNA can be isolated from various sources.When the source of miRNA is primary tumor, miRNA can be extracted, for example, from frozen tissue samples that are routinely prepared and stored in daily clinical practice.

[0079] In a particular embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate of high density array.In one aspect, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or at least 50,000 nucleotide sequences are applied to the substrate.Each sequence may correspond to a different gene, or multiple sequences may be arrayed per gene.The microarrayed genes immobilized on the microchip are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated by incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip specifically hybridize to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser electron microscopy or by another detection method such as a CCD camera. Quantification of hybridization of each arrayed element allows assessment of corresponding mRNA abundance. Using dual-color fluorescence, separately labeled cDNA probes made from two RNA sources are hybridized in pairs to the array. In this way, the relative abundance of transcripts from the two sources corresponding to each specific gene is measured simultaneously. Miniaturization of hybridization allows for easy and rapid assessment of expression patterns of a large number of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts expressed in a few copies per cell and to reproducibly detect at least approximately two-fold differences in expression levels (Schena et al. (1996) Proc. Natl. Acad. Sci. USA 93(2):106-149).Microarray analysis can be performed by commercially available equipment following manufacturer's protocols, including but not limited to Affymetrix GeneChip technology (Affymetrix, Santa Clara, CA), Agilent (Agilent Technologies, Inc., Santa Clara, CA) microarray technology, or Illumina (Illumina, Inc., San Diego, CA) microarray technology.

[0080] The development of microarray methods for large-scale analysis of gene expression makes it possible to systematically search for molecular markers for cancer classification and outcome prediction in various tumor types.

[0081] In some embodiments, the Agilent Whole Human Genome Microarray Kit (Agilent Technologies, Inc., Santa Clara, Calif.) This system is capable of analyzing over 41,000 unique human genes and transcripts, all of which are publicly-annotated. The system is used according to the manufacturer's instructions.

[0082] In some embodiments, the Illumina Whole Genome DASL assay (Illumina Inc., San Diego, Calif.) is used. This system provides a method to simultaneously profile over 24,000 transcripts in a high-throughput manner from minimal RNA input from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissue sources.

[0083] Microarray expression analysis includes identifying genes or gene products that are up-regulated or down-regulated compared to a reference. Identification can be performed using statistical tests to determine the statistical significance of any differential expression observed. In some embodiments, statistical significance is determined using parametric statistical tests. Parametric statistical tests can include, for example, fractional factorial designs, analysis of variance (ANOVA), t-tests, least squares, Pearson correlation, simple linear regression, nonlinear regression, multiple linear regression, or multiple nonlinear regression. Alternatively, parametric statistical tests can include one-way analysis of variance, two-way analysis of variance, or repeated measures analysis of variance. In other embodiments, statistical significance is determined using nonparametric statistical tests. Examples include, but are not limited to, Wilcoxon signed rank test, Mann-Whitney test, Kruskal-Wallis test, Friedman test, Spearman's rank correlation coefficient, Kendall's tau analysis, and nonparametric regression tests. In some embodiments, statistical significance is determined by a p-value of less than about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001.Though the microarray system used in the method of the present invention can assay thousands of transcripts, data analysis only needs to be performed on the transcript of interest, thus reducing the problem of multiple comparisons that are inherent in performing multiple statistical tests.The p-value can also be corrected for multiple comparisons, for example, using Bonferroni correction, its improvements, or other techniques known to those skilled in the art, such as Hochberg correction, Holm-Bonferroni correction, Sidak correction, or Dunnett correction.The degree of differential expression can also be taken into consideration. For example, a gene can be considered to be differentially expressed if the expression fold change compared to the control level is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10 times different in the sample compared to the control.Differential expression takes into account both overexpression and underexpression.A gene or gene product can be considered to be upregulated or downregulated if the differential expression meets a statistical threshold, a fold change threshold, or both.For example, the criteria for identifying differential expression may include both a p-value of 0.001 and a fold change of at least 1.5-fold (upward or downward).Those skilled in the art will understand that such statistical and threshold measures can be adapted to determine differential expression by any of the molecular profiling techniques disclosed herein.

[0084] Various methods of the invention use many kinds of microarrays to detect the presence and potentially the amount of biological entities in a sample. Arrays typically contain addressable components that can detect the presence of an entity in a sample, for example, via a binding event. Microarrays include, but are not limited to, DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays, and SNP microarrays, microRNA arrays, protein microarrays, antibody microarrays, tissue microarrays, cell microarrays (also called transfection microarrays), chemical compound microarrays, and carbohydrate arrays (glycoarrays). DNA arrays typically contain addressable nucleotide sequences that can bind to sequences present in a sample. MicroRNA arrays, such as the MMChips by University of Louisville, or commercially available systems by Agilent, can be used to detect microRNA. Protein microarrays can be used to identify protein-protein interactions, including, but not limited to, identifying substrates of protein kinases, transcription factor protein activation, or to identify targets of bioactive small molecules. Protein arrays can include arrays of nucleotide sequences that bind to various protein molecules, usually antibodies, or proteins of interest. Antibody microarrays include antibodies spotted on a protein chip that are used as capture molecules to detect proteins or other biological substances from samples, for example, from cell or tissue lysate solutions. For example, for diagnostic applications, antibody arrays can be used to detect biomarkers from body fluids, such as serum or urine. Tissue macroarrays include separate tissue cores constructed in an array format to allow multiplexed histological analysis. Cell microarrays, also called transfection microarrays, include various capture agents, such as antibodies, proteins, or lipids, that can interact with cells and facilitate their capture at addressable locations. Compound microarrays include an array of compounds and can be used to detect proteins or other biological substances that bind to the compounds. Carbohydrate arrays (glycoarrays) include an array of carbohydrates and can detect proteins that bind to sugar moieties, for example.Those skilled in the art will appreciate that similar techniques or modifications can be used in accordance with the methods of the present invention.

[0085] Gene expression analysis by massively parallel signature sequencing (MPSS) The method, described by Brenner et al. (2000) Nature Biotechnology 18:630-634, is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate microbeads. First, a microbead library of DNA templates is constructed by in vitro cloning. Then, a planar array of template-containing microbeads is assembled at high density in a flow cell. The free ends of the cloned templates on each microbead are analyzed simultaneously using a fluorescence-based signature sequencing method that does not require separation of DNA fragments. The method has been shown to simultaneously and accurately provide hundreds of thousands of gene signature sequences from a cDNA library in a single run.

[0086] MPSS data has many applications. The expression level of almost all transcripts can be quantitatively measured; the abundance of the signature represents the expression level of the gene in the analyzed tissue. Quantitative methods for analyzing tag frequency and detecting differences between libraries have been published and are incorporated into the public database of SAGE™ data, and are applicable to MPSS data. The availability of complete genome sequences allows direct comparison of signatures to genome sequences, further broadening the utility of MPSS data. Because the targets of MPSS analysis are not preselected (as with microarrays), MPSS data can characterize the full complexity of the transcriptome. This is similar to sequencing millions of ESTs at once, and genome sequence data can be used so that the source of the MPSS signature can be easily identified by computational means.

[0087] Serial Analysis of Gene Expression (SAGE) Serial analysis of gene expression (SAGE) is a method that allows for the simultaneous and quantitative analysis of many gene transcripts without the need to provide individual hybridization probes for each transcript. First, short sequence tags (e.g., about 10-14 bp) are generated that contain enough information to uniquely identify a transcript, provided that the tag is derived from a unique location within each transcript. Many transcripts are then linked together to form a long continuous molecule that can be sequenced to reveal the identity of multiple tags simultaneously. By measuring the abundance of individual tags and identifying the gene that corresponds to each tag, the expression pattern of any transcript population can be quantitatively assessed. See, for example, Velculescu et al. (1995) Science 270:484-487; and Velculescu et al. (1997) Cell 88:243-51.

[0088] DNA copy number profiling Any method capable of determining the DNA copy number profile of a particular sample can be used for molecular profiling according to the present invention, as long as the resolution is sufficient to identify the biomarkers of the present invention.Those skilled in the art will recognize and can use several different platforms to evaluate whole genome copy number changes with sufficient resolution to identify the copy number of one or more biomarkers of the present invention.Some of the platforms and techniques are described in the following embodiments.

[0089] In some embodiments, copy number profile analysis involves the amplification of whole genome DNA by whole genome amplification. Whole genome amplification can use strand-displacing polymerase and random primers.

[0090] In some aspects of these embodiments, the copy number profile analysis involves hybridization of the whole genome amplified DNA with a high density array. In more specific aspects, the high density array has 5,000 or more different probes. In another specific aspect, the high density array has 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 or more different probes. In another specific aspect, each of the different probes on the array is an oligonucleotide having a length of about 15 to 200 bases. In another specific aspect, each of the different probes on the array is an oligonucleotide having a length of about 15-200, 15-150, 15-100, 15-75, 15-60, or 20-55 bases.

[0091] In some embodiments, microarrays are used to aid in the determination of copy number profiles of samples, such as cells from tumors. Microarrays typically include a plurality of oligomers (e.g., DNA or RNA polynucleotides or oligonucleotides, or other polymers) synthesized or deposited on a substrate (e.g., a glass support) in an array pattern. The support-bound oligomers are the "probes" that function to hybridize or bind to sample material (e.g., nucleic acids prepared or obtained from tumor samples) in hybridization experiments. The reverse situation is also applicable: the sample can be bound onto the microarray substrate, and the oligomer probes are present in the hybridization solution. In use, the array surface is contacted with one or more targets under conditions that promote specific high affinity binding of the target to one or more of the probes. In some settings, the sample nucleic acid is labeled with a detectable label, such as a fluorescent tag, so that the hybridized sample and probes are detectable by a scanning device. DNA array technology offers the ability to analyze DNA copy number profiles using a large number (hundreds of thousands) of different oligonucleotides. In some embodiments, the substrate used for the array is surface-derivatized glass or silica, or a polymeric membrane surface (see, for example, Z. Guo, et al., Nucleic Acids Res, 22, 5456-65 (1994); U. Maskos, EM Southern, Nucleic Acids Res, 20, 1679-84 (1992), and EM Southern, et al., Nucleic Acids Res, 22, 1368-73 (1994), each of which is incorporated herein by reference). The modification of the surface of the array substrate can be achieved by a number of techniques.For example, silica or metal oxide surfaces can be derivatized with bifunctional silanes, i.e., silanes with a first functional group that allows covalent attachment to the surface (e.g., Si-halogen or Si-alkoxy groups, as found in --SiCl3 or --Si(OCH3)3, respectively) and a second functional group that can impart the desired chemical and / or physical modification to the surface, to covalently or non-covalently attach ligands and / or polymers or monomers for biological probe arrays. Silylation derivatization and other surface derivatizations known in the art (see, for example, U.S. Patent No. 5,624,711 to Sundberg, U.S. Patent No. 5,266,222 to Willis, and U.S. Patent No. 5,137,765 to Farnsworth, each of which is incorporated herein by reference). Other steps for preparing arrays are described in U.S. Patent No. 6,649,348 to Bass et al., assigned to Agilent Corp., which discloses DNA arrays made by in situ synthesis methods.

[0092] Polymer array synthesis has also been well described in the literature, including WO 00 / 58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, No. 5,550,215, No. 5,571,639, No. 5,578,832, No. 5,593,839, No. 5,599,695, No. 5,624,711, No. 5,631,734, No. 5,795,716, No. 5,831,070, No. 5,837,832, No. 5,856,101, No. 5,858,659, No. Nos. 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846, and 6,428,752 , 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098, PCT Application Nos. PCT / US99 / 00730 (International Publication No. WO99 / 36760) and PCT / US01 / 04285 (International Publication No. WO 01 / 58593), all of which are incorporated by reference in their entirety for all purposes.

[0093] Nucleic acid arrays useful in the present invention include, but are not limited to, those commercially available under the trade name GeneChip™ from Affymetrix (Santa Clara, Calif.). Exemplary arrays are shown on the website at affymetrix.com. Another microarray supplier is Illumina, Inc. of San Diego, Calif., and exemplary arrays are shown on the website at illumina.com.

[0094] In some embodiments, the method of the present invention provides sample preparation.Depending on the microarray and experiment to be carried out, sample nucleic acid can be prepared in several ways by methods known to those skilled in the art.In some aspects of the present invention, before or at the same time as genotyping (analysis of copy number profile), sample can be amplified by several mechanisms.The most common amplification procedure used involves PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,333,675, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the sample can be amplified on an array (see, eg, US Pat. No. 6,300,070, incorporated herein by reference).

[0095] Other suitable amplification methods include ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989); Landegren et al., Science 241, 1077 (1988); and Barringer et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989); and WO88 / 10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874). (1990), and WO90 / 06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245), and nucleic acid based sequence amplification (NABSA) (see, e.g., U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,998,617, and US patent application Ser. No. 09 / 854,317, each of which is incorporated herein by reference.

[0096] Further methods of sample preparation and techniques for reducing the complexity of nucleic acid samples are described in Dong et al., Genome Research 11, 1418 (2001), U.S. Patent Nos. 6,361,947, 6,391,592, and U.S. Patent Application Nos. 09 / 916,135, 09 / 920,491 (U.S. Patent Application Publication No. 20030096235), 09 / 910,292 (U.S. Patent Application Publication No. 20030082543), and 10 / 013,598.

[0097] The method of carrying out polynucleotide hybridization assay has been fully developed in the art.The procedure and conditions of hybridization assay used in the method of the present invention vary according to application and are selected according to known general binding methods, including those mentioned in Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor, NY, 1989);Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987);Young and Davism, PNAS, 80: 1194 (1983). Methods and apparatus for performing repeat and control hybridization reactions are described in U.S. Patent Nos. 5,871,928, 5,874,219, 6,045,996, and 6,386,749 and 6,391,623, each of which is incorporated herein by reference.

[0098] The method of the present invention can also involve the signal detection of hybridization between the ligands after (and / or during) hybridization.See U.S. Patent Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, U.S. Patent Application No. 10 / 389,194, and PCT Application No. PCT / US99 / 06097 (published as WO99 / 47964), each of which is incorporated herein by reference in its entirety for all purposes.

[0099] Methods and apparatus for signal detection and intensity data processing are described, for example, in U.S. Patent Nos. 5,143,854; 5,547,839; 5,578,832; 5,631,734; 5,800,992; 5,834,758; 5,856,092; 5,902,723; 5,936,324; 5,981,956; 6,025,601; 6,090,555; Nos. 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, U.S. patent application Ser. Nos. 10 / 389,194, 60 / 493,495, and PCT application PCT / US99 / 06097 (published as WO99 / 47964), each of which is incorporated by reference in its entirety for all purposes.

[0100] Sequence analysis Molecular profiling according to the present invention includes methods of genotyping one or more biomarkers by determining whether an individual has one or more nucleotide variants (or amino acid variants) in one or more of the genes or gene products. In some embodiments, genotyping one or more genes according to the methods of the present invention can provide further basis for selecting a treatment.

[0101] The biomarkers of the present invention can be analyzed by any method useful for determining changes in nucleic acids or the proteins they encode. According to one embodiment, one skilled in the art can analyze one or more genes for variants, including deletion variants, insertion variants, frameshift variants, nonsense variants, missense variants, and splice variants.

[0102] The nucleic acid used for analyzing one or more genes can be isolated from cells in the sample according to standard methodology (Sambrook et al., 1989). The nucleic acid can be, for example, genomic DNA, or fractionated or total cell RNA, or miRNA obtained from exosomes or cell surface. When using RNA, it may be desirable to convert the RNA into complementary DNA. In one embodiment, the RNA is total cell RNA; in another embodiment, the RNA is polyA RNA; in another embodiment, the RNA is exosomal RNA. Usually, the nucleic acid is amplified. Depending on the format of the assay for analyzing one or more genes, the specific nucleic acid of interest is identified in the sample directly using amplification, or is identified after amplification using a second known nucleic acid. The identified product is then detected. In some applications, detection can be performed by visual means (e.g., ethidium bromide staining of gel). Alternatively, detection may involve indirect identification of the product by chemiluminescence, radioactive scintigraphy of radiolabels, or fluorescent labels, or even by systems that employ electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).

[0103] Various types of defects are known to occur in the biomarkers of the present invention. Changes include, but are not limited to, deletions, insertions, point mutations, and duplications. Point mutations may be silent or may result in stop codons, frameshift mutations, or amino acid substitutions. Mutations may occur inside and outside the coding region of one or more genes and can be analyzed according to the methods of the present invention. Target sites in the nucleic acid of interest may include regions of sequence variation. Examples include, but are not limited to, polymorphisms that exist in different forms such as single nucleotide changes, nucleotide repeats, multiple base deletions (two or more nucleotides deleted from a consensus sequence), multiple base insertions (two or more nucleotides inserted from a consensus sequence), microsatellite repeats (a small number of nucleotide repeats, typically with 5-1000 repeat units), dinucleotide repeats, trinucleotide repeats, sequence rearrangements (including translocations and duplications), chimeric sequences (two sequences from different genetic origins are fused together), and the like. Among sequence polymorphisms, the most frequent polymorphisms in the human genome are single base changes, also called single nucleotide polymorphisms (SNPs). SNPs are abundant, stable and widely distributed throughout the genome.

[0104] Molecular profiling includes methods for identifying the haplotype of one or more genes. A haplotype is a set of genetic determinants located on a single chromosome, and typically includes a specific combination of alleles (all alternative sequences of a gene) in a region of a chromosome. In other words, a haplotype is the topological sequence information of an individual chromosome. Very often, topological SNPs on a chromosome define a haplotype. The combination of haplotypes on a chromosome can determine the genetic profile of a cell. It is the haplotype that determines the association between a specific genetic marker and a disease mutation. Haplotyping can be performed by any method known in the art. Common methods for scoring SNPs include hybridization microarrays or direct gel sequencing, as outlined in Landgren et al., Genome Research, 8:769-776, 1998. For example, only one copy of one or more genes can be isolated from an individual, and the nucleotides at each of the variant positions are determined. Alternatively, allele-specific PCR or similar methods can be used to amplify only one copy of one or more genes in an individual, and determine the SNPs at the variant positions of the present invention.For haplotyping, the Clark method known in the art can also be used.High-throughput molecular haplotyping methods are also disclosed in Tost et al., Nucleic Acids Res., 30(19):e96 (2002), which is incorporated herein by reference.

[0105] Thus, as will be apparent to those skilled in the art of genetics and haplotyping, additional variants in linkage disequilibrium with the variants and / or haplotypes of the invention can be identified by haplotyping methods known in the art. Additional variants in linkage disequilibrium with the variants and / or haplotypes of the invention may also be useful in various applications described below.

[0106] For genotyping and haplotyping purposes, both genomic DNA and mRNA / cDNA can be used, both of which are generally referred to herein as "genes."

[0107] Many techniques for detecting nucleotide variants are known in the art, and any can be used for the method of the present invention. The technique can be protein-based or nucleic acid-based. In either case, the technique used must be sensitive enough to accurately detect small nucleotide or amino acid changes. Most often, a probe labeled with a detectable marker is utilized. Unless otherwise specified in the specific technique described below, any suitable marker known in the art can be used, including but not limited to radioisotopes, fluorescent compounds, biotin detectable with streptavidin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands, and antibodies. See Jablonski et al., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al., Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977).

[0108] In nucleic acid-based detection methods, a target DNA sample, i.e., a sample containing genomic DNA, cDNA, mRNA, and / or miRNA corresponding to one or more genes, must be taken from the individual to be tested. Any tissue or cell sample containing genomic DNA, miRNA, mRNA, and / or cDNA (or parts thereof) corresponding to one or more genes can be used. For this purpose, a tissue sample containing cell nuclei and thus genomic DNA can be taken from the individual. A blood sample can also be useful, except that only white blood cells and other lymphocytes have cell nuclei, while red blood cells do not have nuclei and contain only mRNA or miRNA. Nevertheless, miRNA and mRNA are also useful, since both can be analyzed for the presence of nucleotide variants of their sequences or can serve as templates for cDNA synthesis. Tissue or cell samples can be analyzed directly with little processing. Alternatively, the nucleic acid containing the target sequence can be extracted, purified, and / or amplified before being subjected to the various detection procedures discussed below. In addition to tissue or cell samples, cDNA or genomic DNA derived from a cDNA or genomic DNA library constructed using tissue or cell samples taken from the individual to be tested is also useful.

[0109] Sequence analysis To determine the presence or absence of a particular nucleotide variant, the target genomic DNA or cDNA is sequenced, particularly the region that encompasses the nucleotide variant locus to be detected. Various sequencing techniques, including Sanger and Gilbert chemistry, are generally known and widely used in the art. Pyrosequencing uses a luminescence detection system to monitor DNA synthesis in real time. Pyrosequencing has been shown to be effective in analyzing genetic polymorphisms such as single nucleotide polymorphisms, and can be used in the present invention. See Nordstrom et al., Biotechnol. Appl. Biochem., 31(2):107-112 (2000); Ahmadian et al., Anal. Biochem., 280:103-110 (2000).

[0110] Nucleic acid variants can be detected by an appropriate detection process. Non-limiting examples of methods for detection, quantification, sequencing, etc. include mass detection of mass-modified amplicons (e.g., matrix-assisted laser desorption / ionization (MALDI) mass spectrometry and electrospray (ES) mass spectrometry), primer extension methods (e.g., iPLEX™; Sequenom, Inc.), microsequencing methods (e.g., improvements on primer extension methodology), ligase sequencing (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 2005 / 023363), and methods for detecting, quantifying, sequencing, and the like. 01 / 27326), mismatch sequencing (e.g., U.S. Patent Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), direct DNA sequencing, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blots, GeneChip microarrays, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, TaqMan, molecular beacons, intercalating dyes, FRET primers, AlphaScreen, SNPstream, gene bit analysis (GBA), multiplex minisequencing, S NaPshot, GOOD assay, microarray minisequencing, arrayed primer extension (APEX), microarray primer extension (e.g., microarray sequencing), tag arrays, coded microspheres, template-dependent incorporation (TDI), fluorescence polarization, colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probes, Invader assay, hybridization methods (e.g., hybridization with at least one probe, hybridization with at least one fluorescently labeled probe, etc.), conventional dot blot analysis, single-strand conformation polymorphism analysis (SSCP, e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499; Orita et al., Proc. Natl. Acad. Sci. USA86: 27776-2770 (1989)), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and the techniques described in Sheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706 (1991), White et al., Genomics 12: 301-306 (1992), Grompe et al., Proc. Natl. Acad. Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics 5: 111-117 (1993), cloning and sequencing, electrophoresis, the use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), digital PCR, nanopore sequencing, chips, and combinations thereof. Detection and quantification of alleles or paralogs can be performed using the "closed tube" methods described in U.S. Patent Application Serial No. 11 / 950,395, filed December 4, 2007. In some embodiments, the amount of a nucleic acid species is measured by mass spectrometry, primer extension, sequencing (e.g., any suitable method, e.g., nanopore or pyrosequencing), quantitative PCR (Q-PCR or QRT-PCR), digital PCR, combinations thereof, and the like.

[0111] The term "sequence analysis" as used herein refers to determining a nucleotide sequence, e.g., the nucleotide sequence of an amplification product. The entire sequence or a partial sequence of a polynucleotide, e.g., DNA or mRNA, can be determined, and the determined nucleotide sequence can be referred to as a "read" or a "sequence read". For example, in some embodiments, the linear amplification product can be directly analyzed without further amplification (e.g., by using single molecule sequencing methodology). In certain embodiments, the linear amplification product can be subjected to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing methodology). The read can be subjected to various types of sequence analysis. Any suitable sequencing method can be utilized to detect and measure the amount of nucleotide sequence species, amplified nucleic acid species, or detectable products generated from the above. Examples of specific sequencing methods are described below.

[0112] Sequencing devices or components include devices that can be used by those skilled in the art to determine the nucleotide sequence (e.g., linear and / or exponential amplification products) resulting from the process described herein, and one or more components that are used with such devices. Examples of sequencing platforms include, but are not limited to, 454 platform (Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), Illumina Genomic Analyzer (or Solexa platform) or SOLID system (Applied Biosystems) or Helicos True Single Molecule DNA sequencing technology (Harris TD et al. 2008 Science, 320, 106-109), Pacific Biosciences' Single Molecule Real Time (SMRT™) technology, and nanopore sequencing (Soni GV and Meller A. 2007 Clin Chem 53: 1996-2001). Such platforms allow for highly multiplexed sequencing of many nucleic acid molecules isolated from a specimen in a parallel manner (Dear Brief Funct Genomic Proteomic 2003; 1: 397-416). Each of these platforms allows for single molecule sequencing of clonally propagated or unamplified nucleic acid fragments. Certain platforms involve, for example, sequencing by ligation of dye-modified probes (including cyclic ligation and cleavage), pyrosequencing, and single molecule sequencing. Nucleotide sequence species, amplified nucleic acid species, and detectable products generated therefrom can be analyzed by such sequence analysis platforms.

[0113] Sequencing by ligation is a nucleic acid sequencing method that relies on the sensitivity of DNA ligase to base-pairing mismatches. DNA ligase ligates DNA ends that are correctly base-paired. The ability of DNA ligase to ligate only DNA ends that are correctly base-paired, combined with a mixed pool of fluorescently labeled oligonucleotides or primers, allows for sequencing by fluorescence detection. Longer sequence reads can be obtained by including primers that contain a cleavable bond that can be cleaved after label identification. Cleavage at the linker position removes the label and regenerates the 5' phosphate on the end of the ligated primer, preparing the primer for further rounds of ligation. In some embodiments, the primer can be labeled with more than one fluorescent label, for example, at least 1, 2, 3, 4, or 5 fluorescent labels.

[0114] Sequencing by ligation generally involves the following steps: A clonal bead population can be prepared in an emulsion microreactor containing the target nucleic acid template sequence, amplification reaction components, beads, and primers. After amplification, the templates are denatured and bead enrichment is performed to separate beads with extended templates from undesired beads (e.g., beads with non-extended templates). The templates on selected beads are 3' modified to allow covalent attachment to the slide, and the modified beads can be deposited on a glass slide. The deposition chamber provides the ability to divide the slide into 1, 4, or 8 chambers during the bead loading process. For sequence analysis, primers hybridize to the adapter sequence. A set of probes labeled with four dyes compete for ligation to the sequencing primer. Specificity of probe ligation is achieved by interrogating every fourth and fifth base during a series of ligations. The color at every fifth position is recorded over 5-7 rounds of ligation, detection, and cleavage, with the number of rounds determined by the type of library used. After each round of ligation, a new complementary primer is positioned 1 base in the 5' direction for further rounds of ligation. Five consecutive rounds of primer reset and ligation (5-7 ligation cycles per round) are repeated to generate 25-35 base pairs of sequence for a single tag. In the case of mate pair sequencing, the process is repeated for the second tag.

[0115] Pyrosequencing is a nucleic acid sequencing method based on sequencing by synthesis that relies on the detection of pyrophosphate released upon nucleotide incorporation. In general, sequencing by synthesis involves synthesizing one nucleotide at a time on a DNA strand complementary to the strand whose sequence is being sought. The target nucleic acid can be immobilized on a solid support, hybridized with a sequencing primer, and incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate, and luciferin. Nucleotide solutions are added and removed sequentially. Correct incorporation of a nucleotide releases pyrophosphate, which reacts with ATP sulfurylase in the presence of adenosine 5' phosphosulfate to generate ATP and stimulate the luciferin reaction, which generates a chemiluminescent signal to allow sequencing. The amount of light produced is proportional to the number of bases added. The sequence downstream of the sequencing primer can then be determined. An exemplary system for pyrosequencing involves: ligating an adapter nucleic acid to the nucleic acid under investigation and hybridizing the resulting nucleic acid to beads; amplifying the nucleotide sequence in emulsion; sorting the beads using a picoliter multi-well solid support; and sequencing the amplified nucleotide sequence by pyrosequencing methodology (e.g., Nakano et al., "Single-molecule PCR using water-in-oil emulsion;" Journal of Biotechnology 102: 117-124 (2003)).

[0116] Certain single molecule sequencing embodiments are based on the principle of sequencing by synthesis and utilize single-pair fluorescence resonance energy transfer (single-pair FRET) as the mechanism by which photons are emitted as a result of successful nucleotide incorporation. The emitted photons are often detected using a sensitized or highly sensitive cooled charge-coupled device in conjunction with a total internal reflection microscope (TIRM). A photon is emitted only if the introduced reaction solution contains the correct nucleotide for incorporation into the elongated nucleic acid strand that is synthesized as a result of the sequencing process. In FRET-based single molecule sequencing, energy is transferred via long-range dipole interactions between two fluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5. The donor is excited at its specific excitation wavelength and the excited state energy is non-radiatively transferred to the acceptor dye, which is in turn excited. The acceptor dye finally returns to the ground state by the radiative emission of a photon. The two dyes used in the energy transfer process represent the "single pair" in single-pair FRET. Cy3 is often used as the donor fluorophore and is often incorporated as the first labeled nucleotide. Cy5 is often used as the acceptor fluorophore and is used as the nucleotide label for subsequent nucleotide additions after incorporation of the first Cy3-labeled nucleotide. The fluorophores are typically within 10 nanometers of each other to allow for good energy transfer.

[0117] An example of a system that can be used based on single molecule sequencing generally involves hybridizing a primer to a target nucleic acid sequence to form a complex; binding the complex to a solid phase; repeatedly extending the primer with nucleotides tagged with fluorescent molecules; and taking an image of the fluorescence resonance energy transfer signal after each interaction (e.g., U.S. Pat. No. 7,169,314; Braslavsky et al., PNAS 100(7): 3960-3964 (2003)). Such a system can be used to directly sequence the amplification products (linear amplification products or exponential amplification products) produced by the process described herein. In some embodiments, the amplification products can be hybridized to a primer that contains a sequence complementary to an immobilized capture sequence present on a solid support, such as a bead or a glass slide. Hybridization of the primer-amplification product complex with the immobilized capture sequence immobilizes the amplification product on the solid support for single pair FRET-based sequencing by synthesis. Primers are often fluorescent so that an initial reference image of the slide surface on which the nucleic acids are immobilized can be made. The initial reference image is useful for determining the positions where true nucleotide incorporation occurs. Fluorescent signals detected at array positions not initially identified in the "primer only" reference image are discarded as non-specific fluorescence. After immobilization of the primer-amplification product complex, the bound nucleic acids are often sequenced in parallel by repeated steps of a) polymerase extension in the presence of one fluorescently labeled nucleotide, b) detection of fluorescence using an appropriate microscope, e.g., TIRM, c) removal of the fluorescent nucleotide, and d) restoration to step a with a different fluorescently labeled nucleotide.

[0118] In some embodiments, nucleotide sequencing may be by solid-phase single nucleotide sequencing method and process. Solid-phase single nucleotide sequencing method involves contacting a target nucleic acid with a solid support under conditions in which a single molecule of sample nucleic acid hybridizes to a single molecule of solid support. Such conditions may include providing a solid support molecule and a single molecule of target nucleic acid in a "microreactor". Such conditions may also include providing a mixture in which a target nucleic acid molecule can hybridize to a solid phase nucleic acid on a solid support. Single nucleotide sequencing methods useful in the embodiments described herein are described in U.S. Provisional Patent Application No. 61 / 021,871, filed January 17, 2008.

[0119] In certain embodiments, the nanopore sequencing detection method includes: (a) contacting a sequence-specific detector with a detector under conditions in which the detector specifically hybridizes to the substantially complementary sequence of the target nucleic acid for sequencing ("basic nucleic acid", e.g., a linked probe molecule); (b) detecting a signal from the detector; and (c) determining the sequence of the basic nucleic acid according to the detected signal. In certain embodiments, when the detector interferes with the nanopore structure when the basic nucleic acid passes through the hole, the detector hybridized to the basic nucleic acid is dissociated from the basic nucleic acid (e.g., dissociated successively), and the detector dissociated from the basic nucleic acid is detected. In some embodiments, the detector dissociated from the basic nucleic acid emits a detectable signal, and the detector hybridized to the basic nucleic acid emits a different detectable signal or does not emit a detectable signal. In certain embodiments, nucleotides in a nucleic acid (e.g., a ligated probe molecule) are replaced by a specific nucleotide sequence ("nucleotide representative") corresponding to a specific nucleotide, thereby generating an extended nucleic acid (e.g., U.S. Pat. No. 6,723,513), and a detector hybridizes to the nucleotide representative in the extended nucleic acid, which serves as a base nucleic acid. In such embodiments, the nucleotide representatives can be arranged in a binary or higher order configuration (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001 (2007)). In some embodiments, the nucleic acid is not ligated to generate an extended nucleic acid, but serves directly as a base nucleic acid (e.g., a ligated probe molecule serves as a non-extended base nucleic acid), and a detector is directly contacted with the base nucleic acid. For example, a first detector can hybridize to a first subsequence and a second detector can hybridize to a second subsequence, where the first and second detector each have a detectable label that can be distinguished from one another, and the signals from the first and second detector can be distinguished from one another when they are dissociated from the base nucleic acid.In certain embodiments, the detector comprises a region (e.g., two regions) that hybridizes to the base nucleic acid, which may be about 3 to about 100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 nucleotides in length). The detector may also comprise one or more regions of nucleotides that do not hybridize to the base nucleic acid. In some embodiments, the detector is a molecular beacon. The detector often comprises one or more detectable labels independently selected from those described herein. Each of the detectable labels can be detected by any convenient detection process that can detect the signal (e.g., magnetic, electrical, chemical, optical, etc.) generated by each label. For example, a CD camera can be used to detect signals from one or more distinguishable quantum dots linked to the detector.

[0120] In certain sequence analysis embodiments, the reads can be used to construct larger nucleotide sequences, which can be facilitated by identifying overlapping sequences in different reads and using the identified sequences in the reads. Such sequence analysis methods and software for constructing larger sequences from reads are known to those skilled in the art (e.g., Venter et al., Science 291: 1304-1351 (2001)). In certain sequence analysis embodiments, specific reads, partial nucleotide sequence constructs, and complete nucleotide sequence constructs can be compared between nucleotide sequences within a sample nucleic acid (i.e., internal comparisons) or compared to a reference sequence (i.e., reference comparisons). Internal comparisons can be performed when sample nucleic acids are prepared from multiple samples or from a single sample source that contains sequence variations. Reference comparisons are optionally performed when a reference nucleotide sequence is known and the purpose is to determine whether the sample nucleic acid contains a nucleotide sequence that is substantially similar or the same as the reference nucleotide sequence, or contains a nucleotide sequence that differs from the reference nucleotide sequence. Sequence analysis can be facilitated by using the sequence analysis devices and components described above.

[0121] Primer extension polymorphism detection methods, also referred to herein as "microsequencing" methods, are typically carried out by hybridizing complementary oligonucleotides to the nucleic acid carrying polymorphic sites.In these methods, the oligonucleotides typically hybridize adjacent to the polymorphic sites.The term "adjacent" when used in relation to "microsequencing" methods refers to the 3' end of the extension oligonucleotide, which is sometimes 1 nucleotide from the 5' end of the polymorphic site, often 2 or 3 nucleotides from the 5' end of the polymorphic site, and sometimes 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polymorphic site when the extension oligonucleotide is hybridized to the nucleic acid.The extension oligonucleotide is then extended by one or more nucleotides, often by one, two, or three nucleotides, and the number and / or type of nucleotides added to the extension oligonucleotide determine which one or more polymorphic variants are present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01 / 20039. The extension products can be detected in any manner, such as by fluorescence methods (see, e.g., Chen & Kwok, Nucleic Acids Research 25: 347-353 (1997), and Chen et al., Proc. Natl. Acad. Sci. USA 94 / 20: 10756-10761 (1997)), or by mass spectrometry (e.g., MALDI-TOF mass spectrometry) and other methods described herein.Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Patent Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; 6,194,144; and 6,258,538. Microsequencing detection methods often incorporate an amplification process that proceeds through an extension step. The amplification process typically amplifies a region from a nucleic acid sample that contains a polymorphic site. Amplification can be performed using the above method or, for example, in polymerase chain reaction (PCR), using a pair of oligonucleotide primers, one of which is typically complementary to the region 3' of the polymorphism and the other is typically complementary to the region 5' of the polymorphism. PCR primer pairs can be used in the methods disclosed in, for example, U.S. Patent Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01 / 27327; and WO 01 / 27329. PCR primer pairs can also be used in any commercially available machine that performs PCR, such as any of the GeneAmp™ systems available from Applied Biosystems.

[0122] Other suitable sequencing methods include multiplex polony sequencing (described in Shendure et al., Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome, Sciencexpress, Aug. 4, 2005, pg 1, available at www.sciencexpress.org / 4 Aug. 2005 / Page1 / 10.1126 / science.1117389, incorporated herein by reference), which uses immobilized microbeads, and sequencing in microfabricated picoliter reactors (described in Margulies et al., Genome Sequencing in Microfabricated High-Density Picolitre Reactors, Nature, August 2005, available at www.nature.com / nature (published online 31 Jul. 2005, doi:10.1038 / nature03959, incorporated herein by reference).

[0123] In some embodiments, whole genome sequencing can be used to distinguish alleles of RNA transcripts. Examples of whole genome sequencing methods include, but are not limited to, the nanopore-based sequencing methods described above, sequencing by synthesis, and sequencing by ligation.

[0124] In situ hybridization In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987). In an in situ hybridization assay, cells, for example from a biopsy specimen, are immobilized on a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at moderate temperature to allow annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters. FISH (fluorescence in situ hybridization) uses fluorescent probes that bind only to parts of sequences with which they show a high degree of sequence similarity.

[0125] FISH is a cytogenetic technique used to detect and localize specific polynucleotide sequences in cells. For example, FISH can be used to detect DNA sequences on chromosomes. FISH can also be used to detect and localize specific RNA, such as mRNA, in tissue samples. FISH uses fluorescent probes that bind to specific nucleotide sequences with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out whether and where the fluorescent probes are bound. In addition to detecting specific nucleotide sequences, such as translocations, fusions, breaks, duplications, and other chromosomal abnormalities, FISH can also help define specific gene copy numbers and / or spatial-temporal patterns of gene expression in cells and tissues.

[0126] Comparative genomic hybridization (CGH) uses in situ hybridization kinetics to compare the copy numbers of different DNA or RNA sequences from a sample, or to compare the copy numbers of different DNA or RNA in one sample with the copy numbers of substantially identical sequences in another sample. In many useful applications of CGH, DNA or RNA is isolated from a subject cell or cell population. The comparison can be qualitative or quantitative. Procedures have been described that allow the determination of absolute copy numbers of DNA sequences throughout the genome of a cell or cell population, where the absolute copy numbers are known or determined for one or several sequences. Different sequences are distinguished from each other by the different locations of their binding sites when hybridized to a reference genome, which is usually a metaphase chromosome, but sometimes an interphase nucleus. Copy number information is derived from a comparison of the intensity of hybridization signals between different locations on the reference genome. Methods, techniques, and applications of CGH are known, as described in US Pat. No. 6,335,167 and US patent application Ser. No. 60 / 804,818, the relevant portions of which are incorporated herein by reference.

[0127] Other sequencing methods Nucleic acid variants can also be detected using standard electrophoretic techniques. The detection step can be preceded by an amplification step in some cases, but amplification is not necessary in the embodiments described herein. Examples of methods for detecting and quantifying nucleic acids using electrophoretic techniques can be found in the art. Non-limiting examples include running a sample (e.g., a mixed nucleic acid sample isolated from maternal serum, or, for example, an amplified nucleic acid species) in an agarose or polyacrylamide gel. The gel can be labeled (e.g., stained) with ethidium bromide stain (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001). The presence of a band of the same size as a standard control is indicative of the presence of a target nucleic acid sequence, the amount of which can then be compared to the control based on the intensity of the band, and thus the target sequence of interest can be detected and quantified. In some embodiments, a restriction enzyme that can distinguish between maternal and paternal alleles can be used to detect and quantify target nucleic acid species. In certain embodiments, oligonucleotide probes specific to a target sequence of interest are used to detect the presence of the target sequence of interest. The oligonucleotides can also be used to indicate the amount of a target nucleic acid molecule compared to a standard control based on the intensity of the signal provided by the probe.

[0128] Sequence-specific probe hybridization can be used to detect specific nucleic acid in a mixture or mixed population that contains other nucleic acid species.Under sufficiently stringent hybridization conditions, the probe specifically hybridizes only to substantially complementary sequences.The stringency of hybridization conditions can be relaxed to allow various amounts of sequence mismatch.Several hybridization formats are known in the art, including but not limited to liquid phase, solid phase, or mixed phase hybridization assays. The following articles provide overviews of various hybridization assay formats: Singer et al., Biotechniques 4:230, 1986; Haase et al., Methods in Virology, pp. 189-226, 1984; Wilkinson, In situ Hybridization, Wilkinson ed., IRL Press, Oxford University Press, Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: A Practical Approach, IRL Press, 1987.

[0129] Hybridization complexes can be detected by techniques known in the art. A nucleic acid probe capable of specifically hybridizing to a target nucleic acid (e.g., mRNA or DNA) can be labeled by any suitable method, and the labeled probe can be used to detect the presence of the hybridized nucleic acid. One commonly used detection method is 3 H, 125 I, 35 S, 14 C. 32 P, or 33The radioisotope is autoradiography using a probe labeled with P or the like. The choice of radioisotope depends on the research preference due to the ease of synthesis, stability, and half-life of the selected isotope. Other labels include compounds that bind to anti-ligands or antibodies labeled with fluorophores, chemiluminescent agents, and enzymes (e.g., biotin and digoxigenin). In some embodiments, the probe can be directly conjugated with a label such as a fluorophore, chemiluminescent agent, or enzyme. The choice of label depends on the required sensitivity, ease of conjugation with the probe, stability requirements, and available equipment.

[0130] Alternatively, restriction fragment length polymorphism (RFLP) and AFLP methods can be used for molecular profiling. If the nucleotide variants in the target DNA corresponding to one or more genes cause the disappearance or creation of restriction enzyme recognition sites, the digestion of the target DNA with a specific restriction enzyme will produce altered restriction fragment length patterns. Therefore, the detected RFLP or AFLP will indicate the presence of a specific nucleotide variant.

[0131] Another useful approach is single-strand conformation polymorphism assay (SSCA), which is based on the mobility change of single-stranded target DNA across the nucleotide variant of interest.A single nucleotide change in the target sequence can result in different intramolecular base pairing patterns and thus different secondary structures of single-stranded DNA, which can be detected in non-denaturing gels.See Orita et al., Proc. Natl. Acad. Sci. USA, 86:2776-2770 (1989).Denaturing gel-based techniques such as denaturing concentration fixed gel electrophoresis (CDGE) and denaturing concentration gradient gel electrophoresis (DGGE) detect the difference in the migration rate of mutant sequences compared to wild-type sequences in denaturing gels. See Miller et al., Biotechniques, 5:1016-24 (1999);Sheffield et al., Am. J. Hum. Genet., 49:699-706 (1991);Wartell et al., Nucleic Acids Res., 18:2699-2705 (1990);and Sheffield et al., Proc. Natl. Acad. Sci. USA, 86:232-236 (1989).In addition, double-stranded conformation analysis (DSCA) can also be useful in the present invention.See Arguello et al., Nat. Genet., 18:192-194 (1998).

[0132] The presence or absence of nucleotide variants at a particular locus in one or more genes of an individual can also be detected using the amplification resistance mutation system (ARMS) technique.See European Patent No. 0,332,435;Newton et al., Nucleic Acids Res., 17:2503-2515 (1989);Fox et al., Br. J. Cancer, 77:1267-1274 (1998);Robertson et al., Eur. Respir. J., 12:477-482 (1998).In the ARMS method, a primer is synthesized that matches the nucleotide sequence immediately 5' upstream from the locus to be tested, except that the 3' terminal nucleotide corresponding to the nucleotide at the locus is a predetermined nucleotide.For example, the 3' terminal nucleotide can be identical to the nucleotide at the mutant locus. The primer may be of any suitable length, so long as it hybridizes to the target DNA under stringent conditions only if its 3' terminal nucleotide matches the nucleotide at the locus being tested. Preferably, the primer has at least 12 nucleotides, more preferably about 18-50 nucleotides. If the tested individual has a mutation at the locus, in which a nucleotide matches the 3' terminal nucleotide of the primer, the primer can be further extended when hybridized to the target DNA template, and the primer can initiate a PCR amplification reaction with another suitable PCR primer. In contrast, if the nucleotide at the locus is wild-type, no primer extension can be achieved. Various forms of ARMS techniques developed in the past few years can be used. See, for example, Gibson et al., Clin. Chem. 43:1336-1341 (1997).

[0133] Similar to the ARMS technique is mini-sequencing or single nucleotide primer extension method, which is based on the incorporation of a single nucleotide.An oligonucleotide primer that matches the nucleotide sequence immediately 5' of the locus to be tested is hybridized to target DNA, mRNA or miRNA in the presence of labeled dideoxyribonucleotide.Only if the dideoxyribonucleotide matches the nucleotide of the variant locus to be detected, the labeled nucleotide is incorporated or linked to the primer.Therefore, the identity of the nucleotide of the variant locus can be revealed based on the detection label attached to the incorporated dideoxyribonucleotide.See Syvanen et al., Genomics, 8:684-692 (1990);Shumaker et al., Hum. Mutat., 7:346-354 (1996);Chen et al., Genome Res., 10:549-547 (2000).

[0134] Another set of techniques that are useful in the present invention is the so-called "oligonucleotide ligation assay" (OLA), in which the discrimination between wild-type locus and mutation is based on the ability of two oligonucleotides to anneal close to each other on a target DNA molecule, so that the two oligonucleotides are ligated together by DNA ligase.See Landergren et al., Science, 241:1077-1080 (1988);Chen et al, Genome Res., 8:549-556 (1998);Iannone et al., Cytometry, 39:131-140 (2000).Thus, for example, to detect a single nucleotide mutation at a specific locus in one or more genes, two oligonucleotides can be synthesized, one with a sequence immediately 5' upstream from the locus and whose 3' terminal nucleotide is identical to the nucleotide at the variant locus of the specific gene, and the other with a nucleotide sequence that matches the sequence immediately 3' downstream from the locus of the gene.The oligonucleotides can be labeled for detection purposes. When hybridized to the target gene under stringent conditions, the two oligonucleotides are subjected to ligation in the presence of a suitable ligase. Ligation of the two oligonucleotides indicates that the target DNA has a nucleotide variant at the locus to be detected.

[0135] Detection of subtle genetic changes can also be achieved by various hybridization-based approaches. Allele-specific oligonucleotides are most useful. See Conner et al., Proc. Natl. Acad. Sci. USA, 80:278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989). Oligonucleotide probes (allele-specific) that specifically hybridize to alleles with a particular gene variant at a particular locus, but not to other alleles, can be designed by methods known in the art. The probes can have lengths of, for example, 10 to about 50 nucleotide bases. The target DNA and the oligonucleotide probes can be contacted with each other under sufficiently stringent conditions so that the nucleotide variants can be distinguished from the wild-type gene based on the presence or absence of hybridization. The probes can be labeled to provide a detection signal. Alternatively, allele-specific oligonucleotide probes can be used as PCR amplification primers in "allele-specific PCR," and the presence or absence of a PCR product of the predicted length indicates the presence or absence of a particular nucleotide variant.

[0136] Other useful hybridization-based techniques anneal two single-stranded nucleic acids together, even when mismatches exist due to nucleotide substitution, insertion, or deletion. Various techniques can then be used to detect mismatches. For example, the annealed duplexes can be subjected to electrophoresis. Mismatched duplexes can be detected based on their electrophoretic mobility, which differs from that of perfectly matched duplexes. See Cariello, Human Genetics, 42:726 (1988). Alternatively, in RNase protection assays, RNA probes can be prepared that span the nucleotide variant to be detected and have a detection marker. See Giunta et al., Diagn. Mol. Path., 5:265-270 (1996); Finkelstein et al., Genomics, 7:167-172 (1990); Kinszler et al., Science 251:1366-1370 (1991). The RNA probe can be hybridized with target DNA or mRNA to form a heteroduplex, which is then subjected to ribonuclease RNase A digestion. RNase A digests the RNA probe in the heteroduplex only at the site of mismatch. Digestion can be determined on a denaturing electrophoretic gel based on size change. In addition, mismatch can also be detected by chemical cleavage methods known in the art. For example, see Roberts et al., Nucleic Acids Res., 25:3377-3378 (1997).

[0137] In the mutS assay, a probe can be prepared that matches the gene sequence surrounding the locus where the presence or absence of a mutation is to be detected, except that a certain nucleotide is used at the variant locus. The probe is annealed to the target DNA to form a duplex, and the mutS protein of E. coli is contacted with the duplex. The binding of the mutS protein indicates the presence of a mutation, since the mutS protein only binds to heteroduplex sequences that contain nucleotide mismatches. See Modrich et al., Ann. Rev. Genet., 25:229-253 (1991).

[0138] Based on the above basic technique, which may be useful for detecting mutations or nucleotide variants in the present invention, a wide variety of improvements and modifications have been developed in the art. For example, "sunrise probe" or "molecular beacon" uses fluorescence resonance energy transfer (FRET) properties, resulting in high sensitivity. See Wolf et al., Proc. Nat. Acad. Sci. USA, 85:8790-8794 (1988). Typically, the probe that spans the nucleotide locus to be detected is designed to have a hairpin structure, with one end labeled with a quenching fluorophore and the other end labeled with a reporter fluorophore. In its natural state, one fluorophore is in close proximity to the other fluorophore, so the fluorescence from the reporter fluorophore is quenched by the quenching fluorophore. When the probe hybridizes to the target DNA, the 5' end is separated away from the 3' end, thus regenerating the fluorescent signal. See Nazarenko et al., Nucleic Acids Res., 25:2516-2521 (1997);Rychlik et al., Nucleic Acids Res., 17:8543-8551 (1989);Sharkey et al., Bio / Technology 12:506-509 (1994);Tyagi et al., Nat. Biotechnol., 14:303-308 (1996);Tyagi et al., Nat. Biotechnol., 16:49-53 (1998). To suppress the accumulation of primer dimers, the homotag-assisted non-dimer system (HANDS) can be used in conjunction with the molecular beacon method. See Brownie et al., Nucleic Acids Res., 25:3235-3241 (1997).

[0139] Dye-labeled oligonucleotide ligation assay is a FRET-based method that combines OLA assay with PCR.See Chen et al., Genome Res. 8:549-556 (1998).TaqMan is another FRET-based method for detecting nucleotide variants.TaqMan probes can be oligonucleotides that have the nucleotide sequence of a gene that spans the variant locus of interest and are designed to differentially hybridize with different alleles.The two ends of the probe are labeled with a quenching fluorophore and a reporter fluorophore, respectively.The TaqMan probe is incorporated into a PCR reaction that uses Taq polymerase to amplify the target gene region that includes the locus of interest. Taq polymerase exhibits 5'-3' exonuclease activity but does not have 3'-5' exonuclease activity, so when TaqMan probe is annealed to target DNA template, the 5' end of TaqMan probe is degraded by Taq polymerase during PCR reaction, thus separating reporter fluorophore from quencher fluorophore, and releasing fluorescent signal.See Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280 (1991);Kalinina et al., Nucleic Acids Res., 25:1999-2004 (1997);Whitcombe et al., Clin. Chem., 44:918-923 (1998).

[0140] In addition, the detection in the present invention can also use chemiluminescence-based techniques. For example, an oligonucleotide probe can be designed to hybridize to either wild-type or variant locus, but not to both. The probe is labeled with a highly chemiluminescent acridinium ester. Hydrolysis of the acridinium ester impairs chemiluminescence. Hybridization of the probe to target DNA prevents hydrolysis of the acridinium ester. Thus, the presence or absence of a specific mutation in target DNA is determined by measuring the change in chemiluminescence. See Nelson et al., Nucleic Acids Res., 24:4998-5003 (1996).

[0141] The detection of genetic changes in genes according to the present invention may be based on the "base excision sequence scanning" (BESS) technique. The BESS method is a PCR-based mutation scanning method. BESS T-Scan and BESS G-Tracker, which are similar to the T and G ladder of dideoxy sequencing, are produced. Mutations are detected by comparing the sequences of normal and mutant DNA. See, for example, Hawkins et al., Electrophoresis, 20:1171-1176 (1999).

[0142] Mass spectrometry can be used for molecular profiling according to the present invention. See Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998). For example, in the primer oligobase extension (PROBE™) method, the target nucleic acid is immobilized on a solid support. A primer is annealed to the target immediately 5' upstream from the locus to be analyzed. Primer extension is carried out in the presence of a selected mixture of deoxyribonucleotides and dideoxyribonucleotides. The resulting mixture of newly extended primers is then analyzed by MALDI-TOF. See, for example, Monforte et al., Nat. Med., 3:360-362 (1997).

[0143] In addition, microchip or microarray technology can also be applied to the detection method of the present invention.Essentially, in microchip, a large number of different oligonucleotide probes are immobilized on a substrate or carrier, such as an array on a silicon chip or a glass slide.The target nucleic acid sequence to be analyzed can be contacted with the immobilized oligonucleotide probe on the microchip. See Lipshutz et al., Biotechniques, 19:442-447 (1995);Chee et al., Science, 274:610-614 (1996);Kozal et al., Nat. Med. 2:753-759 (1996);Hacia et al., Nat. Genet., 14:441-447 (1996);Saiki et al., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989);Gingeras et al., Genome Res., 8:435-448 (1998). Alternatively, a plurality of target nucleic acid sequences to be interrogated can be immobilized on a substrate and a series of probes contacted with the immobilized target sequences. See Drmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous microchip technologies have been developed incorporating one or more of the above techniques for detecting mutations. Microchip technology combined with computer analysis tools allows for large-scale rapid screening. The adaptation of microchip technology to the present invention will be apparent to those skilled in the art who are informed of this disclosure.For example, U.S. Patent No. 5,925,525 to Fodor et al; Wilgenbus et al., J. Mol. Med., 77:761-786 (1999); Graber et al., Curr. Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet., 14:441-447 (1996);Shoemaker et al., Nat. Genet., 14:450-456 (1996);DeRisi et al., Nat. Genet., 14:457-460 (1996);Chee et al., Nat. Genet., 14:610-614 (1996);Lockhart et al., Nat. Genet., 14:675-680 (1996);Drobyshev et al., Gene, 188:45-52 (1997).

[0144] As is evident from the above overview of suitable detection techniques, depending on the detection technique used, it may or may not be necessary to amplify the target DNA, i.e., gene, its cDNA, mRNA, miRNA, or part, to increase the number of target DNA molecules. For example, most PCR-based techniques combine amplification of a part of the target with detection of mutations. PCR amplification is well known in the art and is disclosed in U.S. Patent Nos. 4,683,195 and 4,800,159, both of which are incorporated herein by reference. In detection techniques that are not PCR-based, amplification can be achieved, if necessary, by, for example, in vivo plasmid amplification or by purifying target DNA from a large amount of tissue or cell sample. Generally, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2003, 144:1311-1315 ... nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. However, many highly sensitive techniques have been developed that can detect even rare samples with small genetic changes, such as single nucleotide substitutions, without the need to amplify the target DNA in the sample. For example, techniques have been developed to amplify the signal for target DNA, for example, by using branched DNA or dendrimers that can hybridize to target DNA. Branched DNA or dendrimer DNA provides multiple hybridization sites for hybridization probes to bind to and amplify detection signals. Detmer et al., J. Clin. Microbiol., 34:901-907 (1996);Collins et al., Nucleic Acids Res., 25:2979-2984 (1997);Horn et al., Nucleic Acids Res., 25:4835-4841 (1997);Horn et al., Nucleic Acids Res., 25:4842-4849 (1997); Nilsen et al., J. Theor. Biol., 187:273-284 (1997).

[0145] The Invader™ assay is another technique for detecting single nucleotide changes that can be used for molecular profiling according to the present invention. The Invader™ assay uses a novel linear signal amplification technology that improves on the long turnaround times required for typical PCR DNA sequencing-based analyses. See Cooksey et al., Antimicrobial Agents and Chemotherapy 44:1296-1301 (2000). The assay is based on the cleavage of a unique secondary structure formed between two overlapping oligonucleotides that hybridize to a target sequence of interest to form a "flap". Each "flap" then generates thousands of signals per hour. Thus, the results of this technique can be easily deciphered and the method does not require exponential amplification of the DNA target. The Invader™ system utilizes two short DNA probes that hybridize to a DNA target. The structure formed by the hybridization event is recognized by a specific cleavase enzyme, which cleaves one of the probes, releasing a short DNA "flap". Each released "flap" then binds to a fluorescently labeled probe to form another cleavage structure. When the cleavase enzyme cleaves the labeled probe, the probe emits a detectable fluorescent signal. See, e.g., Lyamichev et al., Nat. Biotechnol., 17:292-296 (1999).

[0146] The rolling circle method is another method that avoids exponential amplification. Lizardi et al., Nature Genetics, 19:225-232 (1998) (incorporated herein by reference). For example, Sniper™, a commercial embodiment of this method, is a sensitive high-throughput SNP scoring system designed for precise fluorescent detection of specific variants. For each nucleotide variant, two linear allele-specific probes are designed. The two allele-specific probes are identical except for the 3' base, which is changed to complement the variant site. In the first stage of the assay, the target DNA is denatured and then hybridized with a pair of single-stranded, allele-specific, open circular oligonucleotide probes. Ligation of the probes occurs preferentially if the 3' base exactly complements the target DNA. Subsequent detection of the circularized oligonucleotide probes is by rolling circle amplification, and the amplified probe products are detected by fluorescence. See Clark and Pickering, Life Science News 6, 2000, Amersham Pharmacia Biotech (2000).

[0147] Some other techniques that avoid amplification all together include, for example, surface-enhanced resonance Raman scattering (SERRS), fluorescence correlation spectroscopy, and single molecule electrophoresis. In SERRS, chromophore-nucleic acid conjugate is absorbed on colloidal silver and irradiated with laser light at the resonant frequency of the chromophore. See Graham et al., Anal. Chem., 69:4703-4707 (1997). Fluorescence correlation spectroscopy is based on the spatial-temporal correlation between fluctuating light signals in an electric field and captured single molecules. See Eigen et al., Proc. Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single molecule electrophoresis, the electrophoretic velocity of fluorescently tagged nucleic acid is measured by measuring the time it takes for the molecule to travel a given distance between two laser beams. See Castro et al., Anal. Chem., 67:3181-3186 (1995).

[0148] In addition, allele-specific oligonucleotides (ASOs) can also be used in in situ hybridization using tissue or cell as sample.The oligonucleotide probes that can differentially hybridize with wild-type gene sequence or gene sequence with mutation can be labeled with radioisotope, fluorescent, or other detectable markers.In situ hybridization techniques are well known in the art, and their adaptation to the present invention for detecting the presence or absence of nucleotide variants in one or more genes of a particular individual will be obvious to those skilled in the art who are informed of this disclosure.

[0149] Protein-based detection techniques are also useful for molecular profiling, especially when the nucleotide variants cause amino acid substitutions or deletions or insertions or frameshifts that affect the primary, secondary, or tertiary structure of the protein. Protein sequencing techniques can be used to detect amino acid changes. For example, a protein or a fragment thereof corresponding to a gene can be synthesized by recombinant expression using DNA fragments isolated from the individual to be tested. Preferably, a cDNA fragment of 100-150 base pairs or less that encompasses the polymorphic locus to be determined is used. The amino acid sequence of the peptide can then be determined by conventional protein sequencing methods. Alternatively, HPLC-microscopy tandem mass spectrometry techniques can be used to determine amino acid sequence changes. In this technique, the protein is subjected to proteolytic digestion and the resulting peptide mixture is separated by reversed-phase chromatographic separation. Tandem mass spectrometry is then performed and the data collected therefrom is analyzed. See Gatlin et al., Anal. Chem., 72:757-763 (2000).

[0150] Other protein-based detection molecular profiling techniques include immunoaffinity assays based on antibodies that selectively immunoreact with the proteins encoded by the mutant genes according to the present invention. Methods for producing such antibodies are known in the art. Antibodies can be used to immunoprecipitate specific proteins from liquid samples or to immunoblot proteins separated, for example, by polyacrylamide gels. Immunocytochemical methods can also be used to detect specific protein polymorphisms in tissues or cells. Other well-known antibody-based techniques can also be used, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays (IRMAs), and immunoenzymatic assays (IEMAs), including sandwich assays using, for example, monoclonal or polyclonal antibodies. See, for example, U.S. Patent Nos. 4,376,110 and 4,486,530, both of which are incorporated herein by reference.

[0151] Thus, the presence or absence of one or more genetic nucleotide or amino acid variants in an individual can be determined using any of the detection methods described above.

[0152] Typically, once the presence or absence of one or more gene nucleotide variants or amino acid variants is determined, a physician or genetic counselor or patient or other researcher can be informed of the results. Specifically, the results can be released in a transmittable form that can be communicated or transmitted to other researchers or physicians or genetic counselors or patients. Such forms can be various and can be tangible or intangible. The results regarding the presence or absence of the nucleotide variants of the present invention in the individual being tested can be embodied in explanatory descriptions, figures, photographs, diagrams, images, or any other visual form. For example, gel electrophoresis images of PCR products can be used to explain the results. Diagrams showing where the variants occur in the individual's genes are also useful for showing the test results. Descriptions and visual forms can be recorded on tangible media, such as paper, computer readable media, such as floppy disks, compact disks, etc., or intangible media, such as electronic media in the form of e-mail or website on the Internet or intranet. In addition, the results regarding the presence or absence of a nucleotide variant or amino acid variant in the individual being tested may also be recorded in audio form and transmitted by telephone, facsimile, wireless mobile telephone, Internet telephone, etc., through any suitable medium, such as analog or digital cable line, fiber optic cable, etc.

[0153] Thus, the information and data on the test results can be generated anywhere in the world and sent to different locations. For example, if the genotyping assay is performed abroad, the information and data on the test results can be generated and sent in the above-mentioned transmittable form. The test results in transmittable form can be imported to the United States in this way. Thus, the present invention also encompasses a method for generating information in transmittable form on the genotype of two or more samples suspected of cancer from an individual. The method includes the steps of (1) determining the genotype of DNA from the samples according to the method of the present invention; and (2) embodying the results of the determining step in a transmittable form. The transmittable form is the product of the generating method.

[0154] Data and Analysis The implementation of the present invention may use conventional biological methods, software, and systems. The computer software product of the present invention typically includes a computer readable medium having computer executable instructions for performing the logical steps of the method of the present invention. Suitable computer readable media include floppy disks, CD-ROM / DVD / DVD-ROM, hard disk drives, flash memory, ROM / RAM, magnetic tape, etc. The computer executable instructions may be written in any suitable computer language or combination of several languages. Basic computational biology methods are described, for example, in Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000), and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Genes and Proteins (Wiley & Sons, Inc., 2.sup.nd ed., 2001). See U.S. Patent No. 6,420,108.

[0155] The present invention may also employ various computer program products and software for a variety of purposes, such as probe design, data management, analysis, and instrument operation, see U.S. Patent Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911, and 6,308,170.

[0156] Furthermore, the present invention relates to an embodiment that includes a method of providing genetic information over a network such as the Internet, as shown in US Patent Application Nos. 10 / 197,621, 10 / 063,559 (US Publication No. 20020183936), 10 / 065,856, 10 / 065,868, 10 / 328,818, 10 / 328,872, 10 / 423,403, and 60 / 482,389. For example, one or more molecular profiling techniques can be performed in one location, such as a city, state, country, or continent, and the results can be transmitted to another city, state, country, or continent. Then, treatment selection can be made in whole or in part in the second location. The method of the present invention includes transmitting information between different locations.

[0157] Molecular profiling for treatment selection The method of the present invention provides candidate treatment selection for the subject in need thereof.Molecular profiling can be used to identify one or more candidate treatments for individuals suffering from a disease state in which one or more of the biomarkers disclosed herein are treatment targets.For example, the method can identify one or more chemotherapy treatments for cancer. In one aspect, the invention provides a method for detecting a disease comprising the steps of: performing immunohistochemistry (IHC) analysis on a sample from a subject to determine an IHC expression profile for at least five proteins; performing microarray analysis on the sample to determine a microarray expression profile for at least ten genes; performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least one gene; performing DNA sequencing on the sample to determine a sequencing mutation profile for at least one gene; and detecting i) diseased cells that overexpress or underexpress one or more proteins included in the IHC expression profile, ii) diseased cells that overexpress or underexpress one or more genes included in the microarray expression profile, iii) diseased cells that have no mutations or have one or more mutations in one or more genes included in the FISH mutation profile, and / or iv) diseased cells that have no mutations or have one or more mutations in one or more genes included in the FISH mutation profile. The method includes the steps of comparing the IHC expression profile, the microarray expression profile, the FISH mutation profile and the sequencing mutation profile with a rule database that includes mappings for treatments that have known biological activity on diseased cells that do not have mutations or have one or more mutations in one or more genes that are included in the sequencing mutation profile; and if the comparison with the rule database indicates that the treatment should have biological activity on the diseased cells, and the comparison with the rule database indicates that the treatment is not contraindicated for treating the diseased cells, the treatment is identified.Disease can be cancer.The molecular profiling step can be carried out in any order.In some embodiments, not all of the molecular profiling steps are performed.As a non-limiting example, as described herein, if the quality of the sample does not meet the threshold, microarray analysis is not performed.In another example, sequencing is performed only if FISH analysis meets the threshold.Any relevant biomarker can be evaluated using one or more of the molecular profiling methods described herein or known in the art.The marker only needs to have some direct or indirect association with the treatment to be useful.

[0158] Molecular profiling involves profiling at least one gene (or gene product) for each assay technique performed. Different numbers of genes can be assayed with different techniques. Any marker disclosed herein that is directly or indirectly associated with targeted therapy can be evaluated based on genes, e.g., DNA sequences, and / or gene products, e.g., mRNA or protein. Such nucleic acids and / or polypeptides can be routinely profiled for presence / absence, level or amount, mutations, sequences, haplotypes, rearrangements, copy numbers, etc. In some embodiments, a single gene and / or one or more corresponding gene products are assayed by more than one molecular profiling technique. Genes or gene products (also referred to herein as "markers" or "biomarkers"), e.g., mRNA or protein, are evaluated using applicable techniques (e.g., for evaluating DNA, RNA, protein), including but not limited to FISH, microarray, IHC, sequencing, or immunoassays. Thus, any of the markers disclosed herein can be assayed by a single molecular profiling technique or by multiple methods disclosed herein (e.g., a single marker is profiled by one or more of IHC, FISH, sequencing, microarray, etc.). In some embodiments, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least about 100 genes or gene products are profiled by at least one technique, multiple techniques, or each of FISH, microarray, IHC, and sequencing.In some embodiments, the saturation concentration is at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,0 Each technique profiles 00, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, or at least about 50,000 genes or gene products.The number of markers to be assayed can depend on the technique used.For example, microarray and massively parallel sequencing are useful for high-throughput analysis.

[0159] In some embodiments, a sample from a subject in need thereof is profiled using methods including, but not limited to, IHC expression profiling, microarray expression profiling, FISH mutation profiling, and / or sequencing mutation profiling (such as by PCR, RT-PCR, pyrosequencing) for one or more of the following: ABCC1, ABCG2, ACE2, ADA, ADH1C, ADH4, AGT, androgen receptor, AR, AREG, ASNS, BCL2, BCRP, BDCA1, BIRC5, B-RAF, BRCA1, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDK2, CDW52, CES2, CK 14, CK 17, CK 5 / 6, c-KIT, c-Myc, COX-2, cyclin D1, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, E-cadherin, ECGF1, EGFR, EPHA2, epiregulin, ER, ERBR2, ERCC1, ERCC3, EREG, ESR1, FLT1, folate receptor, FOLR1, FOLR2, FSHB, FSHPRH1, FSHR, FYN, GART, GNRH1, GNRHR1, GSTP1, HCK, HDAC1, Her2 / Neu, HGF, HIF1A, HIG1, HSP90, HSP90AA1, HSPCA, IL 13RA1, IL2RA, KDR, KIT, K-RAS, LCK, LTB, lymphotoxin β receptor, LYN, MGMT, MLH1, MRP1, MS4A1, MSH2, Myc, NFKB1, NFKB2, NFKBIA, ODC1, OGFR, p53, p95, PARP-1, PDGFC, PDGFR, PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLA1, PPARG, PPARGC1, PR, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SPARC MC, SPARCPC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, survivin, TK1, TLE3, TNF, TOP1, TOP2A, TOP2B, TOPO1, TOPO2B, topoisomerase II, TS, TXN, TXNRD1, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YES1, and ZAP70.

[0160] In some embodiments, additional molecular profiling methods are performed. These may include, but are not limited to, PCR, RT-PCR, Q-PCR, SAGE, MPSS, immunoassays, and other techniques for evaluating biological systems described herein or known to those skilled in the art. The selection of genes and gene products to be assayed may be updated as new therapies and new drug targets are identified. Once the expression or mutation of a biomarker is associated with a treatment option, it can be evaluated by molecular profiling. Those skilled in the art will understand that such molecular profiling is not limited to the techniques disclosed herein, but includes any methodology that is conventional for evaluating nucleic acid or protein levels, sequence information, or both. The methods of the present invention may also utilize any improvements to current methods or new molecular profiling techniques developed in the future. In some embodiments, genes or gene products are evaluated by a single molecular profiling technique. In other embodiments, genes and / or gene products are evaluated by multiple molecular profiling techniques. In non-limiting examples, gene sequences can be analyzed by one or more of FISH and pyrosequencing analysis, mRNA gene products can be assayed by one or more of RT-PCR and microarray, and protein gene products can be assayed by one or more of IHC and immunoassays. Those skilled in the art will understand that any combination of biomarkers and molecular profiling techniques that benefit disease treatment is contemplated by the present invention.

[0161] Genes and gene products known to play a role in cancer and that may be assayed by any of the molecular profiling techniques of the invention include, but are not limited to, 2AR, a disintegrin, activator of thyroid and retinoic acid receptors (ACTR), ADAM 11, adipogenesis inhibitory factor (ADIF), alpha6 integrin subunit, alphaV integrin subunit, alpha-catenin, amplified in breast cancer 1 (AIB1), amplified in breast cancer 3 (AIB3), amplified in breast cancer 4 (AIB4), amyloid precursor protein secretase (APPS), AP-2 gamma, APPS, ATP-binding cassette transporter (ABCT), placenta-specific (ABCP), ATP-binding cassette subfamily C member (ABCC1), BAG-1, BASIG IN (BSG), BCEI, B-cell differentiation factor (BCDF), B-cell leukemia 2 (BCL-2), B-cell stimulatory factor-2 (BSF-2), BCL-1, BCL-2 associated X protein (BAX), BCRP, β1 integrin subunit, β3 integrin subunit, β5 integrin subunit, β2 interferon, β-catenin, β-catenin, bone sialoprotein (BSP), breast cancer estrogen-inducible element (BCEI), breast cancer resistance protein (BC RP), breast cancer type 1 (BRCA1), breast cancer type 2 (BRCA2), breast cancer amplified sequence 2 (BCAS2), cadherin, epithelial cadherin-11, cadherin-binding protein, calcitonin receptor (CTR), calcium placental protein (CAPL), calcyclin, CALLA, CAM5, CAPL, carcinoembryonic antigen (CEA), catenin, α1, cathepsin B, cathepsin D, cathepsin K, cathepsin L2, cathepsin O, cathepsin O1, cathepsin V, CD1 0, CD146, CD147, CD24, CD29, CD44, CD51, CD54, CD61, CD66e, CD82, CD87, CD9, CEA, cellular retinol binding protein 1 (CRBP1), c-ERBB-2, CK7, CK8, CK18, CK19, CK20, claudin-7, c-MET, collagenase, fibroblast, collagenase, interstitial, collagenase 3, common acute lymphocytic leukemia antigen (CALLA), connexin 26 (Cx26), connexin 43 (Cx43), cortactin, COX-2, CTLA-8, CTR, CTSD, cyclin D1,Cyclooxygenase-2, cytokeratin 18, cytokeratin 19, cytokeratin 8, cytotoxic T lymphocyte-associated serine esterase 8 (CTLA-8), differentiation inhibitory activity (DIA), DNA amplified in breast cancer 1 (DAM1), DNA topoisomerase IIα, DR-NM23, E-cadherin, EMMPRIN, EMS1, endothelial cell growth factor (ECGR), platelet-derived (PD-ECGF), enkephalinase, epidermal growth factor receptor (EGFR), epithelial ... Membrane antigen (EMA), ER-α, ERBB2, ERBB4, ER-β, ERF-1, erythropoietin-potentiating activity (EPA), ESR1, estrogen receptor-α, estrogen receptor-β, ETS-1, extracellular matrix metalloproteinase inducer (EMMPRIN), fibronectin receptor, β polypeptide (FNRB), fibronectin receptor β subunit (FNRB), FLK-1, GA15.3, GA733.2, galectin-3, γ-catenin, gap junction protein (26 kDa), gap junction protein (43 kDa), gap junction protein alpha-1 (GJA1), gap junction protein beta-2 (GJB2), GCP1, gelatinase A, gelatinase B, gelatinase (72 kDa), gelatinase (92 kDa), gliostatin, glucocorticoid receptor interacting protein 1 (GRIP1), glutathione S-transferase p, GM-CSF, granulocyte chemotactic protein 1 (GCP1), granulocyte-macrophage colony-stimulating factor, growth factor receptor-bound-7 (GRB-7), GSTp, HAP, heat shock cognate protein 70 (HSC70), heat stable antigen, hepatocyte growth factor (HGF), hepatocyte growth factor receptor (HGFR), hepatocyte stimulating factor III (HSF III), HER-2, HER2 / NEU, HERMES antigen, HET, HHM, paraneoplastic humoral hypercalcemia (HHM), ICERE-1, INT-1, intercellular adhesion molecule-1 (ICAM-1), interferon-gamma-inducing factor (IGIF), interleukin-1α (IL-1A), interleukin-1β (IL-1B), interleukin-11 (IL-11), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-6 (IL-6), interleukin-8 (IL-8),Inversely correlated with estrogen receptor expression-1 (ICERE-1), KAI1, KDR, keratin 8, keratin 18, keratin 19, KISS-1, leukemia inhibitory factor (LIF), LIF, lost in inflammatory breast cancer (LIBC), LOT ("lost in transformation"), lymphocyte homing receptor, macrophage colony-stimulating factor, MAGE-3, mammaglobin, maspin, MC56, M-CSF, MDC, MDNCF, MDR, melanoma cell adhesion molecule (MCAM), membrane metalloendopeptidase (MME), membrane-associated neutral endopeptidase (NEP), cysteine-rich protein (MDC), metastasin (MTS-1), MLN64, MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MMP2 ...9, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, MMP28 Escin, monocyte arginine-serpin, monocyte-derived neutrophil chemotactic factor, monocyte-derived plasminogen activator inhibitor, MTS-1, MUC-1, MUC18, mucin-like cancer-associated antigen (MCA), mucin, MUC-1, multidrug resistance protein 1 (MDR, MDR1), multidrug resistance-associated protein-1 (MRP, MRP-1), N-cadherin, NEP, NEU, neutral endopeptider enzyme, neutrophil-activating peptide 1 (NAP1), NM23-H1, NM23-H2, NME1, NME2, nuclear receptor coactivator-1 (NCoA-1), nuclear receptor coactivator-2 (NCoA-2), nuclear receptor coactivator-3 (NCoA-3), nucleoside diphosphate kinase A (NDPKA), nucleoside diphosphate kinase B (NDPKB), oncostatin M (OSM), ornithine decarboxylase (ODC), osteoclast differentiation factor (ODF), osteoclast differentiation factor receptor (ODFR), osteonectin (OSN, ON), osteopontin (OPN), oxytocin receptor (OXTR), p27 / kip1, p300 / CBP cointegrator-associated protein (p / CIP), p53, p9Ka, PAI-1, PAI-2, parathyroid adenomatosis 1 (PRAD1), parathyroid hormone-like hormone (PTHLH), parathyroid hormone-related peptide (PTHrP), P-cadherin, PD-ECGF, PDGF, peanut-reactive urinary mucin (PUM), P-glycoprotein (P-GP), PGP-1, PHGS-2, PHS-2, PIP, plakoglobin,Plasminogen activator inhibitor (type 1), plasminogen activator inhibitor (type 2), plasminogen activator (tissue type), plasminogen activator (urokinase type), platelet glycoprotein IIIa (GP3A), PLAU, pleomorphic adenoma gene-like 1 (PLAGL1), pleomorphic epithelial mucin (PEM), PRAD1, progesterone receptor (PgR), progesterone resistance, prostag Randin endoperoxide synthase-2, prostaglandin G / H synthase-2, prostaglandin H synthase-2, pS2, PS6K, psoriasis, PTHLH, PTHrP, RAD51, RAD52, RAD54, RAP46, receptor-associated coactivator 3 (RAC3), suppressor of estrogen receptor activity (REA), S100A4, S100A6, S100A7, S6K, SART-1, scaffold attachment factor B (SAF -B), scatter factor (SF), secreted phosphoprotein-1 (SPP-1), secreted protein, acidic and cysteine-rich (SPARC), stanniocalcin, steroid receptor coactivator-1 (SRC-1), steroid receptor coactivator-2 (SRC-2), steroid receptor coactivator-3 (SRC-3), steroid receptor RNA activator (SRA), stromelysin-1, stromelysin-3, tenascin-C (TN-C), testis-specific protease 50, thrombospondin I, thrombospondin II, thymidine phosphorylase (TP), thyroid hormone receptor activator molecule 1 (TRAM-1), tight junction protein 1 (TJP1), TIMP1, TIMP2, TIMP3, TIMP4, tissue-type plasminogen activator, TN-C, TP53, tPA, transcription intermediary factor 2 (TRANSCRIPTIONAL INTERMEDIARY FACTOR 2 (TIF2), trefoil factor 1 (TFF1), TSG101, TSP-1, TSP1, TSP-2, TSP2, TSP50, tumor cell collagenase stimulating factor (TCSF), tumor-associated epithelial mucin, uPA, uPAR, urokinase, urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor (uPAR), uvomorulin, vascular endothelial growth factor, vascular endothelial growth factor receptor-2 (VEGFR2),These include vascular endothelial growth factor-A, vascular permeability factor, VEGFR2, very late T cell antigen beta (VLA-β), vimentin, vitronectin receptor alpha polypeptide (VNRA), vitronectin receptor, von Willebrand factor, VPF, VWF, WNT-1, ZAC, ZO-1, and zonula occludens-1.

[0162] Gene products used for IHC expression profiling include, but are not limited to, one or more of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1. IHC profiling of EGFR can also be performed. IHC is also used to detect or test various gene products, including, but not limited to, one or more of the following: EGFR, SPARC, C-kit, ER, PR, androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, thymidylate synthase, Her2 / neu, or TOPO2A. In some embodiments, IHC is used to detect one or more of the following proteins, including but not limited to: ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, or ZAP70.

[0163] Microarray expression profiling can be used to simultaneously measure the expression of one or more genes or gene products, including, but not limited to, the following: ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HIF2A, HIF1B, HIF1C, HIF1D, HIF1E, HIF1F, HIF1G, HIF1H ... SP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, R RM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70. In some embodiments, the genes used for microarray expression profiling are EGFR, SPARC, C-kit, ER, PR, androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, thymidylate synthase, Her2 / neu, TOPO2A, ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOL R2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, or ZAP70.Microarray expression profiling can be carried out using low-density microarrays, expression microarrays, comparative genomic hybridization (CGH) microarrays, single nucleotide polymorphism (SNP) microarrays, proteomics arrays, antibody arrays, or other arrays disclosed herein or known to those skilled in the art.In some embodiments, high-throughput expression arrays are used.Such systems include, but are not limited to, the commercially available systems from Agilent or Illumina, which are described in more detail herein.

[0164] FISH mutation profiling can be used to profile one or more of EGFR and HER2.In some embodiments, FISH is used to detect or test one or more of the following genes, including but not limited to: EGFR, SPARC, C-kit, ER, PR, androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, thymidylate synthase, HER2, or TOPO2A. In some embodiments, FISH is used to detect or test various biomarkers, including but not limited to one or more of the following: ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, EGFR, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SPARC, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, or ZAP70.

[0165] In some embodiments, the genes used for sequencing mutation profiling include one or more of KRAS, BRAF, c-KIT, and EGFR.Sequencing analysis also includes one or more of ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1 , MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70.

[0166] In a related aspect, the present invention provides a method for identifying candidate treatments for a subject in need of such treatment by using molecular profiling of a set of known biomarkers. For example, the method can identify chemotherapeutic drugs for individuals with cancer. The method includes the steps of obtaining a sample from a subject; performing immunohistochemistry (IHC) analysis on the sample to determine an IHC expression profile for at least five of SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1; performing microarray analysis on the sample to determine an IHC expression profile for at least five of ABCC1, ABCG2, ADA, AR ... , ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC 1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT determining a microarray expression profile for at least five of the following: MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, PTGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least one of EGFR and HER2; performing DNA sequencing on the sample to determine a sequencing mutation profile for at least one of KRAS, BRAF, c-KIT, and EGFR;and comparing the IHC expression profile, the microarray expression profile, the FISH mutation profile, and the sequencing mutation profile to a rule database that includes mappings for treatments with known biological activity against i) diseased cells that overexpress or underexpress one or more proteins included in the IHC expression profile, ii) diseased cells that overexpress or underexpress one or more genes included in the microarray expression profile, iii) diseased cells that do not have a mutation or have one or more mutations in one or more genes included in the FISH mutation profile, and / or iv) diseased cells that do not have a mutation or have one or more mutations in one or more genes included in the sequencing mutation profile; and identifying a treatment when the comparison to the rule database indicates that the treatment should have biological activity against the disease and the comparison to the rule database does not indicate a contraindication for the treatment of the disease.The disease may be cancer.The molecular profiling steps can be performed in any order. In some embodiments, not all of the molecular profiling steps are performed. As a non-limiting example, if the sample quality does not meet a threshold, as described herein, microarray analysis is not performed. In some embodiments, IHC expression profiling is performed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the gene products described above. In some embodiments, microarray expression profiling is performed on at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the genes described above.;

[0167] In a related aspect, the present invention provides a method for identifying candidate treatments for a subject in need of such treatment by using molecular profiling of a predefined set of known biomarkers. For example, the method can identify chemotherapeutic agents for an individual with cancer. The method includes the steps of: obtaining a sample from a subject, the sample comprising formalin-fixed paraffin-embedded (FFPE) tissue or fresh frozen tissue, and the sample comprising cancer cells; performing immunohistochemistry (IHC) analysis on the sample to determine an IHC expression profile for at least SPARC, PGP, Her2 / neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCC1, RRM1, BCRP, TOPO1, PTEN, MGMT, and MRP1. performing microarray analysis on the sample to determine whether or not the gene is a marker for at least ABCC1, ABCG2, ADA, AR, ASNS, BCL2, BIRC5, BRCA1, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMT1, DNMT3A, DNMT3B, ECGF1, EGFR, EPHA2, ERBB2, ERCC1, ERCC3, ESR1, FLT1, FOLR2, FYN, GART, GNRH1, GSTP1, HCK, HDAC1, HIF1A, HSP90, HSP100, HSP1100, HSP120, HSP130, HSP140, HSP150, HSP160, HSP170, HSP180, HSP190, HSP191, HSP192, HSP193, HSP194, HSP195, HSP196, HSP197, HSP198, HSP199, HSP199, HSP191, HSP196, HSP199, HSP199, HSP191, HSP192, HSP193, HSP194, HSP195, HSP196, HSP197, HSP198, HSP199, HSP199, HSP199, HSP199, HSP199, HSP199, HSP191, HSP199, HSP191, HSP192, HSP193, HSP194, HSP195, HSP196, HSP197, HSP198, HSP199, HSP199, HSP199, HSP199, HSP199, HSP199, HSP199, HSP199, AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLH1, MS4A1, MSH2, NFKB1, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA1, PTEN, P TGS2, RAF1, RARA, RRM1, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, TK1, TNF, TOP1, TOP2A, TOP2B, TXN The method includes determining a microarray expression profile for RD1, TYMS, VDR, VEGFA, VHL, YES1, and ZAP70; performing fluorescent in situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile for at least EGFR and HER2; and performing DNA sequencing on the sample to determine a sequencing mutation profile for at least KRAS, BRAF, c-KIT, and EGFR.The IHC expression profile, the microarray expression profile, the FISH mutation profile, and the sequencing mutation profile are compared against a rule database, where the rule database includes mappings for treatments with known biological activity against i) diseased cells that over- or under-express one or more proteins included in the IHC expression profile; ii) diseased cells that over- or under-express one or more genes included in the microarray expression profile; iii) diseased cells that have no mutations or have one or more mutations in one or more genes included in the FISH mutation profile; and / or iv) diseased cells that have no mutations or have one or more mutations in one or more genes included in the sequencing mutation profile; and a treatment is identified if the comparison against the rule database indicates that the treatment should have biological activity against the disease; and the comparison against the rule database does not indicate a contraindication for the treatment of the disease. The disease may be cancer. The molecular profiling steps can be performed in any order. In some embodiments, not all of the molecular profiling steps are performed.As a non-limiting example, as described herein, if the quality of the sample does not meet the threshold, microarray analysis is not performed.In some embodiments, the biological material is mRNA, and the quality control test includes A260 / A280 ratio and / or Ct value of RT-PCR using housekeeping gene, such as RPL13a.In some embodiments, if the A260 / A280 ratio is lower than 1.5 or the RPL13a Ct value is higher than 30, the mRNA does not pass the quality control test.In this case, microarray analysis cannot be performed.Alternatively, the microarray result can be attenuated, such as being given lower priority compared to the results of other molecular profiling techniques.

[0168] In some embodiments, molecular profiling is always performed on a specific gene or gene product, and profiling of other genes or gene products is optional. For example, IHC expression profiling can be performed on at least SPARC, TOP2A, and / or PTEN. Similarly, microarray expression profiling can be performed on at least CD52. In other embodiments, in addition to the above genes, additional genes are used to identify treatment. For example, the group of genes used for IHC expression profiling can further include DCK, EGFR, BRCA1, CK 14, CK 17, CK 5 / 6, E-cadherin, p95, PARP-1, SPARC, and TLE3. In some embodiments, the group of genes used for IHC expression profiling further includes Cox-2 and / or Ki-67. In some embodiments, HSPCA is assayed by microarray analysis. In some embodiments, FISH mutation is performed on c-Myc and TOP2A. In some embodiments, sequencing is performed on PI3K.

[0169] The method of the present invention can be performed in any setting where differential expression or mutation analysis is associated with the efficacy of various treatments. In some embodiments, the method is used to identify candidate treatments for subjects with cancer. Under these conditions, the sample used for molecular profiling preferably contains cancer cells. The percentage of cancer in the sample can be determined by methods known to those skilled in the art, for example, using pathology techniques. Cancer cells can also be enriched from the sample, for example, using microdissection techniques. A sample may be required to have a certain threshold of cancer cells before it is used for molecular profiling. The threshold may be at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% cancer cells. The threshold may depend on the analysis method. For example, a technique that reveals expression in individual cells may require a lower threshold than a technique that uses a sample extracted from a mixture of different cells. In some embodiments, the diseased sample is compared to a normal sample taken from the same patient, for example, adjacent non-cancerous tissue.

[0170] Treatment options The system and method of the present invention can be used to select any treatment whose predicted efficacy can be associated with molecular profiling results. The present invention includes the use of molecular profiling results to suggest associations with treatment response. In one embodiment, appropriate biomarkers for molecular profiling are selected based on the tumor type of the subject. Such suggested biomarkers can be used to modify the default list of biomarkers. In other embodiments, molecular profiling is source material independent. In some embodiments, a rule is used to provide a suggested chemotherapy treatment based on the molecular profiling test results. In one embodiment, the rule is created from a summary of peer-reviewed clinical oncology literature. Expert opinion rules can also be used, but are optional. In one embodiment, clinical citations are evaluated for their relevance to the method of the present invention using a hierarchy derived from the evidence scoring system used by the United States Preventive Services Task Force. The "best evidence" can be used as the basis for the rule. The simplest rule is constructed in the form of "if biomarker is positive, then there is one treatment option, otherwise there are two treatment options." Treatment options include no treatment with a particular drug, treatment with a particular drug, or treatment with a combination of drugs. In some embodiments, more complex prescriptions are constructed that include the interaction of two or more biomarkers. In such cases, the more complex interactions are typically supported by clinical studies that analyze the interactions between the biomarkers included in the prescription. Finally, a report can be generated that describes the association between chemotherapy response and biomarkers, and a summary statement of the best evidence that directs the treatment selected. Ultimately, the treating physician decides on the best course of treatment.

[0171] As a non-limiting example, molecular profiling may reveal that the EGFR gene is amplified or overexpressed, thereby indicating the selection of a treatment that may block EGFR activity, such as the monoclonal antibody inhibitors cetuximab and panitumumab, or small molecule kinase inhibitors that are effective in patients with activating mutations in EGFR, such as gefitinib, erlotinib, and lapatinib. Other anti-EGFR monoclonal antibodies in clinical development include zalutumumab, nimotuzumab, and matuzumab. The candidate treatment selected may depend on the setting revealed by molecular profiling. For example, if EGFR is found to have an activating mutation, a kinase inhibitor is often prescribed. Continuing with the exemplary embodiment, molecular profiling may also reveal that some or all of these treatments are likely to be less effective. For example, patients taking gefitinib or erlotinib will eventually develop drug resistance mutations in EGFR. Thus, the presence of drug resistance mutations indicates a contraindication to the selection of a small molecule kinase inhibitor. Those skilled in the art will appreciate that this example can be extended to guide the selection of other candidate therapeutics that act against genes or gene products whose differential expression is revealed by molecular profiling. Similarly, where molecular profiling reveals a particular nucleic acid variant, a candidate drug can be selected that is known to be effective against diseased cells harboring such a variant.

[0172] Cancer therapies that may be identified as candidate treatments by the methods of the present invention include, but are not limited to, 13-cis-retinoic acid, 2-CdA, 2-chlorodeoxyadenosine, 5-azacytidine, 5-fluorouracil, 5-FU, 6-mercaptopurine, 6-MP, 6-TG, 6-thioguanine, Abraxane, Accutane®, actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, aldesleukin, azaci ... Lemtuzumab, ALIMTA, alitretinoin, Alkaban-AQ®, Alkeran®, all-trans retinoic acid, alpha interferon, altretamine, amethopterin, amifostine, aminoglutethimide, anagrelide, Anandron®, anastrozole, arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, arsenic trioxide, asparaginase , ATRA, Avastin®, Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib, Bursufane, Busulfex®, C225, Leucovorin Calcium, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casode x (registered trademark), CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine (registered trademark), Cetuximab, Chlorambucil, Cisplatin, Citrovorum factor, Cladribine, Cortisone, Cosmegen (registered trademark), CPT-11, Cyclophosphamide, Cytadren (registered trademark), Cytarabine, Cytarabine liposomal, Cytosar-U (registered trademark), Cytoxan (registered trademark), Dacarbazine, Dacogen, Dactinomycin, Darbepoetin alfa, Dasatinib, Daunomycin Daunorubicin, Daunorubicin hydrochloride,Daunorubicin liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, Denileukin, Diftitox, DepoCyt™, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard®, Ellence®, Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-Asparaginase, Estramustine, Ethyol Etopophos®, etoposide, etoposide phosphate, Eulexin®, everolimus, Evista®, exemestane, Fareston®, Faslodex®, Femara®, filgrastim, floxuridine, Fludara®, fludarabine, Fluoroplex®, fluorouracil, fluorouracil (cream), fluoxymesterone, flutamide, folinic acid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab Ozogamicin, Gemzar, Gleevec™, Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte Colony Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexalen®, Hexamethylmelanin, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab, Tiuxetan, Idamycin®,Idarubicin, Ifex®, IFN-α, Ifosfamide, IL-11, IL-2, Imatinib Mesylate, Imidazole Carboxamide, Interferon α, Interferon α-2b (PEG conjugate), Interleukin-2, Interleukin-11, Intron A® (Interferon α-2b), Iressa®, Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, Kidrolase (t), Lanacort®, Lapatinib, L-Asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine™, Leuprolide, Leucocristin, Leustatin™, Liposomal Ara-C Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, Mechlorethamine hydrochloride, Medralone®, Medrol®, Megace®, Megestrol, Megestrol acetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate, Methotrexate sodium, Methylprednisolone, Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine, Mutamycin®, Myleran®, My locel™, Mylotarg™, Navelbine™, Nelarabine, Neosar™, Neulasta™, Neumega™, Neupogen™, Nexavar™, Nilandron™, Nilutamide, Nipent™, Nitrogen Mustard, Novaldex™, Novantrone™, Octreotide, Octreotide Acetate, Oncospar™, Oncovin™, Ontak™, Onxal™, Oprelvekin, Orapred™, Orasone™,Oxaliplatin, Paclitaxel, Protein-bound Paclitaxel, Pamidronate, Panitumumab, Panretin®, Paraplatin®, Pediapred®, PEG-interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™, PEG-L-asparaginase, Pemetrexed, Pentostatin, Phenylalanine Mustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, P relone®, procarbazine, PROCRIT®, Proleukin®, Prolife Prospan 20 with Carmustine Implant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®, Rituximab, Roferon-A® (Interferon alpha-2a), Rubex®, Rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim, Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine Tabloid®, thiophosphoamide, Thioplex®, thiotepa, TICE®, Toposar®, topotecan, toremifene, Torisel®, tositumomab, trastuzumab, Treanda®, tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®, VePesid®,Vesanoid®, Viadur™, Vidaza®, vinblastine, vinblastine sulfate, Vincasar Pfs®, vincristine, vinorelbine, vinorelbine tartrate, VLB, VM-26, vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™, Zinecard®, Zoladex®, zoledronic acid, Zolinza, Zometa®, and any combination thereof.

[0173] In some embodiments, a database is created that maps treatments to molecular profiling results. The treatment information may include predicted efficacy of a therapeutic agent on cells with a particular characteristic that can be measured by molecular profiling. Molecular profiling may include differential expression or mutations in a particular gene, protein, or other biomolecule of interest. Through mapping, the results of molecular profiling can be compared against the database to select a treatment. The database may include both positive and negative mappings between treatments and molecular profiling results. In some embodiments, the mapping is created by reviewing the literature regarding the association between biological agents and therapeutic agents. For example, journal articles, patent or patent application publications, scientific presentations, etc. can be reviewed for potential mappings. The mapping may include results of in vivo, e.g., animal or clinical trials, or in vitro experiments, e.g., cell culture. Any mappings found, such as, for example, the cytotoxic effect of a therapeutic agent on cells expressing a certain gene or protein, can be incorporated into the database. In this manner, the database may be continually updated. It will be understood that the methods of the present invention are updated as well.

[0174] Rules for mapping may include various supplementary information. In some embodiments, the database includes prioritization criteria. For example, a treatment with a higher predicted efficacy in a given setting may be preferred over a treatment predicted to have a lower efficacy. Mappings derived from a particular setting, such as clinical trials, may be prioritized over mappings derived from another setting, such as cell culture experiments. Treatments with stronger literature support may be prioritized over treatments supported by more preliminary results. Treatments that are generally applied to the disease type in question, such as cancers of a particular tissue origin, may be prioritized over treatments that are not indicated for that particular disease. Mapping may include both positive and negative correlations between treatments and molecular profiling results. In a non-limiting example, one mapping may suggest the use of a kinase inhibitor such as erlotinib for tumors with activating mutations in EGFR, whereas another mapping may suggest against that treatment if EGFR also has a drug-resistant mutation. Similarly, a treatment may be shown to be effective in cells that overexpress a particular gene or protein, but not effective if the gene or protein is underexpressed.

[0175] The selection of a candidate treatment for an individual may be based on the molecular profiling results by any one or more of the methods described. Alternatively, the selection of a candidate treatment for an individual may be based on the molecular profiling results by two or more of the methods described. For example, the selection of a treatment for an individual may be based on the molecular profiling results by FISH only, IHC only, or microarray analysis only. In other embodiments, the selection of a treatment for an individual may be based on the molecular profiling results by IHC, FISH, and microarray analysis; IHC and FISH; IHC and microarray analysis, or FISH and microarray analysis. The selection of a treatment for an individual may also be based on the molecular profiling results by sequencing or other methods of mutation detection. The molecular profiling results may include mutation analysis with one or more methods such as IHC, immunoassay, and / or microarray analysis. Various combinations and sequential results can be used. For example, treatments can be prioritized according to the results obtained by molecular profiling. In one embodiment, the prioritization is based on the following algorithm: 1) if IHC / FISH and microarray show the same target, it is the first priority; 2) a single positive IHC result is the second priority; or 3) a single positive microarray result is the last priority. Sequencing can also be used to guide the selection. In some embodiments, sequencing reveals drug resistance mutations, so that the resulting drug is not selected even if techniques including IHC, microarray, and / or FISH show differential expression of the target molecule. Any such contraindication, for example, differential expression or mutation of another gene or gene product, may override the selection of treatment.

[0176] An exemplary list of microarray expression results and predicted treatments is shown in Table 1. Molecular profiling is performed to determine whether a gene or gene product in the sample is differentially expressed compared to a control. The control can be any control suitable for the setting, including, but not limited to, the expression level of a control gene such as a housekeeping gene, the expression of the same gene in healthy tissue from the same or other individual, statistical criteria, levels of detection, etc. Those skilled in the art will understand that any applicable molecular profiling technique; for example, the results of microarray analysis PCR, Q-PCR, RT-PCR, immunoassay, SAGE, IHC, FISH, or sequencing can be used to determine the expression status. The expression status of the gene or gene product is used to select agents that are predicted to be effective or ineffective. For example, Table 1 shows that overexpression of the ADA gene or protein presents pentostatin as a possible treatment. On the other hand, underexpression of the ADA gene or protein indicates resistance to cytarabine, suggesting that cytarabine is not the treatment of choice.

[0177] (Table 1) Molecular profiling results and predicted treatment TIFF0007659585000001.tif226170TIFF0007659585000002.tif228170TIFF0007659585000003.tif233170TIFF0007659585000004.tif67170

[0178] Table 2 shows a more comprehensive rule summary for treatment selection. For each biomarker in the table, the type of assay and the assay result are shown. A summary of the effectiveness of various therapeutics given the assay results can be derived from medical literature or other medical knowledge base. The results can be used to guide the selection of specific therapeutics, which are recommended or not recommended. In some embodiments, the table is continuously updated as new literature reports and treatments become available. In this way, the molecular profiling of the present invention will develop and improve over time. The rules in Table 2 can be stored in a database. Once molecular profiling results are obtained, such as differential expression or mutation of a gene or gene product, the results can be compared against the database to guide treatment selection. The set of rules in the database can be updated as new treatments and new treatment data become available. In some embodiments, the rules database is continuously updated. In some embodiments, the rules database is periodically updated. The rules database may be updated at least once a day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 18 months, 2 years, or at least every 3 years. Any relevant correlation or comparison approach may be used to compare the molecular profiling results to the rules database. In one embodiment, a gene or gene product is identified as being differentially expressed by molecular profiling. The rules database is queried to select an entry for the gene or gene product. Selected treatment selection information is extracted from the rules database and used to select a treatment. For example, information for recommending or not recommending a particular treatment may depend on whether the gene or gene product is overexpressed or underexpressed. In some cases, multiple rules and treatments may be derived depending on the results of molecular profiling. In some embodiments, the treatment options are prioritized in a list and presented to the end user. In some embodiments, treatment options are presented without prioritization information.In either case, the individual, e.g., a treating physician or similar caregiver, can choose from among the available treatment options.

[0179] (Table 2) Summary of rules regarding treatment selection TIFF0007659585000005.tif239170TIFF0007659585000006.tif241164TIFF0007659585000007.tif241168TIFF0007659585000008.tif241168TIFF0007659585000009.tif241168TIFF0007659585000010.tif238170TIFF0007659585000011.tif241168TIFF0007659585000012.tif238170TIFF0007659585000013.tif241168TIFF0007659585000014.tif239170TIFF0007659585000015.tif238170TIFF0007659585000016.tif239170TIFF0007659585000017.tif239170TIFF0007659585000018.tif240167TIFF0007659585000019.tif240167TIFF0007659585000020.tif239170TIFF0007659585000021.tif241163TIFF0007659585000022.tif241167TIFF0007659585000023.tif241164TIFF0007659585000024.tif238170TIFF0007659585000025.tif238170TIFF0007659585000026.tif238170TIFF0007659585000027.tif241164TIFF0007659585000028.tif241167TIFF0007659585000029.tif239170TIFF0007659585000030.tif239170TIFF0007659585000031.tif239167TIFF0007659585000032.tif239163TIFF0007659585000033.tif241168TIFF0007659585000034.tif238170TIFF0007659585000035.tif241168TIFF0007659585000036.tif241168TIFF0007659585000037.tif241164TIFF0007659585000038.tif241168TIFF0007659585000039.tif241168TIFF000765958500 0040.tif239170TIFF0007659585000041.tif241164TIFF0007659585000042.tif239170TIFF0007659 585000043.tif241168TIFF0007659585000044.tif241164TIFF0007659585000045.tif239170TIFF00 07659585000046.tif238170TIFF0007659585000047.tif241165TIFF0007659585000048.tif241152.

[0180] The method described herein can be used to provide personalized treatment options, thereby prolonging the survival of a subject. In some embodiments, the subject has been previously treated with one or more therapeutic agents for treating a disease, such as cancer. The cancer may be refractory to one of these agents, for example, by acquiring drug resistance mutations. In some embodiments, the cancer is metastatic. In some embodiments, the subject has not been previously treated with one or more candidate therapeutic agents identified by the method. Regardless of the stage, anatomical location, or anatomical origin of cancer cells, molecular profiling can be used to select candidate therapeutic agents.

[0181] Progression-free survival (PFS) refers to the likelihood that an individual or a group of individuals suffering from a disease, such as cancer, will remain disease-free after the start of a course of treatment. It may refer to the percentage of individuals in a group whose disease is likely to remain stable (e.g., show no signs of progression) after a certain duration. Progression-free survival is an indicator of the effectiveness of a particular treatment. Similarly, disease-free survival (DFS) refers to the likelihood that an individual or a group of individuals suffering from cancer will remain disease-free after the start of a particular treatment. It may refer to the percentage of individuals in a group whose disease is likely to be disease-free after a certain duration. Disease-free survival is an indicator of the effectiveness of a particular treatment. Treatment strategies can be compared based on the PFS or DFS obtained in similar patient groups. When cancer survival is described, disease-free survival is often used together with the term overall survival.

[0182] Candidate therapies selected by molecular profiling according to the invention can be compared to therapies selected without molecular profiling by comparing the progression-free survival (PFS) with the treatment selected by molecular profiling (period B) with the PFS of the most recent treatment when the patient started progressing (period A). See FIG. 32. In one setting, a PFS(B) / PFS(A) ratio of ≧1.3 was used to indicate that the treatment selected by molecular profiling would benefit the patient (Robert Temple, Clinical measurement in drug evaluation. Edited by Wu Ningano and GT Thicker John Wiley and Sons Ltd. 1995;Von Hoff, DD Clin Can Res. 4: 1079, 1999:Dhani et al. Clin Cancer Res. 15: 118-123, 2009). Other methods of comparing the treatment selected by molecular profiling with the treatment selected without molecular profiling include determining the response rate (RECIST) and the percentage of patients who have not progressed or died at 4 months. The term "about" when used in relation to a numerical value of PFS means a variation of + / - 10 percent (10%) for the numerical value. The PFS of the treatment selected by molecular profiling can be extended by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% compared to the treatment selected without molecular profiling. In some embodiments, the PFS of the treatment selected by molecular profiling can be extended by at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least about 1000% compared to the treatment selected without molecular profiling. In still other embodiments, the PFS ratio (PFS on molecular profiling selected therapy or new therapy / PFS on previous therapy or therapy) is at least about 1.3.In still other embodiments, the PFS ratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0. In still other embodiments, the PFS ratio is at least about 3, 4, 5, 6, 7, 8, 9, or 10.

[0183] Similarly, DFS can be compared in patients whose treatment is selected with or without molecular profiling.In some embodiments, DFS of treatment selected by molecular profiling is extended by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% compared to non-molecular profiling selected treatment.In some embodiments, DFS of treatment selected by molecular profiling can be extended by at least 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or at least about 1000% compared to non-molecular profiling selected treatment.In still other embodiments, DFS ratio (DFS of treatment or new treatment selected by molecular profiling / DFS of previous treatment or treatment) is at least about 1.3. In still other embodiments, the DFS ratio is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0. In still other embodiments, the DFS ratio is at least about 3, 4, 5, 6, 7, 8, 9, or 10.

[0184] In some embodiments, although molecular profiling provides valuable patient benefits, the candidate treatment of the present invention does not increase the PFS ratio or DFS ratio in patients.For example, in some cases, no preferred treatment has been identified for patients.In such cases, molecular profiling provides a way to identify candidate treatments when none are currently identified.Molecular profiling can extend PFS, DFS, or life span by at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or 2 years. Molecular profiling can extend PFS, DFS, or life span by at least 2.5, 3, 4, 5, or more years. In some embodiments, the methods of the present invention improve outcomes such that patients are in remission.

[0185] The effectiveness of treatment can also be monitored by other means. Complete remission (CR) includes the complete disappearance of disease: no disease is evident on examination, scan, or other examination. Partial remission (PR) refers to a 30% or greater reduction in the size or number of lesions, although some disease remains in the body. Stable disease (SD) refers to disease in which the size and number of lesions remain relatively unchanged. Generally, a reduction of less than 50% in size or a slight increase is described as stable disease. Progressive disease (PD) means that the disease has increased in size or number on treatment. In some embodiments, molecular profiling according to the present invention results in complete or partial remission. In some embodiments, the method of the present invention results in stable disease. In some embodiments, the present invention can achieve stable disease when non-molecular profiling would lead to progressive disease.

[0186] Computer Systems Conventional data networking, application development, and other functional aspects of the system (and constituent parts of the individual operating components of the system) may not be described in detail herein, but are part of the present invention. Furthermore, the connecting lines shown in the various figures contained herein are intended to illustrate example functional relationships and / or physical connections between the various elements. It should be noted that many alternative or additional functional relationships or physical connections may exist in an actual system.

[0187] The various system components discussed herein may include one or more of the following: a host server or other computer system including a processor for processing digital data; a memory coupled to the processor for storing digital data; an input digitizer coupled to the processor for inputting digital data; an application program stored in the memory and accessible by the processor for directing the processing of the digital data by the processor; a display coupled to the processor and the memory for displaying information derived from the digital data processed by the processor; and a plurality of databases. The various databases used herein may include the following: patient data such as family history, demographic and environmental data, biosample data, previous treatment and protocol data, patient clinical data, molecular profiling data of biosamples, data regarding therapeutic and / or investigational drugs, gene libraries, disease libraries, drug libraries, patient tracking data, file management data, financial management data, billing data, and / or similar data useful in the operation of the system. As one skilled in the art will appreciate, the user computer may include an operating system (e.g., Windows NT, 95 / 98 / 2000, OS2, UNIX, Linux, Solaris, MacOS, etc.) as well as various conventional supporting software and drivers that typically accompany a computer. The computer may include any suitable personal computer, network computer, workstation, minicomputer, mainframe, etc. The user computer may be in a home or medical / business environment with access to a network. In an exemplary embodiment, access is by network or internet via a commercially available web browser software package.

[0188] The term "network" as used herein includes any electrical communication means incorporating both its hardware and software components. Communication between the parties may be accomplished by any suitable communication channel, such as, for example, telephone networks, extranets, intranets, the Internet, points of interaction devices, personal digital assistants (e.g., Palm Pilot, Blackberry, mobile phones, kiosks, etc.), online communications, satellite communications, offline communications, wireless communications, transponder communications, local area networks (LANs), wide area networks (WANs), networked or connected devices, keyboards, mice, and / or any suitable communication or data entry modality. Furthermore, although the present system is frequently described herein as running on the TCP / IP communication protocol, the present system may also be run using IPX, Appletalk, IP-6, NetBIOS, OSI, or any number of existing or future protocols. When the network is in the nature of a public network such as the Internet, it may be beneficial to presume that the network is not secure and open to eavesdroppers. Specific information regarding protocols, standards, and application software utilized in connection with the Internet is generally known to those skilled in the art and need not be described herein. See, for example, DILIP NAIK, INTERNET STANDARDS AND PROTOCOLS (1998); JAVA 2 COMPLETE, various authors, (Sybex 1999); DEBORAH RAY AND ERIC RAY, MASTERING HTML 4.0 (1997); and LOSHIN, TCP / IP CLEARLY EXPLAINED (1997), and DAVID GOURLEY AND BRIAN TOTTY, HTTP, THE DEFINITIVE GUIDE (2002), the contents of which are incorporated herein by reference.

[0189] The various system components may be suitably coupled, independently, individually, or collectively, to a network via data links, which typically include a connection to an Internet Service Provider (ISP) through a local loop, for example, as used in connection with standard modem communications, cable modem, Dish Network, ISDN, Digital Subscriber Line (DSL), or various wireless communication methods. See, for example, GILBERT HELD, UNDERSTANDING DATA COMMUNICATIONS (1996), which is incorporated herein by reference. It should be noted that the network may also be implemented as other types of networks, such as an interactive television (ITV) network. Additionally, the present system contemplates the use, sale, or distribution of any goods, services, or information over any network having similar functionality as described herein.

[0190] "Transmitting" as used herein may include sending electronic data from one system component to another over a network connection. Additionally, "data" as used herein may include generic information such as commands, queries, files, data for storage, etc., in digital or any other form.

[0191] The system is intended for use in connection with web services, utility computing, pervasive and personalized computing, security and identity solutions, autonomous computing, commodity computing, mobile and wireless solutions, open source, biometrics, grid computing, and / or mesh computing.

[0192] Any database discussed herein may include a relational, hierarchical, graphical, or object-oriented structure, and / or any other database organization. Common database products that may be used to implement the database include DB2 by IBM (White Plains, NY), various database products available from Oracle Corporation (Redwood Shores, CA), Microsoft Access or Microsoft SQL Server by Microsoft Corporation (Redmond, Washington), or any other suitable database product. Furthermore, the database may be organized in any suitable manner, for example, as a data table or a lookup table. Each record may be a single file, a set of files, a set of linked data fields, or any other data structure. Association of specific data may be accomplished by any desired data association technique, such as association techniques known or implemented in the art. For example, association may be accomplished either manually or automatically. Automatic association techniques may include, for example, database search, database merge, GREP, AGREP, SQL, using key fields in tables for fast searching, sequential searching across all tables and files, sorting records in a file according to a known order to simplify searching, and / or the like. The association step may be accomplished, for example, by a database merge function using, for example, "key fields" in a preselected database or data sector.

[0193] More specifically, a "key field" divides the database according to high level classes of objects defined by the key field. For example, a particular type of data may be designated as a key field in multiple related data tables, and the data tables may then be linked based on the type of data in the key field. The data corresponding to the key field in each of the linked data tables is preferably the same or of the same type. However, data tables having similar, if not identical, data in the key field may also be linked, for example, by using AGREP. According to one embodiment, any suitable data storage technique may be utilized to store the data without a standard format. Datasets may be stored using any suitable technique, including, for example, storing individual files using the ISO / IEC 7816-4 file structure; implementing a domain where a dedicated file publishes one or more base files containing one or more datasets; using datasets stored in individual files using a hierarchical filing system; datasets stored as records within a single file (including compressed, SQL accessible, hashed via one or more keys, numeric, alphabetic, etc. with a first tuple); binary large objects (BLOBs); stored as ungrouped data elements coded using ISO / IEC 7816-6 data elements; stored as ungrouped data elements coded using ISO / IEC Abstract Syntax Notation (ASN.1), such as in ISO / IEC 8824 and 8825; and / or other proprietary techniques, which may include fractal compression techniques, image compression techniques, etc.

[0194] In one exemplary embodiment, the ability to store a wide variety of information in different formats is facilitated by storing the information as a BLOB. Thus, any binary information may be stored in a storage area associated with a dataset. The BLOB method may store datasets as ungrouped data elements formatted as binary blocks via fixed memory offsets using either fixed storage allocation, circular queue techniques, or best practices for memory management (e.g., least recently used paged memory, etc.). Using the BLOB method, the ability to store various datasets with different formats facilitates storage of data by multiple and unrelated owners of the datasets. For example, a first dataset that may be stored may be provided by a first party, a second dataset that may be stored may be provided by an unrelated second party, and a third dataset that may be stored may be provided by a third party that is unrelated to the first and second parties. Each of these three exemplary datasets may include different information stored using different data storage formats and / or techniques. Furthermore, each dataset may include a subset of data that may also differ from the other subsets.

[0195] As noted above, in various embodiments, data may be stored without regard to a common format. However, in one exemplary embodiment, data sets (e.g., BLOBs) may be annotated in a standard manner when provided for manipulating the data. The annotations may include short headers, trailers, or other suitable indicators associated with each data set that are configured to convey information useful for managing the various data sets. For example, the annotations may be referred to herein as "conditional headers," "headers," "trailers," or "status," and may include an indication of the status of the data set, or may include an identifier that correlates to a particular publisher or owner of the data. Subsequent bytes of data may be used to indicate, for example, the identity of the publisher or owner of the data, a user, a transaction / membership account identifier, etc. Each of these conditional annotations is discussed further herein.

[0196] Dataset annotations may also be used for other types of status information and various other purposes. For example, the dataset annotations may include security information that establishes access levels. The access levels may be configured, for example, to allow only certain individuals, levels of employees, companies, or other entities to access the dataset, or to allow access to certain datasets based on transactions, data issuers or owners, users, etc. Additionally, the security information may restrict / allow only certain actions, such as accessing the dataset, modifying the dataset, and / or deleting the dataset. In one example, the dataset annotation indicates that only the owner or user of the dataset is allowed to delete the dataset, that various identified users may be allowed to access the dataset for reading, and others are completely barred from accessing the dataset. However, other access restriction parameters may also be used that allow various entities to access the dataset with various permission levels as needed. The data, including the header or trailer, may be received by a standalone interaction device configured to add, delete, modify, or augment the data according to the header or trailer.

[0197] Those skilled in the art will also understand that for security reasons, any database, system, device, server, or other component of the system may consist of any combination thereof in a single location or multiple locations, and each database or system may include any of a variety of appropriate security features, such as firewalls, access codes, encryption, decryption, compression, decompression, and / or the like.

[0198] The web client computing device may further comprise an internet browser connected to the internet or an intranet using standard dial-up, cable, DSL, or any other internet protocol known in the art. Transactions originating from the web client may pass through a firewall to prevent unauthorized access from users of other networks. Additionally, additional firewalls may be deployed between various components of the CMS to further enhance security.

[0199] The firewall may include any hardware and / or software appropriately configured to protect the CMS components and / or the enterprise's computing resources from users of other networks. Additionally, the firewall may be configured to limit or restrict access to various systems and components behind the firewall for web clients connecting through the web server. Firewalls may exist in a variety of configurations including stateful inspection, proxy-based, and packet filtering, among others. The firewall may be integrated within the web server or any other CMS component, or may even exist as a separate entity.

[0200] The computers discussed herein may provide a suitable website or other Internet-based graphical user interface accessible by a user. In one embodiment, Microsoft Internet Information Server (IIS), Microsoft Transaction Server (MTS), and Microsoft SQL Server are used in conjunction with a Microsoft operating system, Microsoft NT web server software, Microsoft SQL Server database system, and Microsoft Commerce Server. Additionally, components such as Access or Microsoft SQL Server, Oracle, Sybase, Informix MySQL, Interbase, etc. may be used to provide an Active Data Object (ADO)-enabled database management system.

[0201] Any of the communication, input, storage, database, or display discussed herein may be facilitated through a website having a web page. The term "web page" as used herein is not intended to limit the types of documents and applications that may be used to interact with a user. For example, a typical website may include various formats, Java applets, JavaScript, Active Server Pages (ASP), Common Gateway Interface Script (CGI), Extensible Markup Language (XML), Dynamic HTML, Cascading Style Sheets (CSS), helper applications, plug-ins, etc., in addition to standard HTML documents. The server may include a web service that receives a request from the web server, the request including a URL (http: / / yahoo.com / stockquotes / ge) and an IP address (123.56.789.234). The web server retrieves the appropriate web page and transmits the data or application of the web page to the IP address. A web service is an application that may interact with other applications over a communication medium such as the Internet. Web services are typically based on standards or protocols such as XML, XSLT, SOAP, WSDL, and UDDI. Web services methods are well known in the art and are covered in many standard textbooks, see, for example, ALEX NGHIEM, IT WEB SERVICES: A ROADMAP FOR THE ENTERPRISE (2003), incorporated herein by reference.

[0202] The web-based clinical database for the systems and methods of the present invention preferably has the ability to upload and store clinical data files in native format and is searchable for any clinical parameter. The database is also extensible and can utilize the EAV data model (metadata) to populate clinical annotations from any study for easy integration with other studies. In addition, the web-based clinical database is adaptable and can be XML and XSLT capable of dynamically adding user-customized questions. Additionally, the database includes export capabilities to CDISC ODM.

[0203] Implementers will also appreciate that there are several ways to display data in a browser-based document. Data may be displayed as standard text or in a fixed list, a scrollable list, a drop-down list, an editable text area, a fixed text area, a pop-up window, etc. Similarly, there are several ways available to modify data in a web page, such as, for example, free text entry using a keyboard, selecting menu items, check boxes, option boxes, etc.

[0204] The present system and method may be described herein in terms of functional block components, screenshots, option selections, and various processing stages. It should be understood that such functional blocks may be realized by any number of hardware and / or software components configured to perform specific functions. For example, the present system may use various integrated circuit components, such as memory elements, processing elements, logic elements, look-up tables, etc., that may perform various functions under the control of one or more microprocessors or other control devices. Similarly, the software elements of the present system may be implemented by any programming or scripting language, such as C, C++, Macromedia Cold Fusion, Microsoft Active Server Pages, Java, COBOL, Assembler, PERL, Visual Basic, SQL Stored Procedures, Extensible Markup Language (XML), etc., and various algorithms are implemented by any combination of data structures, objects, processes, routines, or other programming elements. Furthermore, it should be noted that the present system may use any number of conventional techniques for data transmission, signaling, data processing, network control, etc. Still further, the present system may be used to detect or prevent security issues by client-side scripting languages, such as JavaScript, VBScript, etc.For a basic introduction to cryptography and network security, please see any of the following references: (1) "Applied Cryptography: Protocols, Algorithms, And Source Code In C" by Bruce Schneier, published by John Wiley & Sons (second edition, 1995); (2) "Java Cryptography" by Jonathan Knudson, published by O'Reilly & Associates (1998); (3) "Cryptography & Network Security: Principles & Practice" by William Stallings, published by Prentice Hall; all of which are incorporated herein by reference.

[0205] As used herein, the terms "end user," "consumer," "customer," "client," "treating physician," "hospital," or "business" may be used interchangeably and refer to any person, entity, machine, hardware, software, or business, respectively. Each participant is equipped with a computing device to interact with the system and facilitate online data access and data entry. The customer has a computing device in the form of a personal computer, although other types of computing devices may be used, including laptops, notebooks, handheld computers, set-top boxes, cell phones, touch-tone phones, and the like. The owner / operator of the system and method of the present invention has a computing device implemented in the form of a computer server, although other implementations with systems are contemplated, including computing centers depicted as mainframe computers, minicomputers, PC servers, networks of computers located at the same or different geographic locations, and the like. Additionally, the system contemplates the use, sale, or distribution of any goods, services, or information over any network having similar functionality as described herein.

[0206] In one exemplary embodiment, each client customer may be issued an "account" or "account number." As used herein, an account or account number may include any device, code, number, letter, symbol, digital certificate, smart chip, digital signal, analog signal, biometric, or other identifier / indicia (e.g., one or more of an authorization / access code, personal identification number (PIN), Internet code, other identification code, and / or the like) suitably configured to allow a consumer to access, interact with, or communicate with the system. The account number may optionally be located on or associated with a charge card, credit card, debit card, prepaid card, embossed card, smart card, magnetic stripe card, bar code card, transponder, radio frequency card, or associated account. The system may include or interface with any of the aforementioned cards or devices, or a fob having a transponder and an RFID reader in RF communication with the fob. While the system may include an embodiment of a fob, the invention is not so limited. In fact, the system may include any device having a transponder configured to communicate with an RFID reader via RF communication. Exemplary devices may include, for example, a key fob, a tag, a card, a cell phone, a watch, or any such form that may be presented for interrogation. Additionally, the systems, computing units, or devices discussed herein may include "pervasive computing devices," which may include traditional non-computerized devices with embedded computing units. Account numbers may be distributed and stored in any form of plastic, electronic, magnetic, radio frequency, wireless, acoustic, and / or optical device that is capable of transmitting or downloading data from itself to a second device.

[0207] As will be appreciated by those skilled in the art, the system may be embodied as a customization of an existing system, an add-on product, upgraded software, a stand-alone system, a distributed system, a method, a data processing system, a device for data processing, and / or a computer program product. Thus, the system may take the form of an entirely software embodiment, an entirely hardware embodiment, or an embodiment combining both software and hardware aspects. Furthermore, the system may take the form of a computer program product on a computer readable storage medium having computer readable program code means embodied in the storage medium. Any suitable computer readable storage medium may be utilized, including hard disks, CD-ROMs, optical storage devices, magnetic storage devices, and / or the like.

[0208] The present systems and methods are described herein with reference to screenshots, block diagrams, and flowchart illustrations of methods, apparatus (e.g., systems), and computer program products according to various aspects. It will be understood that each functional block of the block diagrams and flowchart illustrations, and combinations of functional blocks in the block diagrams and flowchart illustrations, respectively, can be implemented by computer program instructions.

[0209] Referring now to Figures 2-25, the illustrated process flows and screenshots are merely exemplary and are not intended to limit the scope of the invention described herein. For example, the steps recited in any of the method or process descriptions may be performed in any order and are not limited to the order shown. It will be understood that the following description appropriately refers to the steps and user interface elements shown in Figures 2-25 as well as the various system components described above with reference to Figure 1.

[0210] These computer program instructions can be loaded into a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, where the instructions executing on the computer or other programmable data processing apparatus create means for performing the functions specified in one or more of the flowchart blocks. These computer program instructions can also be stored in a computer readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, where the instructions stored in the computer readable memory create a product that includes instruction means for performing the functions specified in one or more of the flowchart blocks. Computer program instructions can also be loaded into a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce computer-implemented processes, where the instructions executing on the computer or other programmable apparatus provide steps for performing the functions specified in one or more of the flowchart blocks.

[0211] Thus, the functional blocks of the block diagrams and flowchart diagrams support combinations of means for performing a particular function, combinations of steps for performing a particular function, and program instruction means for performing a particular function. It will also be understood that each functional block of the block diagrams and flowchart diagrams, and combinations of functional blocks in the block diagrams and flowchart diagrams, can be executed by either a special purpose hardware-based computer system performing a particular function or step, or an appropriate combination of special purpose hardware and computer instructions. Furthermore, the process flow diagrams and their descriptions may refer to user windows, web pages, websites, web forms, prompts, and the like. The practitioner will understand that the illustrated steps described herein may be included in any number of configurations, including the use of windows, web pages, web forms, pop-up windows, prompts, and the like. It should be further understood that the multiple steps illustrated and described may be integrated into a single web page and / or window, but have been expanded for simplicity. In other cases, steps illustrated and described as a single process step may be divided into multiple web pages and / or windows, but have been integrated for simplicity.

[0212] Advantages, other advantages, and solutions to problems have been described herein with respect to certain embodiments. However, the advantages, benefits, solutions to problems, and any elements that may provide or make any advantages, benefits, or solutions more apparent should not be construed as critical, necessary, or essential features or elements of any or all of the claims of the present invention. Thus, the scope of the present invention should not be limited by anything other than the claims, and in the claims, any reference to an element in the singular is not intended to mean "one and only one" unless expressly stated, but is intended to mean "one or more." All structural, chemical, and functional equivalents to the elements of the above exemplary embodiments known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims of the present invention. Moreover, it is not necessary for an apparatus or method to address each and every problem that the present invention seeks to solve in order to be encompassed by the claims of the present invention. Furthermore, no element, component, or method step in this disclosure is intended to be made available to the public, regardless of whether the element, component, or method step is expressly recited in the claims. No element of the claims herein is to be construed pursuant to the provisions of 35 USC 112, Chapter 6, unless the element is expressly recited using the phrase "means for." As used herein, "comprises," "comprising," or any other variation thereof is intended to cover a non-exclusive inclusion, and a process, method, article, or apparatus comprising a set of elements does not include only such elements, but may also include other elements not expressly recited or inherent to such process, method, article, or apparatus. Furthermore, no element described herein is necessary for the practice of the invention unless expressly recited as "essential" or "critical."

[0213] 1 shows a block diagram of an exemplary embodiment of a system 10 for determining personalized medical interventions for a particular disease state utilizing molecular profiling of a patient's biological specimen. System 10 includes a user interface 12, a host server 14 including a processor 16 for processing data, a memory 18 coupled to the processor, an application program 20 stored within memory 18 and accessible by processor 16 for directing the processing of data by processor 16, a number of internal databases 22, and an external database 24, as well as an interface to a wired or wireless communication network 26 (such as the Internet, for example). System 10 may also include an input digitizer 28 coupled to processor 16 for inputting digital data from data received from user interface 12.

[0214] The user interface 12 includes an input device 30 and a display 32 for inputting data into the system 10 and for displaying information derived from the data processed by the processor 16. The user interface 12 may also include a printer 34 for printing information derived from the data processed by the processor 16, such as a patient report, which may include test results for the target and suggested medical therapy based on the test results.

[0215] The internal databases 22 may include, but are not limited to, patient biosample / specimen information and tracking, clinical data, patient data, patient tracking, file management, research protocols, patient test results with molecular profiling, and billing information and tracking. The external databases 24 may include, but are not limited to, drug libraries, gene libraries, disease libraries, and public and private databases such as UniGene, OMIM, GO, TIGR, GenBank, KEGG, and Biocarta.

[0216] Molecular Profiling Methods Various methods can be used according to the system 10. FIG. 2 shows a flow chart of an exemplary embodiment of a method 50 for determining personalized medical intervention for a particular disease state utilizing molecular profiling of a patient's biological specimen that is not disease specific. To determine medical intervention for a particular disease state using molecular profiling that is not dependent on disease series diagnosis (i.e., not limited to a single disease), at least one test is performed on at least one target from a biological sample of an affected patient in step 52. A target is defined as any molecular finding that can be obtained from a molecular test. For example, a target can include one or more genes, one or more gene expressed proteins, one or more molecular mechanisms, and / or a combination thereof. For example, the expression level of a target can be measured by analyzing the mRNA level of the target or gene, or the protein level of the gene. Tests for finding such targets can include, but are not limited to, fluorescent in situ hybridization (FISH), in situ hybridization (ISH), and other molecular tests known to those skilled in the art. PCR-based methods can also be used, such as real-time PCR or quantitative PCR. Furthermore, in the methods disclosed herein, microarray analysis can be used, such as comparative genomic hybridization (CGH) microarray, single nucleotide polymorphism (SNP) microarray, proteomics array, or antibody array analysis.In some embodiments, the microarray analysis comprises identifying whether a gene is up-regulated or down-regulated with a significance of p<0.001 compared to a reference.Target testing or analysis can also comprise immunohistochemical (IHC) analysis.In some embodiments, the IHC analysis comprises determining whether 30% or more of the sample is stained, whether the staining intensity is +2 or more, or both.

[0217] In addition, the methods disclosed herein also include profiling two or more targets. For example, the expression of multiple genes can be identified. Furthermore, the identification of multiple targets in a sample can be by one method or by various means. For example, the expression of a first gene can be measured by one method, and the expression level of a second gene can be measured by a different method. Alternatively, the same method can be used to detect the expression levels of the first gene and the second gene. For example, the first method can be IHC, and the second method can be by microarray analysis to detect gene expression of the gene.

[0218] In some embodiments, molecular profiling can also include identifying genetic variants such as mutations, polymorphisms (such as SNPs), deletions, or insertions of targets.For example, the identification of SNPs in genes can be determined by microarray analysis, real-time PCR, or sequencing.Other methods disclosed herein can also be used to identify variants of one or more targets.

[0219] Thus, one or more of the following may be performed: IHC analysis in step 54, microanalysis in step 56, and other molecular tests known to those skilled in the art in step 58.

[0220] A biological sample is obtained from an affected patient by taking a biopsy specimen of the tumor, by performing minimally invasive surgery, or, if no recent tumor is available, by taking a sample of the patient's blood, or a cell extract, nuclear extract, cell lysate, or any other biological fluid including, but not limited to, a biological product or substance of biological origin, such as excreta, blood, serum, plasma, urine, sputum, tears, stool, saliva, membrane extract, etc.

[0221] In step 60, a determination is made as to whether one or more of the targets tested in step 52 show altered expression compared to the normal reference for that particular target. In one exemplary method of the present invention, an IHC analysis can be performed in step 54, and a determination is made as to whether any target shows altered expression by IHC analysis in step 64 by determining whether 30% or more of the biological sample cells for the particular target stained +2 or higher. It will be understood by those skilled in the art that staining results may vary depending on the technician performing the test and the type of target being tested, so that staining +1 or higher may indicate altered expression. In another exemplary embodiment of the present invention, a microarray analysis can be performed in step 56, and a determination is made as to whether any target shows altered expression by microarray analysis in step 66 by determining whether the fold change in expression of the particular target relative to the normal tissue of origin reference is significant at p< 0.001 to identify which targets are up-regulated or down-regulated. Altered expression can also be evidenced by the absence of one or more genes, gene expression proteins, molecular mechanisms, or other molecular findings.

[0222] After determining which targets show altered expression in step 60, at least one non-disease specific agent that interacts with each target that has altered expression is identified in step 70. The agent can be any drug or compound that has a therapeutic effect. A non-disease specific agent is a therapeutic agent or compound that has not previously been associated with the treatment of the patient's diagnosed disease that can interact with targets from the patient's biological sample that show altered expression. Some of the non-disease specific agents that have been found to interact with specific targets found in various cancer patients are shown in Table 3 below.

[0223] (Table 3) TIFF0007659585000049.tif72151

[0224] Finally, in step 80, a patient profile report can be provided that includes the patient's test results for various targets and any suggested treatments based on those results. An exemplary patient profile report 100 is shown in Figures 3A-3D. The patient profile report 100 shown in Figure 3A identifies targets tested 102, those tested targets 104 that showed significant changes in expression, and non-disease specific drugs 106 proposed to interact with the targets. The patient profile report 100 shown in Figure 3B identifies the results 108 of immunohistochemical analysis for a particular gene expression protein 110, and whether the gene expression protein is a molecular target 112 by determining whether 30% or more of the tumor cells stained +2 or higher. The report 100 also identifies immunohistochemical tests 114 that were not performed. The patient profile report 100 shown in Figure 3C identifies genes analyzed in the microarray analysis 116, and whether the genes were under- or over-expressed 118 compared to a reference. Finally, the patient profile report 100 shown in Figure 3D identifies the patient's clinical history 120 and the specimens submitted by the patient 122. Molecular profiling techniques can be performed anywhere, for example overseas, and the results can be transmitted over a network to appropriate parties, for example the patient, a physician, a laboratory, or other parties located remotely.

[0225] FIG. 4 shows a flow chart of an exemplary embodiment of a method 200 for identifying a drug therapy / drug that can interact with a target. In step 202, a molecular target that shows an altered expression in some diseased individuals is identified. Then, in step 204, a drug therapy / drug is administered to the diseased individuals. In step 206, any alterations in the molecular target identified in step 202 are identified after administration of the drug therapy / drug to determine whether the drug therapy / drug administered in step 204 interacts with the molecular target identified in step 202. If it is determined that the drug therapy / drug administered in step 204 interacts with the molecular target identified in step 202, rather than approving the drug therapy / drug for a particular disease, the drug therapy / drug can be approved for treating patients who show an altered expression of the identified molecular target.

[0226] 5-14 are flow charts and diagrams showing various parts of the information-based personalized medicine discovery system and method according to the present invention. FIG. 5 is a diagram showing an exemplary clinical decision support system of the information-based personalized medicine discovery system and method according to the present invention. Data obtained through clinical research and clinical care, such as clinical trial data, biomedical / molecular imaging data, genomics / proteomics / chemical libraries / literature / expert curation, biospecimen tracking / LIMS, family history / environmental records, and clinical data, are collected and stored as databases and data marts in a data warehouse. FIG. 6 is a diagram showing the flow of information through the clinical decision support system of the information-based personalized medicine discovery system and method according to the present invention using web services. Users interact with the system by inputting data into the system via form-based input / upload of data sets, formulating queries and running data analysis jobs, and obtaining and evaluating displays of output data. The data warehouse in a web-based system is where data is extracted, transformed, and loaded from various database systems. The data warehouse is also where common formatting, mapping, and transformation occurs. The web-based system also includes a data mart that is created based on the data view of interest.

[0227] A flow chart of an exemplary clinical decision support system of the information-based personalized medicine discovery system and method of the present invention is shown in Figure 7. The clinical information management system includes a laboratory information management system, and the medical information contained in the data warehouse and database includes medical information libraries such as drug libraries, gene libraries, and disease libraries, in addition to literature text mining. Both the information management system and the medical information database and data warehouse for a specific patient are united in a data linking center where diagnostic information and treatment options can be obtained. A financial management system can also be incorporated into the clinical decision support system of the information-based personalized medicine discovery system and method of the present invention.

[0228] FIG. 8 shows an exemplary biological specimen tracking and management system that can be utilized as part of the information-based personalized medicine discovery system and method of the present invention. FIG. 8 shows two host medical centers that transfer specimens to tissue / blood banks. The specimens can go through laboratory analysis before shipping. Research can be performed on the samples via microarray, genotyping, and proteomic analysis. This information can be redistributed to the tissue / blood banks. FIG. 9 shows a flow chart of an exemplary biological specimen tracking and management system that can be utilized with the information-based personalized medicine discovery system and method of the present invention. The host medical center obtains samples from patients and then ships the patient samples to molecular profiling laboratories that can also perform RNA and DNA isolation and analysis.

[0229] A diagram illustrating a method for maintaining a standard clinical vocabulary for use with the informed personalized medicine discovery system and method of the present invention is shown in Figure 10. Figure 10 illustrates how physician findings and patient information related to one physician's patient may be made accessible to another physician so that the physician can utilize the data in making diagnostic and therapeutic decisions for that patient.

[0230] FIG. 11 shows a schematic diagram of an exemplary microarray gene expression database that can be used as part of the information-based personalized medicine discovery system and method of the present invention. The microarray gene expression database includes both external and internal databases accessible via a web-based system. External databases can include, but are not limited to, UniGene, GO, TIGR, GenBank, KEGG. Internal databases can include, but are not limited to, tissue tracking, LIMS, clinical data, and patient tracking. FIG. 12 shows a diagram of an exemplary microarray gene expression database data warehouse that can be used as part of the information-based personalized medicine discovery system and method of the present invention. Laboratory data, clinical data, and patient data can all be housed in the microarray gene expression database data warehouse, which can then be accessed by public / private access and utilized by data analysis tools.

[0231] Another schematic diagram showing the flow of information through the information-based personalized medicine discovery system and method of the present invention is shown in Figure 13. Similar to Figure 7, this schematic diagram includes the clinical information management, medical and literature information management, and financial management of the information-based personalized medicine discovery system and method of the present invention. Figure 14 is a schematic diagram showing an exemplary network of the information-based personalized medicine discovery system and method of the present invention. In order to provide the patient with the proposed treatment or drug based on the various targets identified, the patient, the medical practitioner, the host medical center, and the research institute all share and exchange various information.

[0232] Figures 15-25 are computer screen printouts associated with various portions of the information based personalized medicine discovery system and method shown in Figures 5-14. Figures 15 and 16 show computer screens where physician and insurance company information is entered on behalf of a client. Figures 17-19 show computer screens where information can be entered to order analyses and tests on patient samples.

[0233] Figure 20 is a computer screen showing the results of a microarray analysis of specific genes tested using a patient sample. This information and computer screen is similar to the information detailed in the Patient Profile Report shown in Figure 3C. Figure 22 is a computer screen showing the results of immunohistochemistry testing of a specific patient for various genes. This information is similar to the information contained in the Patient Profile Report shown in Figure 3B.

[0234] FIG. 21 is a computer screen showing options for finding a particular patient, ordering tests and / or results, publishing patient reports, and tracking current cases / patients.

[0235] Figure 23 is a computer screen outlining some of the steps for generating a patient profile report as shown in Figures 3A to 3D. Figure 24 shows a computer screen for ordering immunohistochemistry tests on a patient sample, and Figure 25 shows a computer screen for inputting information regarding the primary tumor site for microarray analysis. It will be understood by those skilled in the art that any number and type of computer screens can be used to input the information required for purposes of utilizing the information-based personalized medicine discovery system and method of the present invention, and to obtain information resulting from utilizing the information-based personalized medicine discovery system and method of the present invention.

[0236] Figures 26-31 show tables showing the frequency of significant changes in expression of specific genes and gene-expressed proteins by tumor type, i.e., the number of times that a gene and / or gene-expressed protein was flagged as a target by tumor type as being significantly over- or under-expressed (see also Examples 1-3). These tables show the total number of times that a gene and / or gene-expressed protein is over- or under-expressed in a particular tumor type, and whether the change in expression was determined by immunohistochemistry analysis (Figures 26, 28) or by microarray analysis (Figures 27, 30). These tables also identify the total number of times that overexpression of any gene-expressed protein occurred in a particular tumor type using immunohistochemistry, and the total number of times that overexpression or underexpression of any gene occurred in a particular tumor type using microarray analysis.

[0237] Thus, the present invention provides a method and system for analyzing diseased tissue using IHC test and gene microarray test according to the IHC and microarray test described above.Patients may be in advanced stages of disease.The biomarker pattern or biomarker signature set can be determined in several tumor types, diseased tissue types, or diseased cells, including fat, adrenal cortex, adrenal gland, adrenal-medulla, appendix, bladder, blood vessel, bone, osteochondral, brain, breast, cartilage, cervix, colon, sigmoid colon, dendritic cell, skeletal muscle, endometrium, esophagus, fallopian tube, fibroblast, gallbladder, kidney, larynx, liver, lung, lymph node, melanocyte, mesothelial lining, myoepithelial cell, osteoblast, ovary, pancreas, parotid gland, prostate, salivary gland, sinus tissue, skeletal muscle, skin, small intestine, smooth muscle, stomach, synovium, joint lining tissue, tendon, testis, thymus, thyroid, uterus, and uterine body.

[0238] The method of the invention is directed to the treatment of cancer of the breast, pancreas, colon and / or rectum, leukemia, skin, bone, prostate, liver, lung, brain, larynx, gallbladder, parathyroid, thyroid, adrenal gland, nervous tissue, head and neck, stomach, bronchus, kidney, basal cell carcinoma, ulcerative and papillary squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, myeloma, giant cell tumor, small cell lung tumor, islet cell carcinoma, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma, enteric ganglion cell tumor, hyperplastic corneal nerve tumor, marfanoid habitus tumor ... tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma, enteric ganglion cell tumor, hyperplastic corneal nerve tumor, marfanoid habitus tumor, myeloma, giant cell tumor, small cell lung tumor, myeloma, giant cell tumor, small cell lung tumor, myeloma, giant cell tumor, small cell lung tumor, myeloma, giant cell tumor, small cell lung tumor, myeloma, giant cell tumor, small cell lung The present invention may be used to select the treatment of any cancer, including, but not limited to, ovarian tumor, Wilms' tumor, seminoma, ovarian tumor, leiomyoma, cervical dysplasia and carcinoma in situ, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, localized skin lesions, mycosis fungoides, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcomas, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforme, leukemia, lymphoma, malignant melanoma, and epidermoid carcinoma.

[0239] Biomarker patterns or biomarker signature sets in several tumor types, affected tissue types, or affected cells, including: paranasal sinuses, middle and inner ear, adrenal glands, appendix, hematopoietic system, bones and joints, spinal cord, breast, cerebellum, cervix, connective soft tissue, uterus, esophagus, eye, nose, eyeball, fallopian tube, extrahepatic bile duct, other oral cavity, intrahepatic bile duct, kidney, appendix-colon, larynx, lips, liver, lung and bronchi, lymph nodes, cerebrum, spinal cord, nasal cartilage, retina, eye, other endocrine glands except unspecified, other female reproductive organs, ovaries, pancreas, penis and scrotum, pituitary gland, pleura, prostate, rectum renal pelvis, ureters, peritoneum, salivary glands, skin, small intestine, stomach, testes, thymus, thyroid, tongue, unknown, bladder, uterus, unspecified, vagina and labia, and vulva, unspecified cancer, may also be determined.

[0240] Therefore, biomarker pattern or biomarker signature set can be used to determine the therapeutic agent or treatment protocol that can interact with said biomarker pattern or signature set.For example, for advanced breast cancer, immunohistochemistry analysis can be used to determine one or more gene expression proteins that are overexpressed.Therefore, biomarker pattern or biomarker signature set can be identified for advanced breast cancer, and therapeutic agent or treatment protocol that can interact with said biomarker pattern or signature set can be identified.

[0241] This example of a biomarker pattern or biomarker signature set for advanced stage breast cancer is only one example of the many biomarker patterns or biomarker signatures for several advanced stage diseases or cancers that can be identified from the tables shown in Figures 26-31. Additionally, by utilizing the method steps of the present invention described above as shown in Figures 1-2 and Figures 5-14, several non-disease specific therapies or treatment protocols can be identified for treating patients with these biomarker patterns or biomarker signature sets.

[0242] The biomarker patterns and / or biomarker signature sets disclosed in the tables shown in Figures 26 and 28 and the tables shown in Figures 27 and 30 can be used for several purposes, including but not limited to, detection of a particular cancer / disease, treatment of a particular cancer / disease, and identification of new drug therapies or protocols for a particular cancer / disease. The biomarker patterns and / or biomarker signature sets disclosed in the tables shown in Figures 26 and 28 and the tables shown in Figures 27 and 30 can also indicate a drug resistance expression profile of a particular tumor type or cancer type. The biomarker patterns and / or biomarker signature sets disclosed in the tables shown in Figures 26 and 28 and the tables shown in Figures 27 and 30 indicate an advanced stage drug resistance profile.

[0243] A biomarker pattern and / or a biomarker signature set may comprise at least one biomarker. In yet other embodiments, a biomarker pattern or signature set may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers. In some embodiments, a biomarker signature set or a biomarker pattern may comprise at least 15, 20, 30, 40, 50, or 60 biomarkers. In some embodiments, a biomarker signature set or a biomarker pattern may comprise at least 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 biomarkers. The analysis of one or more biomarkers may be by one or more methods. For example, the analysis of two biomarkers can be performed using a microarray. Alternatively, one biomarker can be analyzed by IHC and another biomarker can be analyzed by microarray. Any such combination of methods and biomarkers is contemplated herein.

[0244] The one or more biomarkers may include, but are not limited to, Her2 / Neu, ER, PR, c-kit, EGFR, MLH1, MSH2, CD20, p53, cyclin D1, bcl2, COX-2, androgen receptor, CD52, PDGFR, AR, CD25, VEGF, HSP90, PTEN, RRM1, SPARC, survivin, TOP2A, BCL2, HIF1A, AR, ESR1, PDGFRA, KIT, P DGFRB, CDW52, ZAP70, PGR, SPARC, GART, GSTP1, NFKBIA, MSH2, TXNRD1, HDAC1, PDGFC, PTEN, CD33, TYMS, RXRB, ADA, TN F, ERCC3, RAF1, VEGF, TOP1, TOP2A, BRCA2, TK1, FOLR2, TOP2B, MLH1, IL2RA, DNMT1, HSPCA, ERBR2, ERBB2, SSTR1, VHL, V DR, PTGS2, POLA, CES2, EGFR, OGFR, ASNS, NFKB2, RARA, MS4A1, DCK, DNMT3A, EREG, epiregulin, FOLR1, GNRH1, GNRHR1, FSHB, FSHR, FSHPRH1, folate receptor, HGF, HIG1, IL13RA1, LTB, ODC1, PPARG, PPARGC1, lymphotoxin β receptor, Myc, topoisomerase II, TOPO2B, It may be selected from the group consisting of TXN, VEGFC, ACE2, ADH1C, ADH4, AGT, AREG, CA2, CDK2, caveolin, NFKB1, ASNS, BDCA1, CD52, DHFR, DNMT3B, EPHA2, FLT1, HSP90AA1, KDR, LCK, MGMT, RRM1, RRM2, RRM2B, RXRG, SRC, SSTR2, SSTR3, SSTR4, SSTR5, VEGFA, or YES1.

[0245] For example, a biological sample from an individual can be analyzed to determine a biomarker pattern or biomarker signature set that includes biomarkers such as HSP90, survivin, RRM1, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG, or LCK. In other embodiments, the biomarkers SPARC, HSP90, TOP2A, PTEN, survivin, or RRM1 form part of the biomarker pattern or biomarker signature set. In still other embodiments, the biomarkers MGMT, SSTRS3, DNMT3B, VEGFA, SSTR4, RRM2, SRC, RRM2B, HSP90AA1, STR2, FLT1, SSTR5, YES1, BRCA1, RRM1, DHFR, KDR, EPHA2, RXRG, CD52, or LCK are included in the biomarker pattern or biomarker signature set.

[0246] The expression level of HSP90, Survivin, RRM1, SS...

Claims

1. one or more processors; When coupled to and executed by the one or more processors, the method comprises: Obtaining data indicative of the results of performing one or more molecular tests on a plurality of molecular profiling targets in a biological sample obtained from a patient having a particular cancer lineage, the one or more molecular tests comprising at least one of gene expression analysis and nucleic acid sequence analysis, the plurality of molecular profiling targets being selected from the group consisting of EGFR, SPARC, C-kit, ER, PR, androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, thymidylate synthase, Her2 / neu, TOPO2A, ADA, AR, ASNA, obtaining data including BCL2, BRCA2, CD33, CDW52, CES2, DNMT1, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70; comparing the obtained data with corresponding reference values ​​stored in a database in a computer system, where as part of said comparison, the compared reference values ​​are not associated with said particular cancer lineage; identifying at least one therapeutic agent from said comparison based on an indication from said comparison that said at least one therapeutic agent will benefit the patient, said identification being made without regard to association of said at least one therapeutic agent with any particular cancer lineage; and generating a patient profile report indicating that the at least one identified therapeutic agent is a suitable agent for the patient; A computer memory that stores instructions for performing operations including 2. A computer system comprising:

2. The operation is comparing the obtained data with corresponding additional reference values ​​stored in a database of a computer system, where as part of said comparison, the compared additional reference values ​​are associated with one or more particular cancer lineages; identifying, from comparison with the additional reference value, at least one additional therapeutic agent based on an indication from the comparison that the at least one additional therapeutic agent would benefit the patient, wherein the identification is made by considering an association of the at least one additional therapeutic agent with one or more particular cancer lineages. Further comprising: Producing a patient profile report; including said identified at least one additional therapeutic agent in the generated report as a suitable agent for said patient.

2. The system of claim 1, comprising:

3. 2. The system of claim 1, wherein the database is trained without regard to association of a particular reference value with a particular cancer lineage.

4. The operation is Causing administration of at least one of the at least one therapeutic agent to the patient.

13. The system of claim 1, further comprising:

5. The system of claim 1, wherein the at least one identified therapeutic agent is identified solely based on comparing data obtained for the molecular profiling targets, regardless of association with any particular cancer lineage.

6. 10. The system of claim 1, wherein the biological sample comprises tumor tissue.

7. 10. The system of claim 1, wherein the biological sample comprises a bodily fluid.

8. 8. The system of claim 7, wherein the bodily fluid comprises blood, a cell extract, a nuclear extract, a cell lysate, feces, serum, plasma, urine, sputum, tears, stool, saliva, or a membrane extract.

9. 10. The system of claim 1, wherein the reference value is associated with at least one individual who does not have cancer.

10. The system of claim 1, wherein the molecular profiling targets include nucleic acids and / or proteins.

11. 10. The system of claim 1, wherein the one or more molecular tests comprise microarray, genotyping, and / or proteomic analysis of a biological sample from the patient.

12. The operation is Identifying at least one ineffective therapeutic agent that will not benefit the patient from comparing the obtained data with the reference value; and including said identified at least one ineffective therapeutic agent in a generated report.

13. The system of claim 1, further comprising:

13. The operation is Identifying at least one ineffective therapeutic agent that will not benefit the patient from a comparison of the obtained data with additional reference values; and including said identified at least one ineffective therapeutic agent in a generated report.

3. The system of claim 2, further comprising:

14. 2. The system of claim 1, wherein the one or more molecular tests comprise at least one of polymerase chain reaction (PCR), pyrosequencing, real-time PCR, sequencing, next generation sequencing, methylation specific PCR (MSPCR), restriction fragment length polymorphism (RFLP) analysis, immunohistochemistry (IHC), immunoassays, expression microarrays, comparative genomic hybridization (CGH) microarrays, single nucleotide polymorphism (SNP) microarrays, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), and proteomics arrays.

15. 2. The system of claim 1, wherein the one or more molecular tests are configured to identify at least one of a mutation, polymorphism, deletion, insertion, substitution, translocation, fusion, truncation, duplication, amplification, or repeat in a nucleic acid sequence corresponding to at least one of the molecular profiling targets.

16. Obtaining data indicative of the results of performing one or more molecular tests on a plurality of molecular profiling targets in a biological sample obtained from a patient having a particular cancer lineage, the plurality of molecular profiling targets being selected from the group consisting of EGFR, SPARC, C-kit, ER, PR, androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, thymidylate synthase, Her2 / neu, TOPO2A, ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, obtaining data including CES2, DNMT1, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70; comparing the obtained data with corresponding reference values ​​stored in a database in a computer system, where as part of said comparison, the compared reference values ​​are not associated with said particular cancer lineage; identifying at least one therapeutic agent from the comparison based on an indication from the comparison that the at least one therapeutic agent will benefit the patient, wherein the identification is made regardless of association of the at least one therapeutic agent with any particular cancer lineage; and generating a patient profile report indicating that the at least one identified therapeutic agent is a suitable agent for the patient; 4. A computer-implemented method comprising:

17. When executed by one or more computers: Obtaining data indicative of the results of performing one or more molecular tests on a plurality of molecular profiling targets in a biological sample obtained from a patient having a particular cancer lineage, the plurality of molecular profiling targets being selected from the group consisting of EGFR, SPARC, C-kit, ER, PR, androgen receptor, PGP, RRM1, TOPO1, BRCP1, MRP1, MGMT, PDGFR, DCK, ERCC1, thymidylate synthase, Her2 / neu, TOPO2A, ADA, AR, ASNA, BCL2, BRCA2, CD33, CDW52, obtaining data including CES2, DNMT1, ERBB2, ERCC3, ESR1, FOLR2, GART, GSTP1, HDAC1, HIF1A, HSPCA, IL2RA, KIT, MLH1, MS4A1, MASH2, NFKB2, NFKBIA, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLA, PTEN, PTGS2, RAF1, RARA, RXRB, SSTR1, TK1, TNF, TOP1, TOP2A, TOP2B, TXNRD1, TYMS, VDR, VEGF, VHL, and ZAP70; comparing the obtained data with corresponding reference values ​​stored in a database in a computer system, where as part of said comparison, the compared reference values ​​are not associated with said particular cancer lineage; identifying at least one therapeutic agent from said comparison based on an indication from said comparison that said at least one therapeutic agent will benefit the patient, said identification being made without regard to association of said at least one therapeutic agent with any particular cancer lineage; and generating a patient profile report indicating that the at least one identified therapeutic agent is a suitable agent for the patient; instructions for causing the one or more computers to perform operations including One or more computer readable storage media having stored thereon.

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