`
`RELATED APPLICATIONS
`
`This application is a continuation of US. Patent Application No. 14/832,860, filed August 21, 2015, which
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`application is a continuation of US. Patent Application No. 14/551,345, filed November 24, 2014 and now
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`abandoned, which application is a continuation of US. Patent Application No. 14/ 175,781, filed February 7,
`
`2014 and now US. Patent No. 9,092,392 which issued July 28, 2015, which is a continuation of US. Patent
`
`Application No. 12/658,770, filed February 12, 2010 and now US. Patent No. 8,768,629 which issued July 1,
`
`2014 and which claims the benefit of US. Provisional Applications 61/15 1,75 8, filed on February 11, 2009,
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`61/170,565, filed on April 17, 2009, 61/217,289, filed on May 28, 2009, 61/229,686, filed on July 29, 2009,
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`61/279,970, filed on October 27, 2009, 61/261,709, filed on November 16, 2009, and 61/294,440, filed on
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`January 12, 2010, all of which applications are incorporated herein by reference in their entirety.
`
`BACKGROUND
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`[0001] Disease states in patients are typically treated with treatment regimens or therapies that are selected
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`based on clinical based criteria, that is, a treatment therapy or regimen is selected for a patient based on the
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`determination that the patient has been diagnosed with a particular disease (which diagnosis has been made
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`from classical diagnostic assays). Although the molecular mechanisms behind various disease states have
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`been the subject of studies for years, the specific application of a diseased individual’s molecular profile in
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`determining treatment regimens and therapies for that individual has been disease specific and not widely
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`pursued.
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`[0002] Some treatment regimens have been determined using molecular profiling in combination with
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`clinical characterization of a patient such as observations made by a physician (such as a code from the
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`International Classification of Diseases, for example, and the dates such codes were determined), laboratory
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`test results, X-rays, biopsy results, statements made by the patient, and any other medical information
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`typically relied upon by a physician to make a diagnosis in a specific disease. However, using a combination
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`of selection material based on molecular profiling and clinical characterizations (such as the diagnosis of a
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`particular type of cancer) to determine a treatment regimen or therapy presents a risk that an effective
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`treatment regimen may be overlooked for a particular individual since some treatment regimens may work
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`well for different disease states even though they are associated with treating a particular type of disease state.
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`[0003] Patients with refractory and metastatic cancer are of particular concern for treating physicians. The
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`majority of patients with metastatic cancer eventually run out of treatment options for their tumors. These
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`patients have very limited options after their tumor has progressed on standard front line and second line (and
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`sometimes third line and beyond) therapies. Although these patients may participate in Phase I and Phase II
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`clinical trials for new anticancer agents, they must usually meet very strict eligibility criteria to do so. Studies
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`have shown that when patients participate in these types of trials, the new anticancer agent may give response
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`rates of anywhere from 5% to 10% on average in Phase I settings to 12% in Phase II settings. These patients
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`also have the option of electing to receive the best supportive care to treat their symptoms.
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`[0004] There has recently been an eXplosion of interest in developing new anticancer agents that are more
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`targeted against a cell surface receptor or an upregulated or amplified gene product. This approach has met
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`with some success (e.g. trastuzumab against HER2/neu in breast cancer cells, rituXimab against CD20 in
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`lymphoma cells, bevacizamab against VEGF, and cetuXimab against EGFR). However, patients’ tumors still
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`eventually progress on these therapies. If a larger number of targets or molecular findings such as molecular
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`mechanisms, genes, gene eXpressed proteins, and/or combinations of such were measured in a patient’s
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`tumor, one may find additional targets or molecular findings that can be eXploited by using specific
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`therapeutic agents. Identifying multiple agents that can treat multiple targets or underlying mechanisms
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`would provide cancer patients with a viable therapeutic alternative to those treatment regimens which
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`currently eXist.
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`[0005] Molecular profiling analysis identifies one or more individual profiles that often drive more informed
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`and effective personalized treatment options, which can result in improved patient care and enhanced
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`treatment outcomes. The present invention provides methods and systems for identifying treatments for these
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`individuals by molecular profiling a sample from the individual.
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`SUMMARY OF THE INVENTION
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`[0006] The present invention provides methods and system for molecular profiling, using the results from
`
`molecular profiling to identify treatments for invidiuals. In some embodiments, the treatments were not
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`identified intially as a treatment for the disease.
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`[0007]
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`In an aspect, the invention provides a method of identifying a candidate treatment for a subject in
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`need thereof, comprising: performing an immunohistochemistry (IHC) analysis on a sample from the subject
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`to determine an IHC eXpression profile on at least five proteins, performing a microarray analysis on the
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`sample to determine a microarray eXpression profile on at least ten genes, performing a fluorescent in-situ
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`hybridization (FISH) analysis on the sample to determine a FISH mutation profile on at least one gene,
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`performing DNA sequencing on the sample to determine a sequencing mutation profile on at least one gene,
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`and comparing the IHC eXpression profile, microarray expression profile, FISH mutation profile and
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`sequencing mutation profile against a rules database. The rules database comprises a mapping of treatments
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`whose biological activity is known against cancer cells that: i. overeXpress or undereXpress one or more
`
`proteins included in the IHC eXpression profile, ii. overeXpress or undereXpress one or more genes included in
`
`the microarray eXpression profile, iii. have no mutations, or one or more mutations in one or more genes
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`included in the FISH mutation profile, and/or iv. have no mutations, or one or more mutations in one or more
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`genes included in the sequencing mutation profile. The candidate treatment is identified if: i. the comparison
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`step indicates that the treatment should have biological activity against the cancer; and ii. the comparison step
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`does not contraindicate the treatment for treating the cancer.
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`[0008]
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`In some embodiments, the IHC expression profiling comprises assaying one or more of SPARC,
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`PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCCl, RRMl, BCRP, TOPOl, PTEN,
`
`MGMT, and MRP1.
`
`[0009]
`
`In some embodiments, the microarray eXpression profiling comprise assaying one or more of
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`ABCCl, ABCGZ, ADA, AR, ASNS, BCL2, BIRC5, BRCAl, BRCA2, CD33, CD52, CDA, CES2, DCK,
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`DHFR, DNMTl, DNMT3A, DNMT3B, ECGFl, EGFR, EPHA2, ERBB2, ERCCl, ERCC3, ESRl, FLTl,
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`FOLR2, FYN, GART, GNRHl, GSTPl, HCK, HDACl, HIFlA, HSP90AA1, IL2RA, HSP90AA1, KDR,
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`KIT, LCK, LYN, MGMT, MLHl, MS4A1, MSH2, NFKBl, NFKB2, OGFR, PDGFC, PDGFRA, PDGFRB,
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`PGR, POLAl, PTEN, PTGS2, RAFl, RARA, RRMl, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC,
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`SSTRl, SSTR2, SSTR3, SSTR4, SSTR5, TKl, TNF, TOPl, TOP2A, TOP2B, TXNRDl, TYMS, VDR,
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`VEGFA, VHL, YESl, and ZAP70.
`
`[0010]
`
`In some embodiments, the FISH mutation profiling comprises assaying EGFR and/or HER2.
`
`[0011]
`
`In some embodiments, the sequencing mutation profiling comprises assaying one or more of KRAS,
`
`BRAF, c-KIT and EGFR.
`
`[0012]
`
`In another aspect, the invention provides a method of identifying a candidate treatment for a subject
`
`in need thereof, comprising: performing an immunohistochemistry (IHC) analysis on a sample from the
`
`subject to determine an IHC eXpression profile on at least five of: SPARC, PGP, Her2/neu, ER, PR, c-kit, AR,
`
`CD52, PDGFR, TOP2A, TS, ERCCl, RRMl, BCRP, TOPOl, PTEN, MGMT, and MRP1, performing a
`
`microarray analysis on the sample to determine a microarray eXpression profile on at least five of: ABCCl,
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`ABCGZ, ADA, AR, ASNS, BCL2, BIRC5, BRCAl, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR,
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`DNMTl, DNMT3A, DNMT3B, ECGFl, EGFR, EPHA2, ERBB2, ERCCl, ERCC3, ESRl, FLTl, FOLR2,
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`FYN, GART, GNRHl, GSTPl, HCK, HDACl, HIFlA, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK,
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`LYN, MGMT, MLHl, MS4A1, MSH2, NFKBl, NFKBZ, OGFR, PDGFC, PDGFRA, PDGFRB, PGR,
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`POLAl, PTEN, PTGS2, RAFl, RARA, RRMl, RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTRl,
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`SSTR2, SSTR3, SSTR4, SSTR5, TKl, TNF, TOPl, TOP2A, TOP2B, TXNRDl, TYMS, VDR, VEGFA,
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`VHL, YES 1, and ZAP70, performing a fluorescent in-situ hybridization (FISH) analysis on the sample to
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`determine a FISH mutation profile on EGFR and/or HER2, performing DNA sequencing on the sample to
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`determine a sequencing mutation profile on at least one of KRAS, BRAF, c-KIT and EGFR, and comparing
`
`the IHC expression profile, microarray eXpression profile, FISH mutation profile and sequencing mutation
`
`profile against a rules database. The rules database comprises a mapping of treatments whose biological
`
`activity is known against cancer cells that: i. overeXpress or undereXpress one or more proteins included in the
`
`IHC eXpression profile, ii. overeXpress or undereXpress one or more genes included in the microarray
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`eXpression profile, iii. have no mutations, or one or more mutations in one or more genes included in the
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`FISH mutation profile; and/or iv. have no mutations, or one or more mutations in one or more genes included
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`in the sequencing mutation profile. The 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 does not
`
`contraindicate the treatment for treating the cancer. In some embodiments, the IHC expression profiling is
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`performed on at least 50%, 60%, 70%, 80% or 90% of the biomarkers listed. In some embodiments, the
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`microarray expression profiling is performed on at least 50%, 60%, 70%, 80% or 90% of the biomarkers
`
`listed.
`
`[0013]
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`In a third aspect, the invention provides a method of identifying a candidate treatment for a cancer in
`
`a subject in need thereof, comprising: performing an immunohistochemistry (IHC) analysis on a sample from
`
`the subject to determine an IHC expression profile on at least the group of proteins consisting of: SPARC,
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`PGP, Her2/neu, ER, PR, c-kit, AR, CD52, PDGFR, TOP2A, TS, ERCCl, RRMl, BCRP, TOPOl, PTEN,
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`MGMT, and MRP1, performing a microarray analysis on the sample to determine a microarray expression
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`profile on at least the group of genes consisting of ABCCl, ABCG2, ADA, AR, ASNS, BCL2, BIRC5,
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`BRCAl, BRCA2, CD33, CD52, CDA, CES2, DCK, DHFR, DNMTl, DNMT3A, DNMT3B, ECGFl, EGFR,
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`EPHA2, ERBB2, ERCCl, ERCC3, ESRl, FLTl, FOLR2, FYN, GART, GNRHl, GSTPl, HCK, HDACl,
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`HIFlA, HSP90AA1, IL2RA, HSP90AA1, KDR, KIT, LCK, LYN, MGMT, MLHl, MS4A1, MSH2, NFKBl,
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`NFKBZ, OGFR, PDGFC, PDGFRA, PDGFRB, PGR, POLAl, PTEN, PTGS2, RAFl, RARA, RRMl,
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`RRM2, RRM2B, RXRB, RXRG, SPARC, SRC, SSTRl, SSTR2, SSTR3, SSTR4, SSTR5, TKl, TNF, TOPl,
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`TOP2A, TOP2B, TXNRDl, TYMS, VDR, VEGFA, VHL, YESl, and ZAP70, performing a fluorescent in-
`
`situ hybridization (FISH) analysis on the sample to determine a FISH mutation profile on at least the group of
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`genes consisting of EGFR and HER2, performing DNA sequencing on the sample to determine a sequencing
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`mutation profile on at least the group of genes consisting of KRAS, BRAF, c-KIT and EGFR, and comparing
`
`the IHC expression profile, microarray expression profile, FISH mutation profile and sequencing mutation
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`profile against a rules database. The rules database comprises a mapping of treatments whose biological
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`activity is known against cancer cells that: i. overexpress or underexpress one or more proteins included in the
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`IHC expression profile, ii. overexpress or underexpress one or more genes included in the microarray
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`expression profile, iii. have zero or more mutations in one or more genes included in the FISH mutation
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`profile, and/or iv. have zero or more mutations in one or more genes included in the sequencing mutation
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`profile. The candidate treatment is identified if: i. the comparison step indicates that the treatment should
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`have biological activity against the cancer, and ii. the comparison step does not contraindicate the treatment
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`for treating the cancer.
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`[0014]
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`In some embodiments of the methods of the invention, the sample comprises formalin-fixed paraffin-
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`embedded (FFPE) tissue, fresh frozen (FF) tissue, or tissue comprised in a solution that preserves nucleic acid
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`or protein molecules. In some embodiments, any one of the microarray analysis, the FISH mutational analysis
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`or the sequencing mutation analysis is not performed. For example, a method may not be performed unless
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`the sample passes a quality control test. In some embodiments, the quality control test comprises an
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`A260/A280 ratio or a Ct value of RT-PCR of RPLl3a mRNA. For example, the quality control test can
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`require an A260/A280 ratio < 1.5 or the RPLl3a Ct value is > 30.
`
`[0015]
`
`In some embodiments, the microarray expression profiling is performed using a low density
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`microarray, an expression microarray, a comparative genomic hybridization (CGH) microarray, a single
`
`nucleotide polymorphism (SNP) microarray, a proteomic array or an antibody array.
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`[0016] The methods of the invention can require assaying of certain markers, including additional markers.
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`In some embodiments, the IHC expression profiling is performed on at least SPARC, TOP2A and/or PTEN.
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`The microarray expression profiling can be performed on at least CD52. The IHC expression profiling further
`
`consists of assaying one or more of DCK, EGFR, BRCAl, CK 14, CK 17, CK 5/6, E-Cadherin, p95, PARP-l,
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`SPARC and TLE3. In some embodiments, the IHC expression profiling fithher consists of assaying Cox-2
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`and/or Ki-67. In some embodiments, the microarray expression profiling fithher consists of assaying
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`HSPCA. In some embodiments, the FISH mutation profiling fithher consists of assaying c-Myc and/or
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`TOP2A. The sequencing mutation profiling can comprise assaying PI3K.
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`[0017] A number of genes and gene products can be assayed according to the methods of the invention. For
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`example, the genes used for the IHC expression profiling, the microarray expression profiling, the FISH
`
`mutation profiling, and the sequencing mutation profiling independently comprise one or more of ABCCl,
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`ABCG2, ACE2, ADA, ADHlC, ADH4, AGT, Androgen receptor, AR, AREG, ASNS, BCL2, BCRP,
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`BDCAl, BIRC5, B-RAF, BRCAl, BRCA2, CA2, caveolin, CD20, CD25, CD33, CD52, CDA, CDK2,
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`CDW52, CES2, CK 14, CK 17, CK 5/6, c-KIT, c-Myc, COX-2, Cyclin D1, DCK, DHFR, DNMTl,
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`DNMT3A, DNMT3B, E-Cadherin, ECGFl, EGFR, EPHA2, Epiregulin, ER, ERBR2, ERCCl, ERCC3,
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`EREG, ESRl, FLTl, folate receptor, FOLRl, FOLR2, FSHB, FSHPRHl, FSHR, FYN, GART, GNRHl,
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`GNRHRl, GSTPl, HCK, HDACl, Her2/Neu, HGF, HIFlA, HIGl, HSP90, HSP90AA1, HSPCA, IL13RA1,
`
`IL2RA, KDR, KIT, K-RAS, LCK, LTB, Lymphotoxin Beta Receptor, LYN, MGMT, MLHl, MRPl,
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`MS4A1, MSH2, Myc, NFKBl, NFKB2, NFKBIA, ODCl, OGFR, p53, p95, PARP-l, PDGFC, PDGFR,
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`PDGFRA, PDGFRB, PGP, PGR, PI3K, POLA, POLAl, PPARG, PPARGCl, PR, PTEN, PTGS2, RAFl,
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`RARA, RRMl, RRM2, RRM2B, RXRB, RXRG, SPARC, SPARC MC, SPARC PC, SRC, SSTRl, SSTR2,
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`SSTR3, SSTR4, SSTR5, Survivin, TK1, TLE3, TNF, TOPl, TOP2A, TOP2B, TOPOl, TOPO2B,
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`Topoisomerase II, TS, TXN, TXNRDl, TYMS, VDR, VEGF, VEGFA, VEGFC, VHL, YESl and ZAP70.
`
`[0018]
`
`In some embodiments, the microarray expression analysis comprises identifying whether a gene is
`
`upregulated or downregulated relative to a reference with statistical significance. The statistical significance
`
`can be determined at a p-value ofless than or equal to 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001. The p-
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`value can also be corrected for multiple comparisons. Correction for multiple comparisons can include
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`Bonneferoni’s correction or a modification thereof.
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`[0019]
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`In some embodiments, the IHC analysis comprises determining whether 30% or more of said sample
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`is +2 or greater in staining intensity.
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`[0020] The rules contained within the rules database used by the methods of the invention can be based on
`
`the efficacy of various treatments particular for a target gene or gene product. The rules database can
`
`comprise the rules listed herein in Table 1 and/or Table 2.
`
`[0021]
`
`In some embodiments of the methods of the invention, a prioritized list of candidate treatments are
`
`identified. Prioritizing can include ordering the treatments from higher priority to lower priority according to
`
`treatments based on microarray analysis and either IHC or FISH analysis; treatments based on IHC analysis
`
`but not microarray analysis; and treatments based on microarray analysis but not IHC analysis.
`
`[0022]
`
`In some embodiments of the methods of the invention, the candidate treatment comprises
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`administration of one or more candidate therapeutic agents. The one or more candidate therapeutic agents can
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`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,
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`Cisplatin, carboplatin, Cyclophosphamide, Cyclophosphamide/Vincristine, cytarabine, dasatinib, decitabine,
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`Doxorubicin, Epirubicin, epirubicin, Erlotinib, Etoposide, exemestane, fluoropyrimidines, Flutamide,
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`fiilvestrant, Gefitinib, Gefitinib and Trastuzumab, Gemcitabine, gonadorelin, Goserelin, hydroxyurea,
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`Imatinib, Irinotecan, Ixabepilone, Lapatinib, Letrozole, Leuprolide, liposomal doxorubicin,
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`medroxyprogesterone, megestrol, methotrexate, mitomycin, nab-paclitaxel, octreotide, Oxaliplatin, Paclitaxel,
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`Panitumumab, pegaspargase, pemetrexed, pentostatin, sorafenib, sunitinib, Tamoxifen, Tamoxifen-based
`
`treatment, Temozolomide, topotecan, toremifene, Trastuzumab, VBMCP/Cyclophosphamide, Vincristine, or
`
`any combination thereof. The one or more candidate therapeutic agents can also be 5FU, bevacizumab,
`
`capecitabine, cetuximab, cetuximab + gemcitabine, cetuximab + irinotecan, cyclophospohamide,
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`diethylstibesterol, doxorubicin, erlotinib, etoposide, exemestane, fluoropyrimidines, gemcitabine, gemcitabine
`
`+ etoposide, gemcitabine + pemetrexed, irinotecan, irinotecan + sorafenib, lapatinib, lapatinib + tamoxifen,
`
`letrozole, letrozole -- capecitabine, mitomycin, nab-paclitaxel, nab-paclitaxel + gemcitabine, nab-paclitaxel +
`
`
`
`trastuzumab, oxaliplatin, oxaliplatin + 5FU + trastuzumab, panitumumab, pemetrexed, sorafenib, sunitinib,
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`sunitinib, sunitinib -- mitomycin, tamoxifen, temozolomide, temozolomide + bevacizumab, temozolomide +
`
`sorafenib, trastuzumab, vincristine, or any combination thereof.
`
`[0023]
`
`In embodiments of the methods of the invention, the sample comprises cancer cells. The cancer can
`
`be a metastatic cancer. The cancer can be refractory to a prior treatment. The prior treatment can be the
`
`standard of care for the cancer. Sometimes, the subject has been previously treated with one or more
`
`therapeutic agents to treat a cancer. Sometimes, the subject has not previously been treated with one or more
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`candidate therapeutic agents identified.
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`[0024]
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`In some embodiments, the cancer comprises a prostate, lung, melanoma, small cell
`
`(esopha/retroperit), cholangiocarcinoma, mesothelioma, head and neck (SCC), pancreas, pancreas
`
`neuroendocrine, small cell, gastric, peritoneal pseudomyxoma, anal Canal (SCC), vagina (SCC), cervical,
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`renal, eccrine seat adenocarinoma, salivary gland adenocarinoma, uterine soft tissue sarcoma (uterine), GIST
`
`(Gastric), or thyroid-anaplastic cancer. In some embodiments, the cancer comprises a cancer of the accessory,
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`sinuses, middle and inner ear, adrenal glands, appendix, hematopoietic system, bones and joints, spinal cord,
`
`breast, cerebellum, cervix uteri, connective and soft tissue, corpus uteri, esophagus, eye, nose, eyeball,
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`fallopian tube, extrahepatic bile ducts, mouth, intrahepatic bile ducts, kidney, appendix-colon, larynx, lip,
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`liver, lung and bronchus, lymph nodes, cerebral, spinal, nasal cartilage, retina, eye, oropharynx, endocrine
`
`glands, female genital, ovary, pancreas, penis and scrotum, pituitary gland, pleura, prostate gland, rectum
`
`renal pelvis, ureter, peritonem, salivary gland, skin, small intestine, stomach, testis, thymus, thyroid gland,
`
`tongue, unknown, urinary bladder, uterus, vagina, labia, and vulva. In some embodiments, the sample
`
`comprises cells selected from the group consisting of adipose, adrenal cortex, adrenal gland, adrenal gland —
`
`medulla, appendix, bladder, blood, blood vessel, bone, bone cartilage, brain, breast, cartilage, cervix, colon,
`
`colon sigmoid, dendritic cells, skeletal muscle, enodmetrium, esophagus, fallopian tube, fibroblast,
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`gallbladder, kidney, larynx, liver, lung, lymph node, melanocytes, mesothelial lining, myoepithelial cells,
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`osteoblasts, ovary, pancreas, parotid, prostate, salivary gland, sinus tissue, skeletal muscle, skin, small
`
`intestine, smooth muscle, stomach, synovium, joint lining tissue, tendon, testis, thymus, thyroid, uterus, and
`
`uterus corpus. In some embodiments, the cancer comprises a breast, colorectal, ovarian, lung, non-small cell
`
`lung cancer, cholangiocarcinoma, mesothelioma, sweat gland, or GIST cancer.
`
`[0025] Progression free survival (PFS) or disease free survival (DFS) for the subject can be extended using
`
`the methods of the invention. For example, the PFS or DFS can be 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 prior treatment. In addition, the patient’s lifespan can be extended using the
`
`methods of the invention to select a candidate treatment. For example, the patient’s lifespan can 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 by at least 5 years.
`
`INCORPORATION BY REFERENCE
`
`[0026] All publications and patent applications mentioned in this specification are herein incorporated by
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`reference to the same extent as if each individual publication or patent application was specifically and
`
`individually indicated to be incorporated by reference.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0027] 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:
`
`[0028] FIG. 1 illustrates a block diagram of an exemplary embodiment of a system for determining
`
`individualized medical intervention for a particular disease state that utilizes molecular profiling of a patient’s
`
`biological specimen that is non disease specific.
`
`[0029] FIG. 2 is a flowchart of an exemplary embodiment of a method for determining individualized
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`medical intervention for a particular disease state that utilizes molecular profiling of a patient’s biological
`
`specimen that is non disease specific.
`
`[0030] FIGS. 3A through 3D illustrate an exemplary patient profile report in accordance with step 80 of
`
`FIG. 2.
`
`[0031] FIG. 4 is a flowchart of an exemplary embodiment of a method for identifying a drug therapy/agent
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`capable of interacting with a target.
`
`[0032] FIGS. 5-14 are flowcharts and diagrams illustrating various parts of an information-based
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`personalized medicine drug discovery system and method in accordance with the present invention.
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`[0033] FIGS. 15-25 are computer screen print outs associated with various parts of the information-based
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`personalized medicine drug discovery system and method shown in FIGS. 5-14.
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`[0034] FIGS. 26A-26H represent a table that shows the frequency of a significant change in expression of
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`gene expressed proteins by tumor type.
`
`[0035] FIGS. 27A-27H represent a table that shows the frequency of a significant change in expression of
`
`certain genes by tumor type.
`
`[0036] FIGS. 28A-28O represent a table that shows the frequency of a significant change in expression for
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`certain gene expressed proteins by tumor type.
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`[0037] FIG. 29 is a table which shows biomarkers (gene expressed proteins) tagged as targets in order of
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`frequency based on FIG. 28.
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`[0038] FIGS. 30A-300 represent a table that shows the frequency of a significant change in expression for
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`certain genes by tumor type.
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`[0039] FIG. 31 is a table which shows genes tagged as targets in order of frequency based on FIG. 30.
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`[0040] FIG. 32 illustrates progression free survival (PFS) using therapy selected by molecular profiling
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`(period B) with PFS for the most recent therapy on which the patient has just progressed (period A). If
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`PFS(B) / PFS(A) ratio 2 1.3, then molecular profiling selected therapy was defined as having benefit for
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`patient.
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`[0041] FIG. 33 is a schematic of methods for identifying treatments by molecular profiling if a target is
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`identified.
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`[0042] FIG. 34 illustrates the distribution of the patients in the study as performed in Example 1.
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`[0043] FIG. 35 is graph depicting the results of the study with patients having PFS ratio 2 1.3 was 18/66
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`(27%).
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`[0044] FIG. 36 is a waterfall plot of all the patients for maximum % change of summed siameters of target
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`lesions with respect to baseline diameter.
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`[0045] FIG. 37 illustrates the relationship between what clinician selected as what she/he would use to treat
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`the patient before knowing what the molecular profiling results suggested. There were no matches for the 18
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`patients with PFS ratio 2 1.3.
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`[0046] FIG. 38 is a schematic of the overall survival for the 18 patients with PFS ratio 2 1.3 versus all 66
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`patients.
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`[0047] FIG. 39 shows an example output of microarray profiling results and calls made using a cutoff value.
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`[0048] FIGS. 40A-40J illustrate an exemplary patient report based on molecular profiling.
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`DETAILED DESCRIPTION OF THE INVENTION
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`[0049] The present invention provides methods and systems for identifying targets for treatments by using
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`molecular profiling. The molecular profiling approach provides a method for selecting a candidate treatment
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`for an individual that could favorably change the clinical course for an individual with a condition or disease,
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`such as cancer. The molecular profiling approach can provide clinical benefit for individuals, such as
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`providing a longer progression free survival (PFS), longer disease free survival (DFS), longer overall survival
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`(OS) or extended lifespan when treated using molecular profiling approaches than using conventional
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`approaches to selecting a treatment regimen. Molecular profiling can suggest candidate treatments when a
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`disease is refractory to current therapies, e.g., after a cancer has developed resistance to a standard-of—care
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`treatment.
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`[0050] Molecular profiling can be performed by any known means for detecting a molecule in a biological
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`sample. Profiling can be performed on any applicable biological sample. The sample typically comes from
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`an individual with a suspected or known disease or disorder, such as, but not limited to, a biopsy sample from
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`a cancer patient. Molecular profiling of the sample can also be performed by any number of techniques that
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`assess the amount or state of a biological factor, such as a DNA sequence, an mRNA sequence or a protein.
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`Such techniques include without limitation immunohistochemistry (IHC), in situ hybridization (ISH),
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`fluorescent in situ hybridization (FISH), various types of microarray (mRNA expression arrays, protein
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`arrays, etc), various types of sequencing (Sanger, pyrosequencing, etc), comparative genomic hybridization
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`(CGH), NextGen sequencing, Northern blot, Southern blot, immunoassay, and any other appropriate
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`technique under development to assay the presence or quantity of a biological molecule of interest. Any one
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`or more of these methods can be used concurrently or subsequent to each other.
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`[0051] Molecular profiling is used to select a candidate treatment for a disorder in a subject. For example,
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`the candidate treatment can be a treatment known to have an effect on cells that differentially express genes as
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`identified by molecular profiling techniques. Differential expression can include either overexpression and
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`underexpression of a biological product, e.g., a gene, mRNA or protein, compared to a control. The control
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`can include similar cells to the sample but without the disease. The control can be derived from the same
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`patient, e.g., a normal adjacent portion of the same organ as the diseased cells, or the control can be derived
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`from healthy tissues from other patients. The control can be a control found in the same sample, e. g. a
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`housekeeping gene or a product thereof (e.g., mRNA or protein). For example, a control nucleic acid can be
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`one which is known not to differ depending on the cancerous or non-cancerous state of the cell. The
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`expression level of a control nucleic acid can be used to normalize signal levels in the test and reference
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`populations. Exemplary control genes include, but are not limited to, e.g., B-actin, glyceraldehyde 3-
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`phosphate dehydrogenase and ribosomal protein Pl. Multiple controls or types of controls can be used. The
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`source of differential expression can vary. For example, a gene copy number may be increased in a cell,
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`thereby resulting in increased expression of the gene. Altemately, transcription of the gene may be modified,
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`e.g., by chromatin remodeling, differential methylation, differential expression or activity of transcription
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`factors, etc. Translation may also be modified, e.g., by differential expression of factors that degrade mRNA,
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`translate mRNA, or silence translation, e.g., microRNAs or siRNAs. In some embodiments, differential
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`expression comprises differential activity. For example, a protein may carry a mutation that increases the
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`activity of the protein, such as constitutive activation, thereby contributing to a diseased state. Molecular
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`profiling that reveals changes in activity can be used to guide treatment selection.
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`[0052] When multiple drug targets are revealed as differentially expressed by molecular profiling, decision
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`rules can be put in place to prioritize the selection of certain treatments. Any such rule can be used that helps
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`prioritize treatment can be used to prioritize treatments, e.g., direct results of molecular profiling, anticipated
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`efficacy, prior history with the same or other treatments, expected side effects, availability, cost, drug-drug
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`interactions, and other factors considered by a treating physician. The physician can ultimately decide on the
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`course of treatment. Accordingly, molecular profiling can select candidate treatments based on individual
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`characteristics of diseased cells, e.g., tumor cells, and other personalized factors in a subject in need of
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`treatment, as opposed to relying on a traditional one-size fits all approach taken to target therapy against a
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`certain indication. In some cases, the recommended treatments are those not typically used to treat the disease
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`or disorder inflicting the subject. In some cases, the recommended treatments are used after standard-of—care
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`therapies are no longer providing adequate efficacy.
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`[0053] Nucleic acids include deoxyribonucleotides or ribonucleotides and polymers thereof in either single-
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`or double-stranded form, and complements thereof. Nucleic acids can contain known nucleotide analogs or
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`modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally
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`occurring, which have similar binding properties as the refere