KR20070022694A - Gene Expression Markers for Predicting Response to Chemotherapy - Google Patents

Abstract

The present invention provides a set of genes whose expression is important for prognosis of cancer. In particular, the present invention provides gene expression information useful for predicting whether a cancer patient is likely to have a favorable therapeutic response to chemotherapy.

Cancer, Prognosis, Genes, Gene Expression Information, Chemotherapy, Therapeutic Response

Description

Gene Expression Markers for Predicting Response to Chemotherapy

The present invention provides a set of genes whose expression is important for prognosis of cancer. In particular, the present invention provides gene expression information useful for predicting whether a cancer patient is likely to have a favorable therapeutic response to chemotherapy.

Tumor doctors include different combinations of chemotherapy drugs that are characterized as a "standard of care" and numerous combinations of drugs that do not qualify for labeling for a particular cancer but have evidence of efficacy in that cancer. There are numerous treatment options available. The best possibility for good treatment outcomes should specify the optimal cancer treatment available to the patient and this designation needs to be made as soon as possible after diagnosis. In particular, it is important to determine the likelihood of patient response to “treatment criteria” chemotherapy because chemotherapeutic agents such as anthracycline and taxanes have limited efficacy and are toxic. Thus, identification of the most responsive or least responsive patient can, through smarter patient selection, increase the net benefits that these drugs must provide and reduce net mortality and toxicity.

Currently, diagnostic tests used in clinical practice are single analytes and thus do not obtain the potential value of known relationships between many different markers. In addition, diagnostic tests are not quantitative, mainly dependent on immunohistochemistry. This method yields different results in different laboratories, in part because the reagents are not standardized, and in part because the interpretation is subjective and cannot be easily quantified. RNA-based testing was not frequently used due to the problem of RNA degradation over time and the fact that fresh tissue samples for analysis were difficult to obtain from patients. Tissues immobilized and embedded in paraffin are more readily available, and methods for detecting RNA in fixed tissues have been established. However, these methods typically do not allow the study of large numbers of genes (DNA or RNA) from small amounts of material. Thus, traditionally, fixed tissues are rarely used except for immunohistochemical detection of proteins.

In recent years, several groups have published studies on the classification of various cancer types by microarray gene expression analysis (see, for example, Golub et. al ., Science 286: 531-537 (1999), Bhattacharjae et al ., Proc . Natl. Acad . Sci . USA 98: 13790-13795 (2001), Chen-Hsiang et. al ., Bioinformatics 17 (Suppl . 1): S316-S322 (2001), and Ramaswamy et. al ., Proc . Natl. Acad . Sci . USA 98: 15149-15154 (2001)]. Certain classifications of human breast cancer based on gene expression patterns have also been reported (Martin et. al ., Cancer Res . 60: 2232-2238 (2000), West et al ., Proc . Natl . Acad . Sci . USA 98: 11462-11467 (2001), Sorlie et. al ., Proc . Natl . Acad . Sci . USA 98: 10869-10874 (2001), and Yan et. al ., Cancer Res . 61: 8375-8380 (2001)]). However, these studies focus on improving and refine the already established classification of various types of cancer, including breast cancer in the majority of cases, and do not provide new insights into the relationship of genes that are differentially expressed in general, and These findings are not linked to treatment strategies to improve clinical outcomes.

Although modern molecular biology and biochemistry have uncovered hundreds of genes whose activity affects tumor cell behavior, their differentiation status, and resistance or sensitivity to certain therapeutic agents, with a few exceptions, the status of these genes It is not routinely used to make clinical decisions about drug treatment. One notable exception is the use of estrogen receptor (ER) protein expression in breast carcinoma in selecting patients for treatment with anti-estrogen drugs such as tamoxifen. Another exceptional example is the use of ErbB2 (Her2) protein expression in breast carcinoma for treatment with Her2 antagonist drug Herceptin® (Genentech, Inc., South San Francisco, CA). To choose a patient.

Despite recent advances, the challenges of cancer treatment remain to personalize tumor treatment in order to target specific treatment regimens for pathologically distinct tumor types and ultimately maximize outcomes. Thus, there is a need for tests that simultaneously provide predictive information about patient response to various treatment options. This is especially true of breast cancer, whose biology is little understood. A few subgroups of breast cancer, such as ErbB2 positive subgroups and subclasses characterized by little or no gene expression of estrogen receptors (ER) and a few additional transcription factors (Perou et al. al ., Nature 406: 747-752 (2000)] do not reflect the cellular and molecular heterogeneity of breast cancer and do not allow the design of therapeutic strategies to maximize patient response.

Breast cancer is the most common type of cancer among women in the United States and the leading cause of cancer deaths among women ages 40-59. Therefore, there is a particular need for clinically confirmed breast cancer trials that predict patient response to chemotherapy.

Summary of the Invention

The present invention provides a set of genes useful for predicting the response to chemotherapy in cancer, for example breast cancer patients. The present invention also provides clinically confirmed cancers, such as breast cancer tests, that predict patient response to chemotherapy using multi-gene RNA analysis. The present invention employs the use of stored paraffin-embedded biopsy materials for the analysis of all markers in the relevant gene set and is therefore compatible with the most widely available types of biopsy materials.

In one aspect, the invention relates to a chemistry of a subject diagnosed with cancer comprising determining the expression level of one or more prognostic RNA transcripts or expression products thereof in a biological sample comprising cancer cells obtained from the subject diagnosed with cancer. A method of predicting a response to therapy wherein the prognostic RNA transcripts comprise TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A.Catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat G.Catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Hepsin (Hepsin); CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; A transcript of at least one gene selected from the group consisting of TK1, ErbB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, CD68, GSTM1, BCL2, ESR1

(a) ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1; ERBB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2 and CD68; Or if at least one expression unit of the corresponding expression product has increased, the subject is predicted to have an increased response to chemotherapy,

(b) TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; GSTM1, BCL2, ESR1; Or if one or more expression units of the corresponding expression product has increased, it is predicted that the subject has the potential for reduced response to chemotherapy.

In certain embodiments, the prognostic RNA transcript in the method comprises TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; And a transcript of at least one gene selected from the group consisting of TK1.

In other embodiments, the reaction is a complete pathological reaction.

In a preferred embodiment, the subject is a human patient.

The cancer can be any type of cancer, but is preferably a solid tumor such as breast cancer, ovarian cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer and lung cancer.

If the tumor is breast cancer, it may be for example invasive breast cancer or stage II or III breast cancer.

In certain embodiments, the chemotherapy is adjuvant chemotherapy.

In another embodiment, the chemotherapy is neoadjuvant chemotherapy.

Neoadjuvant chemotherapy can be administered, for example, with taxane derivatives such as docetaxel and / or paclitaxel, and / or other anticancer agents such as members of anthracycline anticancer agents, doxorubicin, topoisomerase inhibitors, and the like. It may include.

The method may comprise the determination of two or more, or five or more, or ten or more or fifteen or more expression levels of the prognostic transcripts or expression products thereof listed above.

The biological sample may be, for example, a tissue sample comprising cancer cells, wherein the tissue may be fixed and embedded in paraffin, or fresh or frozen.

In certain embodiments, the tissue is obtained from microneedle, core or other type of biopsy.

In another embodiment, the tissue sample is obtained by microneedle aspiration, bronchial lavage or coronary biopsy.

The expression level of the prognostic RNA transcript (s) can be determined, for example, by RT-PCR or other PCR-based methods, immunohistochemistry, proteomics techniques, or any other method known in the art or combinations thereof. Can be measured.

In one embodiment, the assay for measuring the prognostic RNA transcripts or expression products thereof is provided in the form of kit (s).

In another aspect, the invention relates to an array comprising a polynucleotide hybridizing to multiple species of the following genes: TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; TK1, ErbB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, CD68, GSTM1, BCL2, ESR1.

In one embodiment, the array comprises polynucleotides that hybridize to multiple species of the following genes: TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; TK1.

In other embodiments, the array comprises polynucleotides that hybridize to multiple species of the following genes: ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1; ERBB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2 and CD68.

In another embodiment, the array comprises polynucleotides that hybridize to multiple species of the following genes: ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1.

In another embodiment, the array comprises polynucleotides that hybridize to multiple species of the following genes: TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; GSTM1, BCL2, ESR1.

In other embodiments, the array comprises polynucleotides that hybridize to multiple species of the following genes: TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C.

In various embodiments, the array comprises at least 5, or at least 10, or at least 15 or at least 10 polynucleotides.

In certain embodiments, the array comprises polynucleotides that hybridize to all of the genes listed above.

In another particular embodiment, the array comprises more than one polynucleotide that hybridizes to the same gene.

In other embodiments, at least one of the polynucleotides comprises an intron-based sequence, wherein expression of the intron-based sequence correlates with expression of the corresponding exon sequence.

In various embodiments, the polynucleotides can be cDNA or oligonucleotides.

In another aspect, the invention

(a) TBP in cancer cells obtained from patients to produce personalized genomic profiles; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNXI; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; Determining normalized expression of an RNA transcript or expression product of a gene or gene set selected from the group consisting of TK1, ErbB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, CD68, GSTM1, BCL2, ESR1; And

(b) generating a report summarizing the data obtained by the gene expression analysis

It relates to a method of making a personalized genomic profile for a patient, comprising.

In specific embodiments, ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1; ERBB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2 and CD68; Or if the expression of one or more of the corresponding expression products is determined to be increased, the report includes a prediction that the subject has the potential to increase response to chemotherapy. In this case, in certain embodiments, the method comprises the additional step of treating the patient with a chemotherapeutic agent.

In the above method, TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; GSTM1, BCL2, ESR1; Or if the expression of one or more of the corresponding expression products is determined to be increased, the report includes a prediction that the subject has the potential to reduce the response to chemotherapy.

In another aspect, the invention

(a) ACTB, BAG1, BCL2, CCNB1, CD68, SCUBE2, CTSL2, ESR1, GAPD, GRB7, GSTM1, GUSB, ERBB2, MKI67, MYBL2, PGR, RPLPO, STK6, MMP11, BIRC5, TFRC, or expression products thereof Determining the expression level of RNA transcripts, and

(b) calculating a recurrence score (RS)

It relates to a method of determining the likelihood of a patient's response to chemotherapy, including.

In one embodiment, patients with RS> 50 are in the top 50 percentiles of patients who are likely to respond to chemotherapy.

In another embodiment, patients with RS <35 are in the lower 50 percentile of patients who are likely to respond to chemotherapy.

In further embodiments, the RS comprises (i) a growth factor subset: GRB7 and HER2; (ii) estrogen receptor subsets: ER, PR, Bcl2, and CEGP1; (iii) proliferation subsets: SURV, Ki.67, MYBL2, CCNB1, and STK15; And (iv) invasive subsets: CTSL2, and STMY3, wherein a gene of any of subsets (i) to (iv) is co-expressed with said gene in said tumor and has a Pearson correlation coefficient> 0.40 Generate a subset of genes);

(c) Subsets (i) to (iv) are weighted for each breast cancer recurrence to calculate the recurrence score (RS) of the subject.

The method described above measures RNA transcripts of CD68, GSTM1 and BAG1 or expression products thereof, or corresponding replacement genes or expression products thereof, and includes the contribution of the gene or replacement gene to breast cancer recurrence in RS calculations. It may further comprise a step.

RS can be determined using, for example, the following equation:

RS = (0.23 to 0.70) x GRB7 axis threshold-(0.17 to 0.55) x ER axis + (0.52 to 1.56) x growth axis threshold + (0.07 to 0.21) x infiltration axis + (0.03 to 0.15) x CD68-(0.04 To 0.25) x GSTM1-(0.05 to 0.22) x BAG1

[Wherein,

(i) GRB7 axis = (0.45-1.35) × GRB7 + (0.05-0.15) × HER2;

(ii) if the GRB7 axis <-2, then the GRB7 axis threshold = -2,

     If the GRB7 axis> -2, then the GRB7 axis threshold = GRB7 axis;

(iii) the ER axis = (Est1 + PR + Bcl2 + CEGP1) / 4;

(iv) axis of proliferation = (SURV + Ki.67 + MYBL2 + CCNB1 + STK15) / 5;

(v) if propagation axis <-3.5, propagation axis threshold = -3.5,

    If propagation axis ≧ −3.5, then propagation axis threshold = proliferation axis;

(vi) infiltration axis = (CTSL2 + STMY3) / 2,

Wherein individual contributions of the genes in (iii), (iv) and (vi) are weighted with a coefficient between 0.5 and 1.5, with higher RS indicating an increased likelihood of recurrence of breast cancer.

In another embodiment, RS is obtained using the following formula:

RS (range, 0-100) = + 0.47 × HER2 group score

                        -0.34 × ER group score

                        + 1.04 × Growth Score

                        + 0.10 × infiltration group score

                        + 0.05 × CD68

                        0.08 × GSTM1

                        0.07 × BAG1

1 shows the relationship between likelihood of patient response to chemotherapy and recurrence score (RS) based on results from clinical trials with pathologically complete response endpoints.

Table 1 shows a list of genes whose expression correlates positively or negatively with breast cancer response to adriamycin and taxane neoadjuvant chemotherapy. Results are obtained from clinical trials with pathologic complete response endpoints. Statistical analysis used a univariate generalized linear model with a probit link function.

Table 2 provides a list of genes whose expression predicts breast cancer response to chemotherapy. Results are obtained from retrospective clinical trials. The table includes sequences for the forward and reverse primers (indicated by "f" and "r") and probes (indicated by "p"), and the accession numbers for the genes used for PCR propagation.

Table 3 shows the amplicon sequences used for PCR propagation of the indicated genes.

A. Definition

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et. al . , Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994)] and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992). )]) Provides general guidance for many of the terms used in this application to those skilled in the art.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. In fact, the invention is not limited to the methods and materials described in any way. For the purposes of the present invention, the following terms are defined below.

The term “microarray” refers to the regular arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

The term "polynucleotide", when used in the singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, polynucleotides as defined herein include, but are not limited to, DNA comprising one- and two-stranded DNA, one- and two-stranded regions, one- and two-stranded RNA, one-and RNAs comprising two-stranded regions, single-stranded or more typically two-stranded, or hybrid molecules comprising DNA and RNA comprising one- and two-stranded regions. Also, as used herein, the term “polynucleotide” refers to a three-stranded region comprising RNA or DNA or both RNA and DNA. The strands in this region can be from the same molecule or from different molecules. A zone may comprise all of one or more molecules, but more specifically includes only one zone of some of the molecules. One of the molecules of the triple-helix region is an oligonucleotide. The term "polynucleotide" specifically includes cDNA. The term includes DNA (including cDNA) and RNA containing one or more modified bases. Thus, a DNA or RNA having a backbone modified for stability or for other reasons is a "polynucleotide" as intended herein. In addition, DNA or RNA comprising an unusual base such as inosine or a modified base such as tritium base is included within the term "polynucleotide" as defined herein. In general, the term "polynucleotide" refers to all chemically, enzymatically and / or metabolically modified forms of unmodified polynucleotides, as well as DNA and RNA characteristics of cells and viruses, including simple and complex cells. It includes a chemical form having a.

The term “oligonucleotide” refers to a relatively short polynucleotide, including but not limited to one-stranded deoxyribonucleotides, one- or two-stranded ribonucleotides, RNA: DNA hybrids and two-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods using, for example, commercially available automated oligonucleotide synthesizers. However, oligonucleotides can be prepared by a variety of other methods, including in vitro recombinant DNA-mediated techniques, and by expression of DNA in cells and organisms.

The terms “differentially expressed gene”, “differential gene expression” and their synonyms used interchangeably refer to disease, particularly cancer, such as breast cancer, as compared to its expression in normal or control subjects. Refers to genes that are activated at higher or lower levels among subjects with a disease. The term also includes genes whose expression is activated at higher or lower levels in different stages of the same disease. It will also be appreciated that differentially expressed genes may be activated or inhibited at the nucleic acid level or the protein level, or may undergo other splicing to result in different polypeptide products. Such differences can be demonstrated, for example, by changes in mRNA levels, surface expression, secretion or other distribution of the polypeptide. Differential gene expression is a comparison of expression between two or more genes or their gene products, or a comparison of expression ratios between two or more genes or their gene products, or even two differently processed genes of the same gene. Comparison of products (these may differ between a normal subject and a disease, in particular a subject suffering from cancer, or between various stages of the same disease). Differential expression is, for example, a quantitative, as well as qualitative difference in the pattern of transient or cell expression in a gene or its expression product between normal and diseased cells, or between cells undergoing different disease events or disease stages. Includes all of them. For the purposes of the present invention, "differential gene expression" is at least about 2 times, preferably at least about 4 times, between the expression of a given gene in normal and diseased subjects or at various stages of disease development in a diseased subject, More preferably at least about 6 times and most preferably at least about 10 times.

The term “standardized” with respect to a gene transcript or gene expression product refers to the level of the transcript or gene expression product relative to the average level of the transcript / product of the reference gene set, wherein the reference genes are throughout the patient, tissue or treatment. Selected based on their minimal variation (“housekeeping genes”), or reference genes are all of the genes tested. In the latter case, generally referred to as "global normalization", it is important that the total number of genes tested is relatively large, preferably greater than 50. Specifically, the term 'standardized' with respect to RNA transcripts refers to the level of transcription relative to the average of the levels of transcription of a set of reference genes. More specifically, the mean level of RNA transcript as measured by TaqMan® RT-PCR refers to the Ct value—mean Ct value of the reference gene transcript set.

The terms "expression threshold" and "defined expression threshold" are used interchangeably and above this level refer to the level of the gene or gene product at which the gene or gene product is used as a predictive marker for patient response or drug resistance. . Thresholds are typically defined experimentally from clinical studies. The expression threshold may be selected for maximum sensitivity (eg to detect all responders to one drug), or maximum selectivity (eg to select only responders for one drug), or minimum error.

The phrase “gene amplification” refers to the process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. Replicated regions (extension of amplified DNA) are often referred to as "amplicons". Often, the amount of messenger RNA (mRNA) produced, ie gene expression, is also increased in proportion to the number of copies made of a particular gene.

The term “prognosis” is used herein to refer to the prediction of the likelihood of death from cancer or the progression of neoplastic diseases such as breast cancer, including recurrence, metastatic spread and drug resistance. The term “prediction” herein refers to the likelihood that a patient will respond favorably or adversely to a drug or set of drugs and also the extent of these responses, or that the patient will not have cancer recurrence after surgical removal of major tumors and / or chemotherapy Used to say the likelihood of survival for a certain period of time. The prediction method of the present invention can be used clinically to determine treatment by selecting the most appropriate treatment technique for any particular patient. The predictive methods of the present invention are directed to whether a patient is advantageously responsive to a treatment regimen, such as a surgical procedure, chemotherapy with a given drug or drug combination, and / or radiation therapy, or surgery and / or chemotherapy. It is a valuable means of predicting the long term survival of the patient after the end of therapy or other treatment techniques.

The term “long term” survival is used herein to refer to surviving at least 3 years, more preferably at least 8 years, most preferably at least 10 years after surgery or other treatment.

The term "tumor" as used herein refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all cancerous and cancerous cells and tissues.

The terms "cancer" and "cancerous" describe or refer to physiological conditions in mammals that are typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urethra, thyroid cancer, kidney cancer, carcinoma, melanoma, and brain cancer. It is not limited to.

The "oncology" of cancer includes all the phenomena that harm a patient's well-being. This includes, but is not limited to, abnormal or uncontrollable cell growth, metastasis, disruption of normal functioning of neighboring cells, release of abnormal levels of cytokines or other secretory products, inhibition or exacerbation of neoplastic or immunological responses, neoplasia, precancerous conditions , Malignancies, infiltration of surrounding or distant tissues or organs, such as lymph nodes.

"Patient response" includes, but is not limited to, (1) some degree of inhibition of tumor growth, including slowing and complete growth inhibition; (2); Reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibit (ie, reduce, slow or complete stop) of infiltration of tumor cells into adjacent peripheral organs and / or tissues; (5) inhibition (ie, reduction, slowing down or complete stopping) of metastasis; (6) augmentation of anti-tumor immune response (which may, but need not necessarily) lead to tumor regression or rejection; (7) relief to some extent of one or more symptoms associated with the tumor; (8) increased survival after treatment; And / or (9) any endpoint that represents a benefit for the patient, including a reduced mortality rate in a given period after treatment.

"New adjuvant anticancer therapy" is an additional or adjuvant therapy given prior to the main (main) therapy. Neoadjuvant cancer therapy includes, for example, chemotherapy, radiation therapy, and hormonal therapy. Thus, chemotherapy may be administered prior to surgery to shrink the tumor to make the surgery more effective, or in the case of previously inoperable tumors.

The “stringency” of the hybridization reaction is easily determined by one of ordinary skill in the art and is an experimental calculation that generally depends on probe length, wash temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally relies on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less so. For further details and explanations on the stringency of the hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Strict conditions" or "high stringency conditions" as defined herein typically include (1) low ionic strength, for example, at 50 ° C. for 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate wash and Using high temperatures; (2) denaturant at 42 ° C. during hybridization, for example formamide, for example 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / Using 750 mM sodium chloride, 75 mM sodium citrate with 50 mM sodium phosphate buffer, pH 6.5; Or (3) 50% formamide at 42 ° C., 5 × SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution , Sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, 0.2 × SSC (sodium chloride / sodium citrate) and 50% formamide (at 55 ° C.) at 42 ° C. ), Followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 ° C.

"Moderately stringent conditions" may be the same as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and less stringent washing solutions and hybridizations than those described above. The use of conditions (eg, temperature, ionic strength and% SDS). Examples of moderately stringent conditions include 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × denhardt solution, 10% dextran sulfate at 37 ° C. , And overnight incubation in a solution comprising 20 mg / ml denatured sheared salmon sperm DNA followed by washing of the filter in 1 × SSC at about 37-50 ° C. Those skilled in the art will appreciate how to adjust the temperature, ionic strength, etc. required to adopt factors such as probe length and the like.

In the context of the present invention, reference to "one or more", "two or more", "five or more", etc., among the genes listed in any particular gene set, refers to any one or any and all combinations of the listed genes. Means water.

B. Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombination techniques), microbiology, cell biology, and biochemistry (which are within the ordinary skill in the art). These techniques are described in ([. "Molecular Cloning: A Laboratory Manual", 2 nd edition (Sambrook et al, 1989)], [. "Oligonucleotide Synthesis" (MJ Gait, ed, 1984)], [ "Animal Cell Culture" (RI Freshney, ed., 1987)], "Methods in Enzymology" (Academic Press, Inc.), "" Handbook of Experimental Immunology ", 4 ^th edition (DM Weir & CC Blackwell, eds., Blackwell Science Inc , 1987)], "Gene Transfer Vectors for Mammalian Cells" (JM Miller & MP Calos, eds., 1987)], "Current Protocols in Molecular Biology" (FM Ausubel et al., Eds., 1987)] And ["PCR: The Polymerase Chain Reaction", (Mullis et al., Eds., 1994)).

1. Create a gene expression profiling (Profiling)

Gene expression profiling methods include methods based on hybridization analysis of polynucleotides, methods based on sequencing polynucleotides, and methods based on proteomics. The most commonly used methods known in the art for the quantification of mRNA expression in a sample are Northern blotting and in situ hybridization (Parker & Barnes, Methods). in Molecular Biology 106: 247-283 (1999)]; RNAse protection assay (Hod, Biotechniques 13: 852-854 (1992)); And PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) (Weis et al ., Trends in Genetics 8: 263-264 (1992)]. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, can be used. Representative methods for sequencing-based gene expression analysis include gene expression analysis by serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS). .

2. PCR -based Gene Expression Profiling Method

a. Reverse Transcriptase PCR ( RT - PCR )

One of the most sensitive and most flexible quantitative PCR-based gene expression profiling methods is RT-PCR, which compares mRNA levels in different sample populations in normal and tumor tissues with or without drug treatment. It can be used to characterize gene expression patterns, determine closely related mRNAs, and analyze RNA structure.

The first step is the isolation of mRNA from the target sample. Starting materials are typically total RNA isolated from human tumors or tumor cell lines and corresponding normal tissue or cell lines, respectively. Thus, RNA, along with pooled DNA from a healthy donor, may be a tumor or tumor of various major tumors (breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thyroid, testes, ovaries, uterus, etc. Cell lines). If the source of mRNA is the primary tumor, the mRNA can be extracted, for example, from frozen or stored paraffin-embedded and immobilized (eg formalin-fixed) tissue samples.

General methods for mRNA extraction are known in the art and described in Ausubel et. al . , Current Protocols of Molecular Biology , John Wiley and Sons (1997), are described in standard textbooks of molecular biology. Methods of RNA extraction from tissue embedded in paraffin are described, for example, in Rupp and Locker, Lab. Invest . 56: A67 (1987) and De Andres et. al ., BioTechniques 18: 42044 (1995). In particular, RNA isolation can be performed according to the manufacturer's instructions using a commercial kit, such as a purification kit from Qiagen, a buffer set and a protease. For example, total RNA from cells in culture can be isolated using Qiagen RN easy mini-columns. Other commercially available RNA isolation kits include the MasterPure ^™ Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wisconsin) and Paraffin Block RNA Isolation Kit (Ambion, Inc.). (Ambion, Inc.). Complete RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumors can be isolated, for example, by cesium chloride density gradient centrifugation.

Since RNA cannot be used as a template for PCR, the first step in gene expression profiling by RT-PCR is reverse transcription of the RNA template into cDNA, followed by exponential amplification into its PCR reaction. The two most commonly used reverse transcriptases are avian myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney rat leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically first antigen-stimulated using specific primers, random hexamers, or oligo-dT primers, depending on the environment and goal of expression profiling. For example, the extracted RNA can be reverse-transcribed using the GeneAmp RNA PCR Kit (Perkin Elmer, California, USA) according to the manufacturer's instructions. The derived cDNA can then be used as a template in subsequent PCR reactions.

Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically uses Taq DNA polymerase, which has 5'-3 'nuclease activity, but has 3'-5' read protection. There is a lack of proofreading endonuclease activity. Thus, Takman® PCR typically utilizes 5'-nuclease activity that hybridizes hybridization probes bound to its target amplicons of Taq or Tth polymerase, but any that have equivalent 5 'nuclease activity. May be used. Two oligonucleotide primers are used to generate representative amplicons of the PCR reaction. The third oligonucleotide or probe is designed to detect a nucleotide sequence located between two PCR primers. The probe is non-extensible by Taq DNA polymerase enzyme and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quench dye when the two dyes are placed together as close as they are on the probe. During the amplification reaction, Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resulting probe fragments dissociate in solution and have no quenching effect of the second fluorophore on the signal from the released reporter dye. One molecule of reporter dye is released from each of the synthesized new molecules, and detection of the unquenched reporter dye provides a basis for quantitative interpretation of the data.

TAKMAN® RT-PCR is a commercially available instrument, such as the ABI Prism 7700 ^™ Sequence Detection System ^™ (Perkin-Elmer-Applied Biosystems, USA). Foster City, CA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred 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, laser, charge-coupled device (CCD), camera and computer. The system amplifies the sample in a 96-well format on a thermocycler. During amplification, laser-induced fluorescence signals are collected in real time via fiber optic cables for all 96 wells. The system includes software for running the device and for analyzing the data.

5'-nuclease assay data is initially expressed as Ct, or threshold cycle. As discussed above, the fluorescence value is recorded every cycle and represents the amount of product amplified to that point in the amplification reaction. The point when the fluorescence signal is first recorded as statistically significant is the threshold cycle (C _t ).

In order to minimize the variability effects and errors between samples, RT-PCR is generally performed using reference RNA, which is ideally expressed at some level between different tissues, and is not affected by experimental treatment. . The RNA most often used to standardize gene expression patterns is mRNA for the live gene glyceraldehyde-3-phosphate-dehydrogenase (GAPD) and β-actin (ACTB).

A more recent change in RT-PCR technology is real time quantitative PCR, which measures PCR product accumulation via double-labeled fluorogenic probes (ie, Taqman® probes). Real-time PCR is compatible with both quantitative competitive PCR (wherein internal competitors for each target sequence are used for standardization) and quantitative comparison PCR using standardized genes included in the sample or saline genes for RT-PCR. For further details, see for example Held et. al ., Genome Research 6: 986-994 (1996).

b. Mass array (MassARRAY) system

In the massarray-based gene expression profiling method developed by Sequenom, Inc. (San Diego, Calif.), CDNA obtained after isolation and reverse transcription of RNA is synthesized from a synthetic DNA molecule (competitor) (this is a single Spiked to the targeting cDNA region at all positions except base) and used as internal standard. The cDNA / competitor mixture is PCR amplified and post-PCR shrimp alkaline phosphatase (SAP) enzyme treatment is added to cause dephosphorylation of the remaining nucleotides. After inactivation of alkaline phosphatase, PCR products from competitors and cDNAs are primer stretched, which produces separate mass signals for competitor- and cDNA-derived PCR products. After purification, these products are metered onto an array of chips already loaded with the components necessary for analysis using matrix-assisted laser desorption ionization flow time mass spectrometry (MALDI-TOF MS) analysis. The cDNA present in the reaction is then quantified by analyzing the peak area ratio in the resulting mass spectrum. For further details, see, eg, Ding and Cantor, Proc . Natl . Acad . Sci . USA 100: 3059-3064 (2003).

c. Other PCR -Based Methods

Additional PCR-based techniques are described, for example, in parallax displays (Liang and Pardee, Science 257: 967-971 (1992)); Amplified fragment length polymorphism (iAFLP) (Kawamoto et al., Genome Res . 12: 1305-1312 (1999); BeadArray ^™ technology (Illumina, San Diego, CA) (Oliphant et al., Discovery of Markers for Disease (Supplement to Biotechniques ), June 2002] and Ferguson et al., Analytical Chemistry 72: 5618 (2000)])); Beads for gene expression detection using a commercially available Luminex ¹⁰⁰ LabMAP system and multicolor-coded microspheres (Luminex Corp., Austin, Texas) for rapid assay for gene expression Arrays for Detection of Gene Expression (BADGE) (Yang et al., Genome Res . 11: 1888-1898 (2001); And high coat expression profiling (HiCEP) analysis (Fukumura et al., Nucl . Acids . Res. 31 (16) e94 (2003)).

3. Microarray

Differential gene expression can be determined or confirmed using microarray techniques. Thus, expression profiles of breast cancer-related genes can be measured in fresh or paraffin embedded tumor tissue using microarray technology. In this method, sequences of interest (including cDNA and oligonucleotides) are plated or arranged on a microchip substrate. The arranged sequences are then hybridized with specific DNA probes from the cell or tissue of interest. As in the RT-PCR method, the source of mRNA is typically human RNA or tumor cell lines, and total RNA isolated from the corresponding normal tissues or cell lines. Thus RNA can be isolated from various major tumors or tumor cell lines. If the source of mRNA is a major tumor, the mRNA may be, for example, frozen or stored paraffin-embedded and fixed (eg formalin-fixed) tissue samples (which are routinely prepared and preserved with daily clinical practice). Can be extracted from.

In certain embodiments of microarray technology, PCR amplified inserts of cDNA clones are applied onto the substrate in a dense array. Preferably, 10,000 or more nucleotide sequences are added to the substrate. Microarrayed genes immobilized on microchips with 10,000 elements each are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissue of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by in-focus laser microscopy or by another detection method, such as a CCD camera. Quantification of hybridization of each arranged element allows for evaluation of the corresponding mRNA excess. In the case of dual color fluorescence, separately labeled cDNA probes generated from two RNA sources hybridize to each pair in the array. Thus, the relative excess of transcripts from two sources corresponding to each specified gene is determined simultaneously. Miniaturization scale hybridization provides convenient and rapid evaluation of expression patterns for large numbers of genes. This method has been shown to have the necessary sensitivity to detect rare transcripts (which are expressed in a few copies per cell) and reproducibly with at least approximately 2-fold differences in expression (Schena et. al ., Proc . Natl . Acad . Sci . USA 93 (2): 106-149 (1996)]. Microarray analysis can be performed by commercially available equipment according to the manufacturer's protocol, for example using Affymetrix GenChip technology or Insight's microarray technology.

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

4. Continuous Analysis of Gene Expression ( SAGE )

Serial analysis of gene expression (SAGE) is a method that allows for simultaneous and quantitative analysis of large numbers of gene transcripts without the need to provide separate hybridization probes for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains enough information to uniquely identify a transcript, with the tag being obtained from a unique location within each transcript. Many transcripts are then linked together to form a long series of molecules (sequenced to represent the identity of multiple tags simultaneously). The expression pattern of any transcript population can be quantitatively assessed by measuring the excess of an individual tag and identifying the gene corresponding to each tag. For more details, see, eg, Velculescu et. al ., Science 270: 484-487 (1995) and Velculescu et al ., Cell 88: 243-51 (1997).

5. Gene Expression Analysis by Massively Parallel Signature Sequencing ( MPSS )

Brenner et al ., Nature Biotechnology 18: 630-634 (2000) is a sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads. First, microbead libraries of DNA templates are constructed by in vitro cloning. This is followed by the assembly of a planar array of template-containing microbeads in high density (typically 3 × 10 ⁶ microbeads / cm) flow cells. The free end of the cloned template on each microbead is analyzed simultaneously using a fluorescence-based signature sequencing method that does not require DNA fragment separation. This method has been shown to provide hundreds and thousands of gene signature sequences from yeast cDNA libraries simultaneously and accurately in one operation.

6. Immunohistochemistry

Immunohistochemical methods are also suitable for detecting the expression of prognostic markers of the present invention. Thus, expression is detected using antibodies or antisera, preferably polyclonal antisera and most preferably monoclonal antibodies specific for each marker. Antibodies can be detected, for example, by direct labeling of the antibodies themselves with radiolabels, fluorescent labels, hapten labels such as biotin, or enzymes such as horse radish peroxidase or alkaline phosphatase. Alternatively, an unlabeled primary antibody is used in combination with a labeled secondary antibody comprising an antiserum, polyclonal antiserum or monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are known in the art and are commercially available.

7. Proteomics

The term “proteome” is defined as the entirety of a protein present in a sample (eg, tissue, organism or cell culture) at a particular time period. Proteomics in particular involves the study of the overall change in protein expression in a sample (also called "expression proteomics"). Proteomics typically include the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of individual proteins recovered from the gel, such as mass spectrometry or N-terminal sequencing, and (3) analysis of data using bioinformatics. Proteomics methods are valuable appendices to other methods of gene expression profiling and can be used alone or in combination with other methods to detect the product of prognostic markers of the present invention.

8. General description of mRNA isolation, purification and amplification

Representative protocol steps for profiling gene expression using immobilized, paraffin-embedded tissue as an RNA source, including mRNA isolation, purification, primer extension and amplification, are described in various published magazine articles (eg, literature (TE Godfrey et al. J. Molec . Diagnostics 2: 84-91 [2000] and K. Specht et al., Am. J. Pathol . 158: 419-29 [2001]). . In summary, a representative method begins with cutting about 10 μm thick sections of paraffin-embedded tumor tissue samples. RNA is then extracted and proteins and DNA are removed. After analysis of RNA concentration, RNA repair and / or amplification steps may be included if necessary, followed by RT-PCR after RNA is reverse transcribed using a gene specific promoter. Finally, the data is analyzed to determine the best treatment option (s) available to the patient based on the characteristic gene expression patterns identified in the observed tumor samples.

9. Cancer Chemotherapy

Chemotherapeutic agents used to treat cancer can be divided into several groups depending on their mechanism of action. Some chemotherapeutic agents directly damage DNA and RNA. By disrupting the replication of DNA, the chemotherapeutic agent completely stops replication or results in the production of nonsense DNA or RNA. These categories include, for example, cisplatin (Platinol®), daunorubicin (Cerubidine®), doxorubicin (Adriamycin®), and etoposide (VePesid®). ) Is included. Another group of cancer chemotherapeutic agents interfere with the formation of nucleotides or deoxynucleotides, thereby blocking RNA synthesis and cell replication. Examples of drugs in this class include methotrexate (Abitrexate®), mercaptopurine (Purinethol®), fluorouracil (Adrucil®), and hydroxyurea (hydrarea) (Hydrea) ®). The third class of chemotherapeutic agents affects the synthesis and degradation of mitotic spindles, thereby disrupting cell division. Examples of drugs in this class include vinblastine (Velban®), vincristine (Oncovin®) and taxanes such as pacitaxel (Taxol®), and tocetaxel ( Taxotere®) Tocetaxel is currently administered locally in patients with locally advanced or metastatic breast cancer after a previous chemotherapy failure and after failure of previous platinum-based chemotherapy or It is allowed to treat patients with metastatic small cell lung cancer.

A common problem associated with chemotherapy is the high toxicity of chemotherapeutic agents such as anthracycline and taxanes, which limits the clinical benefits of this treatment approach.

Most patients receive chemotherapy immediately after surgical removal of the tumor. This approach is commonly referred to as adjuvant therapy. However, chemotherapy may also be administered before surgery, as is called so-called neoadjuvant cancer treatment. Although the use of neoadjuvant chemotherapy originated in the treatment of advanced and inoperable breast cancer, it has also been used in the treatment of other types of cancer. The efficacy of neoadjuvant chemotherapy has been tested in several clinical trials. The multi-center National Surgical Adjuvant Breast and Bowel Project B-18 (NSAB B-18) Exam (Fisher et al., J. Clin . Oncology 15: 2002-2004 (1997) and Fisher et al., J. Clin . Oncology 16: 2672-2685 (1998)]), the neoadjuvant cancer therapy is a combination of adriamycin and cyclophosphamide. Was performed together (“AC regimen”). In another clinical trial, neoadjuvant cancer therapy was administered using a combination of 5-fluorouracil, epirubicin and cyclophosphamide ("FEC regimen") (van Der Hage et al., J. Clin . Oncol . 19: 4224-4237 (2001). Newer clinical trials also used taxane-containing neoadjuvant chemotherapy regimens. See, eg, Holmes et al., J. Natl . Cancer Inst . 83: 1797-1805 (1991) and Moliterni et al., Seminars in Oncology , 24: S 17-10-S-17-14 (1999)]. For further information on neoadjuvant chemotherapy for breast cancer, see Cleator et al., Endocrine- Related Cancer 9: 183-195 (2002).

10. Clinical application of cancer gene sets, assayed gene subsequences , and gene expression data

An important aspect of the present invention is the use of the measured expression of specific genes by breast cancer tissue to provide prognostic information. For this purpose, it is essential to correct (standardize) the amount of RNA tested, variations in RNA quality used, and differences in other factors, such as machine and operator differences. Therefore, assays typically measure and incorporate the use of reference RNA, including those transcribed from known salivary genes such as GAPD and ACTB. Accurate methods for standardizing gene expression data are provided in "User Bulletin # 2" for the ABI PRISM 7700 Sequence Detection System (Applied Biosystems; 1997). Alternatively, normalization can be based on the mean or median signal (Ct) of the assayed genes or all of their many subsets (full standardization approach). In the studies described in the Examples below, a so-called central standardization strategy was used, which used a subset of screened genes selected based on lack of correlation with clinical outcome for standardization.

11. Response scores for relapse and therapy and their application

Pending Application No. filed July 10, 2003. 60 / 486,302 describes a computational-based prognosis trial to determine the likelihood of cancer recurrence and / or the likelihood that a patient will respond well to a treatment technique. Features of the operation that distinguish this from other cancer prognostic methods include 1) a unique set of test mRNAs (or corresponding gene expression products) used to measure recurrence, 2) specific weights used to combine expression data, and 3) thresholds used to divide patients into groups with different levels of risk, eg, low, medium and high risk groups. This operation yields a numerical recurrence score (RS) or a response score (RTS) for therapy if the patient's response to the treatment is evaluated.

The test requires laboratory assays to determine the levels of specified mRNAs or expression products thereof, but is fixed or paraffin embedded tumor biopsies that are either fresh or frozen tissue or already collected and stored from patients. Test specimens are available in very small quantities. Thus, the test can be non-invasive. It is also compatible with several different methods of tumor tissue harvested, for example, via core biopsy or microneedle aspiration.

According to this method, the cancer recurrence score (RS) is

(a) generating a gene or protein expression profile with a biological sample comprising cancer cells obtained from the subject;

(b) quantifying the expression levels (ie mRNA or protein levels) of a plurality of individual genes to determine expression values for each gene;

(c) generate a subset of gene expression values, each comprising an expression value for genes linked by cancer-related biological function and / or by co-expression;

(d) multiply the expression level of each gene in one subset by a coefficient reflecting its relative contribution to cancer recurrence or therapy response in the subset and add the multiplied value to yield a value for the subset;

(e) multiply that value of each subset by a coefficient that reflects its contribution to cancer recurrence or therapy response;

(f) is determined by summing the values for each subset multiplied by the coefficients to obtain a recurrence score (RS) or therapy response (RTS) score, wherein

The contribution of each subset that does not linearly correlate with cancer recurrence or therapy response is included only above a predetermined threshold value,

Increased expression of the specified genes gives a negative value to subsets that reduce the risk of cancer recurrence and positive expression to the subsets where expression of the specified genes increases the risk of cancer recurrence.

In a specific embodiment, RS is

(a) Among biological samples containing tumor cells obtained from the subject, GRB7, HER2, EstRl, PR, Bcl2, CEGP1, SURV, Ki.67, MYBL2, CCNB1, STK15, CTSL2, STMY3, CD68, GSTM1, and BAG1 Or determine the degree of expression of their expression products;

(b) is determined by calculating the recurrence score (RS) by the formula:

RS = (0.23 to 0.70) x GRB7 axis threshold-(0.17 to 0.51) x ER axis + (0.53 to 1.56) x growth axis threshold + (0.07 to 0.21) x infiltration axis + (0.03 to 0.15) x CD68-(0.04 To 0.25) x GSTM1-(0.05 to 0.22) x BAG1

[Wherein,

(i) GRB7 axis = (0.45-1.35) × GRB7 + (0.05-0.15) × HER2;

(ii) if the GRB7 axis <-2, then the GRB7 axis threshold = -2,

     If the GRB7 axis> -2, then the GRB7 axis threshold = GRB7 axis;

(iii) the ER axis = (Est1 + PR + Bcl2 + CEGP1) / 4;

(iv) axis of proliferation = (SURV + Ki.67 + MYBL2 + CCNB1 + STK15) / 5;

(v) if propagation axis <-3.5, propagation axis threshold = -3.5,

    If propagation axis ≧ −3.5, then propagation axis threshold = proliferation axis;

(vi) infiltration axis = (CTSL2 + STMY3) / 2,

Wherein the values for all individual genes not specifically indicated in the range can vary between about 0.5 and 1.5, with higher RS indicating an increased likelihood of recurrence of the cancer.

Further details of the invention will be described in the following non-limiting examples.

In invasive breast cancer New supportive cancer  Retrospective Study of Chemotherapy: Paraffin-embedded Core Biopsy  Gene Expression Profile of Tissue

This was a joint study involving Genomic Health, Inc. (Redwood City, Calif.), And Institute Tumori (Milan, Italy). The main purpose of this study was to explore the correlation between pathologic complete response (pCR) to neoadjuvant chemotherapy in locally advanced breast cancer and molecular profile before treatment.

Patient Inclusion Criteria :

Histological diagnosis of invasive breast cancer (operation date 1998-2002); Skin infiltration and / or N2 axillary condition and / or ipsilateral benign clavicle lymph node; Core biopsies, neoadjuvant chemotherapy and surgical resections performed in Istituto Nazionale Tumori (Milan, Italy); Consent to know and sign that a biological material from histological diagnosis or diagnostic procedure is to be used in the study; And histopathological evaluation indicating an appropriate amount of tumor tissue for inclusion in the study.

Exclusion Criteria:

Distant metastasis; No tumor block available from initial core biopsy or from surgical resection; Or no or little tumor in the block (less than 5% of the total tissue on the slide) when the pathologist assessed by observing the H & E slide.

Study design

89 evaluable patients (from a set of 96 clinically evaluable patients) were identified and studied. Levels of 384 mRNA species were measured by RT-PCR, indicating the product of the candidate cancer-related gene selected in the biomedical research literature. Only one gene was lost due to inappropriate signals.

Patient characteristics were as follows: mean age: 50 years old; Tumor grade: 24% equivalent, 55% medium, and 21% poor; 63% of patients were ER positive {by immunohistochemistry}; 70% of patients had benign lymph nodes.

All patients received first neoadjuvant chemotherapy: Doxorubicin + Taxol 3 weeks / 3 cycles followed by Taxol® (paclitaxel) 1 week / 12 cycles. Tumors were surgically removed after the end of chemotherapy. Core tumor biopsy specimens were obtained prior to commencement of chemotherapy and used as RNA sources for RT-PCR assays.

Materials and methods

Fixed and paraffin embedded (FPE) tumor tissues were obtained from biopsies before and after chemotherapy. Core biopsies were performed before chemotherapy. In that case, pathologists selected the most representative primary tumor blocks to provide nine 10 micron fragments for RNA analysis. Specifically, a total of nine sections (10 microns thick each) were prepared and pooled in three Costa Brand Microcentrifuge Tubes (polypropylene, 1.7 mL tubes, transparent; three sections in each tube) and pooled. It was.

Messenger RNA was extracted using the MasterPure ^™ RNA Purification Kit (Epicentre Technologies) and quantified by RiboGreen® fluorescence method (Molecular probes). Molecular assays of quantitative gene expression were performed using ABI Prism 7900 ^™ Sequence Detection System ^™ (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA). The ABI Prism 7900 ^™ consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies the sample in a 384-well format on a thermocycler. During amplification, laser-induced fluorescence signals are collected in real time for all 384 wells and detected in the CCD. The system includes software for running the device and for analyzing the data.

Analysis and results

Tumor tissue was analyzed for 384 genes. Threshold cycle (C _T ) values for each patient were normalized based on the mean of one subset of screened genes for a particular patient selected based on lack of correlation with clinical outcome (central standardization strategy). Favorable Response to Chemotherapy Patients were defined as pathological complete response (pCR). Patients were formally assessed for response upon completion of all chemotherapy.

Clinical complete response (cCR) requires complete disappearance of all clinically detectable disease by physical observation or diagnostic breast imaging.

Pathologically complete response (pCR) requires the absence of residual breast cancer upon histological observation of biopsied breast tissue, lumpectomy or mastectomy specimens after primary chemotherapy. There may be residual coronary carcinoma (DCIS) in the epithelium. There may be no residual cancer in the segment. Eleven (12%) of 89 evaluable patients had pathologic complete response (pCR). Seven of these patients were ER negative.

Partial clinical response was defined as at least 50% reduction in tumor area (sum of the products of the longest vertical diameter) or at least 50% reduction in the sum of the products of the longest vertical diameter of the multiple lesions in the breast and armpit . No disease area was increased by> 25% and no new lesions appeared.

The analysis was performed by comparing the relationship between normalized gene expression and the dual outcome of pCR or no pCR. Univariate generalization models were used with the probit or logit link functions. See, eg, Van K. Borooah, LOGIT and PROBIT, Ordered Multinominal Models, Sage University Paper, 2002.

Table 1 provides a correlation of gene expression and pathological response and lists 86 genes with a p-value of <0.1 for differences between groups. The second column (subject is “direction”) indicates whether increased expression correlates with a decrease or increase in the likelihood of response to chemotherapy. The statistical significance of the predicted value for each gene is provided by the P-value (right column).

Based on the data described in Table 1, the following genes, namely: ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; Increased expression of TK1 correlates with increased likelihood of complete pathological response to treatment; The following genes: TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBPS; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCNDI; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; Increased expression of Bcl2 correlates with a reduced likelihood of complete pathological response to treatment.

The relationship between the recurrence risk calculation (described in U.S. Application No. 60 / 486,302 in the same year) and pCR was also investigated. The calculation includes the measured levels of 21 mRNA species. Sixteen mRNAs (named below) were candidate clinical markers and the remaining five (ACTB, GAPD, GUSB, RPLPO, and TFRC) were reference genes. Baseline-standardized expression measurements range from 0 to 15, where one unit increase reflects a 2 fold increase in RNA.

Recurrence scores (RS) were calculated from four sets of marker genes (four genes of the estrogen receptor group-ESRI, PGR, BCL2, and SCUBE2; five genes of the proliferation set-MKI67, MYBL2, BIRC5, CCNB 1, and STK6; Quantitative expression of two genes of the HER2 set-ERBB2 and GRB7; two genes of the invasion group-MMP11 and CTSL2) and three other individual genes-GSTM1, BAG1, and CD68.

Although the genes and multiplication coefficients used in the equations may vary, in representative embodiments, the following equations may be used to calculate RS:

RS (range, 0-100) = + 0.47 × HER2 group score

                        -0.34 × ER group score

                        + 1.04 × Growth Score

                        + 0.10 × infiltration group score

                        + 0.05 × CD68

                        0.08 × GSTM1

                        0.07 × BAG1

Applying this operation to the study of clinical and gene expression data sets yields a continuous curve for RS versus pCR values, as shown in FIG. 1. The observation of these data shows that patients with RS> 50 are in the upper 50 percentile of patients in terms of the likelihood of response to chemotherapy, and patients with RS <35 are in the lower 50 Shows in percentile.

All references cited throughout this specification are expressly incorporated herein by reference.

While the invention has been described with particular emphasis on certain embodiments, it will be apparent to those skilled in the art that changes and variations in specific methods and techniques are possible. Accordingly, the invention includes all modifications that fall within the spirit and scope of the invention as defined by the following claims.

                                SEQUENCE LISTING <110> GENOMIC HEALTH, INC.       BAKER, JOFFRE B.       SHAK, STEVEN       GIANNI, LUCA <120> GENE EXPRESSION MARKERS FOR PREDICTING   RESPONSE TO CHEMOTHERAPY    <130> 39740-0017 <140> PCT / US2005 / 011760 <141> 2005-04-07 <160> 340 FastSEQ for Windows Version 4.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 1 cgttccgatc ctctatactg cat 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 2 aggtccctgt tggccttata gg 22 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 3 atgcctacag caccctgatg tcgca 25 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 4 tgcagactgt accatgctga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 5 ggccagcacc ataatcctat 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 6 ctgcacacgg ttctaggctc cg 22 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 7 ctgcatgtga ttgaataaga aacaaga 27 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 8 tgtggacctg atccctgtac ac 22 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 9 tgaccacacc aaagcctccc tgg 23 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 10 acgaattgtc ggtgaggtct 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 11 gtccatgctg aaatcattgg 20 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 12 caggatacca cagtcctgga gaccc 25 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 13 cgcttctatg gcgctgagat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 14 tcccggtaca ccacgttctt 20 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 15 cagccctgga ctacctgcac tcgg 24 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 16 tcctgccacc cttcaaacc 19 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 17 ggcggtaaat tcatcatcga a 21 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 18 caggtcacgt ccgaggtcga caca 24 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 19 ggacagcagg aatgtgtttc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 20 acccactcga tttgtttctg 20 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 21 cattggctcc ccgtgacctg ta 22 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer        <400> 22 gggtcaggtg cctcgagat 19 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer        <400> 23 ctgctcactc ggctcaaact c 21 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe        <400> 24 tgggcccaga gcatgttcca gatc 24 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer        <400> 25 cgttgtcagc acttggaata caa 23 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer        <400> 26 gttcaacctc ttcctgtgga ctgt 24 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe        <400> 27 cccaattaac atgacccggc aaccat 26 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer        <400> 28 cctggagggt cctgtacaat 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer        <400> 29 ctaattgggc tccatctcg 19 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe        <400> 30 catcatggga ctcctgccct tacc 24 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer        <400> 31 cagatggacc tagtacccac tgaga 25 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer        <400> 32 cctatgattt aagggcattt ttcc 24 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe        <400> 33 ttccacgccg aaggacagcg at 22 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 34 gcatgttcgt ggcctctaag a 21 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 35 cggtgtagat gcacagcttc tc 22 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 36 aaggagacca tccccctgac ggc 23 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 37 cgtcaggacc caccatgtct 20 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 38 ggttaattgg tgacatcctc aaga 24 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 39 cgcggccgag acatggcttg 20 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 40 tgtatttcaa gacctctgtg cactt 25 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 41 ttagcctgag gaattgctgt gtt 23 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 42 tttatgaacc tgccctgctc ccaca 25 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 43 agatgaagtg gaaggcgctt 20 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 44 tgcctctgta atcggcaact g 21 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 45 caccgcggcc atcctgca 18 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 46 gggcgtggaa cagtttatct 20 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 47 cacggtgaag gtttcgagt 19 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 48 agacatctgc cccaagaagg acgt 24 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 49 tggattggag ttctgggaat g 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 50 gcttgcactc cacaggtaca ca 22 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 51 actggccgtg gcactggaca aca 23 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 52 aaacgagcag tttgccatca g 21 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 53 gttggtgatg ttccgaagca 20 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 54 cctcaccggc atagactgga agcg 24 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 55 tgacaatcag cacacctgca t 21 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 56 tgtgactaca gccgtgatcc tta 23 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 57 caggccctct tccgagcggt 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 58 ctgaaggagc tccaagacct 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 59 caaaaccgct gtgtttcttc 20 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 60 tgctgatgtg ccctctcctt gg 22 <210> 61 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 61 gtggccatcc agctgacc 18 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 62 cagtggtagg tgatgttctg gga 23 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 63 tcctgcgcct gatgtccacc g 21 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 64 cagccaagaa ctggtatagg agct 24 <210> 65 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 65 aaactggctg ccagcattg 19 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 66 tctcctagcc agacgtgttt cttgtccttg 30 <210> 67 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 67 cgacagttgc gatgaaagtt ctaa 24 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 68 ggctgctaga gaccatggac at 22 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 69 cctcctcctg ttgctgccac taatgct 27 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 70 aacttcaagg tcggagaagg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 71 tggctaaact cctgcacttg 20 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 72 ccgtccacgg tctcctcctc a 21 <210> 73 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Foward Primer <400> 73 gtgctacgcc accctgtt 18 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 74 caggggcttc tcgtagatgt 20 <210> 75 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 75 ccgatgttca cgcctttggg tc 22 <210> 76 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Foward Primer <400> 76 ggtgcctact ccattgtgg 19 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 77 gtggagccct tcttcctctt 20 <210> 78 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 78 tactccagca ggcacacaaa cacg 24 <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 79 gggagaacgg gatcaatagg at 22 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 80 gcatcagcca gtcctcaaac t 21 <210> 81 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 81 ctcattgggc accagcaggt ttcc 24 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 82 tccaattcca gcatcactgt 20 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 83 ggcagtgaag gcgataaagt 20 <210> 84 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 84 agaaaagctg tttgtctccc cagca 25 <210> 85 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 85 tgcacagagg gtgtgggtta c 21 <210> 86 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 86 tcttcatctg atttacaagc tgtacatg 28 <210> 87 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 87 caatgcttcc aacaatttgt ttgcttgcc 29 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 88 actccctcta cccttgagca 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 89 caggcctcag ttccttcagt 20 <210> 90 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 90 cagaagaaca gctcagggac ccct 24 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 91 tggtccatcg ccagttatca 20 <210> 92 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 92 tgttctagcg atcttgcttc aca 23 <210> 93 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 93 atctgtatgc ggaacctcaa aagagtccct 30 <210> 94 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 94 cggttatgtc atgccagata cac 23 <210> 95 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 95 gaactgagac ccactgaaga aagg 24 <210> 96 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 96 cctcaaaggt actccctcct cccgg 25 <210> 97 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 97 tggctcttaa tcagtttcgt tacct 25 <210> 98 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 98 caaggcatat cgatcctcat aaagt 25 <210> 99 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 99 tgtcccacga ataatgcgta aattctccag 30 <210> 100 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 100 gtccaggtgg atgtgaaaga 20 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 101 cggccaggat acacatctta 20 <210> 102 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 102 cagcaggccc tcaaggagct g 21 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 103 acggatcaca gtggaggaag 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 104 ctcatccgtc gggtcatagt 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 105 cgctggctca cccctacctg 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 106 ccagcaccat tgttgaagat 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 107 agtctcttgg gcatcgagtt 20 <210> 108 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 108 ccccagacca agtgtgaata catgct 26 <210> 109 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 109 gcactttggg attctttcca ttat 24 <210> 110 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 110 gcatgtaaga agaccctcac tgaa 24 <210> 111 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 111 acaacattct cggtgcctgt aacaaagaa 29 <210> 112 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 112 ggattgtaga ctgtcaccga aattc 25 <210> 113 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 113 ggctattcct cattttctct acaaagtg 28 <210> 114 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 114 cctccaggag gctaccttct tcatgttcac 30 <210> 115 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 115 ccagtggagc gcttccat 18 <210> 116 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 116 ctctctgggt cgtctgaaac aa 22 <210> 117 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 117 tcggccactt catcaggacg cag 23 <210> 118 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 118 ggataattca gacaacaaca ccatct 26 <210> 119 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 119 tgaagtaatc agccacagac tcaat 25 <210> 120 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 120 tcaattgtaa cattctcacc caggccttg 29 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 121 gaagcgcaga tcatgaagaa 20 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 122 ctcctcagac accactgcat 20 <210> 123 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 123 ctgaagcacg acaagctggt ccag 24 <210> 124 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 124 tcagcagcaa gggcatcat 19 <210> 125 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 125 ggtggttttc ttgagcgtgt act 23 <210> 126 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 126 cgcccgcagg cctcatcct 19 <210> 127 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 127 caaaggagct cactgtggtg tct 23 <210> 128 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 128 gagtcagaat ggcttattca cagatg 26 <210> 129 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 129 tgttccaacc actgaatctg gacc 24 <210> 130 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 130 ttgggaaata tttgggcatt 20 <210> 131 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 131 agaagctagg gtggttgtcc 20 <210> 132 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 132 ttgggacatt gtagacttgg ccagac 26 <210> 133 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <133> 133 gcatgggaac catcaacca 19 <210> 134 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 134 tgaggagttt gccttgattc g 21 <210> 135 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 135 ccatggacca acttcactat gtgacagagc 30 <210> 136 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 136 ctccgacgtg gctttcca 18 <210> 137 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 137 cgtaattggc agaattgatg aca 23 <210> 138 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 138 tggcccacag catctatgga atccc 25 <139> <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 139 ccatctgcat ccatcttgtt 20 <210> 140 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 140 ggccaccagg gtattatctg 20 <210> 141 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 141 ctccccaccc ttgagaagtg cct 23 <210> 142 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 142 aggctgctgg aggtcatctc 20 <210> 143 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 143 cttcctgcgg ccacagtct 19 <210> 144 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 144 ccagaggccg tttcttggcc g 21 <210> 145 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 145 tccatgatgg ttctgcaggt t 21 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 146 tgagcagcac catcagtaac g 21 <210> 147 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 147 ccccggacag tggctctgac g 21 <210> 148 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 148 aacgactgct actccaagct caa 23 <210> 149 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 149 ggatttccat cttgctcacc tt 22 <210> 150 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 150 tgcccagcat cccccagaac aa 22 <210> 151 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 151 gcatggtagc cgaagatttc a 21 <210> 152 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 152 tttccggtaa tagtctgtct catagatatc 30 <210> 153 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 153 cgcgtcatac caaaatctcc gattttga 28 <210> 154 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 154 cctgaacctt ccaaagatgg 20 <210> 155 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 155 accaggcaag tctcctcatt 20 <210> 156 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 156 ccagattgga agcatccatc tttttca 27 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 157 agccatcact ctcagtgcag 20 <210> 158 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 158 actgcagagt cagggtctcc 20 <210> 159 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 159 caggtcctat cgtggcccct ga 22 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Foward Primer <400> 160 ccacagctca ccttctgtca 20 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 161 cctcagtgcc agtctcttcc 20 <210> 162 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 162 tccatcccag ctccagccag 20 <210> 163 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 163 agagatcgag gctctcaagg 20 <210> 164 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 164 ggccttttac ttcctcttcg 20 <210> 165 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 165 tggttcttct tcatgaagag cagctcc 27 <210> 166 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 166 tgagtggaaa agcctgacct atg 23 <210> 167 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 167 ggactccatc tcttcttggt caa 23 <210> 168 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 168 tgaagtcatc agctttgtgc caccacc 27 <210> 169 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 169 gacttttgcc cgctaccttt c 21 <210> 170 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 170 gccactaact gcttcagtat gaagag 26 <210> 171 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 171 acagctcatt gttgtcacgc cgga 24 <210> 172 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 172 tgatggtcct atgtgtcaca ttca 24 <210> 173 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 173 tgggacagga aacacaccaa 20 <210> 174 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 174 caggtttcat accaacacag gcttcagcac 30 <175> 175 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 175 cgctcagcca gatgcaatc 19 <210> 176 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 176 gcactgagat cttcctattg gtgaa 25 <210> 177 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 177 tgccccagtc acctgctgtt a 21 <210> 178 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 178 gtgaaatgaa acgcaccaca 20 <210> 179 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 179 gaccctgctc acaaccagac 20 <210> 180 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 180 cagccctttg gggaagctgg 20 <210> 181 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 181 ccaacgcttg ccaaatcct 19 <210> 182 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 182 acggtagtga cagcatcaaa actc 24 <210> 183 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 183 aaccagctct ctgtgacccc aatt 24 <210> 184 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 184 tgattaccat catggctcag a 21 <210> 185 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 185 cttgtgaaaa tgccatccac 20 <210> 186 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 186 tcccaattgt cgcttcttct gcag 24 <210> 187 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Sequence <400> 187 ccgccctcac ctgaagaga 19 <210> 188 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Sequence <400> 188 ggaataagtt agccgcgctt ct 22 <210> 189 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 189 cccagtgtcc gccaaggagc g 21 <210> 190 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 190 ggccaggatc tgtggtggta 20 <210> 191 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 191 ctccacgtcg tggacattga 20 <210> 192 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 192 ctctggcctt ccgagaaggt acc 23 <210> 193 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 193 ggctgtggct gaggctgtag 20 <210> 194 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 194 ggagcattcg aggtcaaatc a 21 <210> 195 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 195 ttcccagagt gtctcacctc cagcagag 28 <210> 196 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 196 gcatcaggct gtcattatgg 20 <210> 197 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 197 agtagttgtg ctgcccttcc 20 <210> 198 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 198 tgtccttacc tgtgggagct gtaaggtc 28 <210> 199 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 199 ctgacacttg ccgcagagaa 20 <210> 200 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 200 aggtggtcct tggtctggaa 20 <210> 201 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 201 ccctttctca cccacctcat ctgcac 26 <210> 202 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 202 cgcttgccta actcatactt tcc 23 <210> 203 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 203 ccattcagac tgcgccactt 20 <210> 204 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 204 tccacgcagc gtggcactg 19 <210> 205 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 205 gcgacagctc ctctagttcc a 21 <206> 206 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 206 ttcccgaagt ctccgcccg 19 <210> 207 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 207 ggaacaccag cttgaatttc ct 22 <210> 208 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 208 ggtgtcagat ataaatgtgc aaatgc 26 <210> 209 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 209 ttcgatattg ccagcagcta taaa 24 <210> 210 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 210 tgctgtcctg tcggtctcag tacgttca 28 <210> 211 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 211 tgtggatgct ggattgattt 20 <210> 212 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 212 aagcagcact tcctggtctt 20 <210> 213 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 213 ccactggtgc agctgctaaa tagca 25 <210> 214 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 214 agtgggagac acctgacctt 20 <210> 215 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 215 tgatctgggc attgtactcc 20 <210> 216 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 216 ttgatcttct gctcaatctc agcttgaga 29 <210> 217 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 217 cccgttcggt ctgaggaa 18 <210> 218 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 218 gagcactcaa ggtagccaaa gg 22 <210> 219 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 219 tccggttcgc catgtcccg 19 <210> 220 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 220 aacagagaca ttgccaacca 20 <210> 221 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 221 gtgatttgcc caggaagttt 20 <210> 222 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 222 ttggatctgc ttgctgtcca aacc 24 <210> 223 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 223 cgcgagcccc tcattataca 20 <210> 224 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 224 cactcgccgt tgacatcct 19 <210> 225 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 225 ctccccacag cgcatcgagg aa 22 <210> 226 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 226 cagtggagac cagttgggta gtg 23 <210> 227 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 227 ccttgaagag cgtcccatca 20 <210> 228 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 228 cacacatgca gagcttgtag cgtaccca 28 <210> 229 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 229 gggctcagct ttcagaagtg 20 <210> 230 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 230 acatgttcag ctggtccaca 20 <210> 231 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 231 tggcagtttt cttctgtcac caaaa 25 <210> 232 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 232 tcacatgcca ctttggtgtt 20 <210> 233 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 233 cttgcaggaa gcggctatac 20 <210> 234 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 234 tcctgggaga gattgaccag ca 22 <210> 235 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 235 gcccgaaacg ccgaatata 19 <210> 236 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 236 cgtggctctc ttatcctcat gat 23 <210> 237 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 237 taccgcagca aaccgcttgg g 21 <210> 238 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 238 gccgggaaga ccgtaattgt 20 <210> 239 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 239 cagcggcacc aggttcag 18 <210> 240 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 240 caaatggctt cctctggaag gtccca 26 <210> 241 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 241 tgctgttgct gagtctgttg 20 <210> 242 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 242 cttgcctggc ttcacagata 20 <210> 243 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 243 ccagtcccca gaagaccatg tctg 24 <210> 244 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 244 tgtggtgagg aaggagtcag 20 <210> 245 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 245 cccagagagt gggtcagc 18 <210> 246 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 246 ctgtgactgt ctccagggct tcca 24 <210> 247 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 247 tggcttcagg agctgaatac c 21 <210> 248 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 248 tgctgtcgtg atgagaaaat agtg 24 <210> 249 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 249 caggcacaca caggtgggac acaaat 26 <210> 250 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 250 gtatcaggac cacatgcagt acatc 25 <210> 251 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 251 tgtcggaatt gatactggca tt 22 <210> 252 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 252 ttgatgcctg tcttcgcgcc ttct 24 <210> 253 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Forward Primer <400> 253 tttccaaaca tcagcgagtc 20 <210> 254 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Reverse Primer <400> 254 aacaggagcg cttgaaagtt 20 <210> 255 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide Probe <400> 255 acggtgcttc tccctctcca gtg 23 <210> 256 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 256 cgttccgatc ctctatactg catcccaggc atgcctacag caccctgatg tcgcagccta 60 taaggccaac agggacct 78 <210> 257 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 257 tgcagactgt accatgctga ccattgccca tcgcctgcac acggttctag gctccgatag 60 gattatggtg ctggcc 76 <210> 258 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 258 ctgcatgtga ttgaataaga aacaagaaag tgaccacacc aaagcctccc tggctggtgt 60 acagggatca ggtccaca 78 <210> 259 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 259 acgaattgtc ggtgaggtct caggatacca cagtcctgga gaccctatcc aatgatttca 60 gcatggac 68 <210> 260 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 260 cgcttctatg gcgctgagat tgtgtcagcc ctggactacc tgcactcgga gaagaacgtg 60 gtgtaccggg a 71 <210> 261 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 261 tcctgccacc cttcaaacct caggtcacgt ccgaggtcga cacaaggtac ttcgatgatg 60 aatttaccgc c 71 <210> 262 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 262 ggacagcagg aatgtgtttc tccatacagg tcacggggag ccaatggttc agaaacaaat 60 cgagtgggt 69 <210> 263 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 263 gggtcaggtg cctcgagatc gggcttgggc ccagagcatg ttccagatcc cagagtttga 60 gccgagtgag cag 73 <210> 264 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 264 cgttgtcagc acttggaata caagatggtt gccgggtcat gttaattggg aaaaagaaca 60 gtccacagga agaggttgaa c 81 <210> 265 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 265 cctggagggt cctgtacaat ctcatcatgg gactcctgcc cttacccagg ggccacagag 60 cccccgagat ggagcccaat tag 83 <210> 266 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 266 cagatggacc tagtacccac tgagatttcc acgccgaagg acagcgatgg gaaaaatgcc 60 cttaaatcat agg 73 <210> 267 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 267 gcatgttcgt ggcctctaag atgaaggaga ccatccccct gacggccgag aagctgtgca 60 tctacaccg 69 <210> 268 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 268 cgtcaggacc caccatgtct gccccatcac gcggccgaga catggcttgg ccacagctct 60 tgaggatgtc accaattaac c 81 <210> 269 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 269 tgtatttcaa gacctctgtg cacttattta tgaacctgcc ctgctcccac agaacacagc 60 aattcctcag gctaa 75 <210> 270 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 270 agatgaagtg gaaggcgctt ttcaccgcgg ccatcctgca ggcacagttg ccgattacag 60 aggca 65 <210> 271 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 271 gggcgtggaa cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac 60 cgtg 64 <210> 272 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 272 tggattggag ttctgggaat gtactggccg tggcactgga caacagtgtg tacctgtgga 60 gtgcaagc 68 <210> 273 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 273 aaacgagcag tttgccatca gacgcttcca gtctatgccg gtgaggctgc tgggccacag 60 ccccgtgctt cggaacatca ccaac 85 <210> 274 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 274 tgacaatcag cacacctgca ttcaccgctc ggaagagggc ctgagctgca tgaataagga 60 tcacggctgt agtcaca 77 <210> 275 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 275 ctgaaggagc tccaagacct cgctctccaa ggcgccaagg agagggcaca tcagcagaag 60 aaacacagcg gttttg 76 <210> 276 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 276 gtggccatcc agctgacctt cctgcgcctg atgtccaccg aggcctccca gaacatcacc 60 taccactg 68 <210> 277 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 277 cagccaagaa ctggtatagg agctccaagg acaagaaaca cgtctggcta ggagaaacta 60 tcaatgctgg cagccagttt 80 <210> 278 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 278 cgacagttgc gatgaaagtt ctaatctctt ccctcctcct gttgctgcca ctaatgctga 60 tgtccatggt ctctagcagc c 81 <210> 279 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 279 aacttcaagg tcggagaagg ctttgaggag gagaccgtgg acggacgcaa gtgcaggagt 60 ttagcca 67 <210> 280 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 280 gtgctacgcc accctgttcg gacccaaagg cgtgaacatc gggggcgcgg gctcctacat 60 ctacgagaag cccctg 76 <210> 281 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 281 ggtgcctact ccattgtggc gggcgtgttt gtgtgcctgc tggagtaccc ccgggggaag 60 aggaagaagg gctccac 77 <210> 282 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 282 gggagaacgg gatcaatagg atcggaaacc tgctggtgcc caatgagaat tactgcaagt 60 ttgaggactg gctgatgc 78 <210> 283 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 283 tccaattcca gcatcactgt ggagaaaagc tgtttgtctc cccagcatac tttatcgcct 60 tcactgcc 68 <210> 284 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 284 tgcacagagg gtgtgggtta caccaatgct tccaacaatt tgtttgcttg cctcccatgt 60 acagcttgta aatcagatga aga 83 <210> 285 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 285 actccctcta cccttgagca agggcagggg tccctgagct gttcttctgc cccatactga 60 aggaactgag gcctg 75 <210> 286 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 286 tggtccatcg ccagttatca catctgtatg cggaacctca aaagagtccc tggtgtgaag 60 caagatcgct agaaca 76 <210> 287 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 287 cggttatgtc atgccagata cacacctcaa aggtactccc tcctcccggg aaggcaccct 60 ttcttcagtg ggtctcagtt c 81 <210> 288 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 288 tggctcttaa tcagtttcgt tacctgcctc tggagaattt acgcattatt cgtgggacaa 60 aactttatga ggatcgatat gccttg 86 <210> 289 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 289 gtccaggtgg atgtgaaaga tccccagcag gccctcaagg agctggctaa gatgtgtatc 60 ctggccg 67 <210> 290 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 290 acggatcaca gtggaggaag cgctggctca cccctacctg gagcagtact atgacccgac 60 ggatgag 67 <210> 291 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 291 ccagcaccat tgttgaagat ccccagacca agtgtgaata catgctcaac tcgatgccca 60 agagact 67 <210> 292 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 292 gcactttggg attctttcca ttatgattct ttgttacagg caccgagaat gttgtattca 60 gtgagggtct tcttacatgc 80 <210> 293 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 293 ggctattcct cattttctct acaaagtggc ctcagtgaac atgaagaagg tagcctcctg 60 gaggagaatt tcggtgacag tctacaatcc 90 <210> 294 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 294 ccagtggagc gcttccatga cctgcgtcct gatgaagtgg ccgatttgtt tcagacgacc 60 cagagag 67 <210> 295 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 295 ggataattca gacaacaaca ccatctttgt gcaaggcctg ggtgagaatg ttacaattga 60 gtctgtggct gattacttca 80 <210> 296 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 296 gaagcgcaga tcatgaagaa gctgaagcac gacaagctgg tccagctcta tgcagtggtg 60 tctgaggag 69 <210> 297 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 297 tcagcagcaa gggcatcatg gaggaggatg aggcctgcgg gcgccagtac acgctcaaga 60 aaaccacc 68 <210> 298 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 298 caaaggagct cactgtggtg tctgtgttcc aaccactgaa tctggacccc atctgtgaat 60 aagccattct gactc 75 <210> 299 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 299 ttgggaaata tttgggcatt ggtctggcca agtctacaat gtcccaatat caaggacaac 60 caccctagct tct 73 <210> 300 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 300 gcatgggaac catcaaccag caggccatgg accaacttca ctatgtgaca gagctgacag 60 atcgaatcaa ggcaaactcc tca 83 <210> 301 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 301 ctccgacgtg gctttccagt ggcccacagc atctatggaa tcccatctgt catcaattct 60 gccaattacg 70 <210> 302 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 302 ccatctgcat ccatcttgtt tgggctcccc acccttgaga agtgcctcag ataataccct 60 ggtggcc 67 <210> 303 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 303 aggctgctgg aggtcatctc cgtgtgtgat tgccccagag gccgtttctt ggccgccatc 60 tgccaagact gtggccgcag gaag 84 <210> 304 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 304 tccatgatgg ttctgcaggt ttctgcggcc ccccggacag tggctctgac ggcgttactg 60 atggtgctgc tca 73 <210> 305 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 305 aacgactgct actccaagct caaggagctg gtgcccagca tcccccagaa caagaaggtg 60 agcaagatgg aaatcc 76 <210> 306 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 306 gcatggtagc cgaagatttc acagtcaaaa tcggagattt tggtatgacg cgagatatct 60 atgagacaga ctattaccgg aaa 83 <210> 307 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 307 cctgaacctt ccaaagatgg ctgaaaaaga tggatgcttc caatctggat tcaatgagga 60 gacttgcctg gt 72 <210> 308 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 308 agccatcact ctcagtgcag ccaggtccta tcgtggcccc tgaggagacc ctgactctgc 60 agt 63 <210> 309 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 309 ccacagctca ccttctgtca ggtgtccatc ccagctccag ccagctccca gagaggaaga 60 gactggcact gagg 74 <210> 310 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 310 agagatcgag gctctcaagg aggagctgct cttcatgaag aagaaccacg aagaggaagt 60 aaaaggcc 68 <210> 311 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 311 tgagtggaaa agcctgacct atgatgaagt catcagcttt gtgccaccac cccttgacca 60 agaagagatg gagtcc 76 <210> 312 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 312 gacttttgcc cgctaccttt cattccggcg tgacaacaat gagctgttgc tcttcatact 60 gaagcagtta gtggc 75 <210> 313 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 313 tgatggtcct atgtgtcaca ttcatcacag gtttcatacc aacacaggct tcagcacttc 60 ctttggtgtg tttcctgtcc ca 82 <210> 314 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 314 cgctcagcca gatgcaatca atgccccagt cacctgctgt tataacttca ccaataggaa 60 gatctcagtg c 71 <210> 315 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 315 gtgaaatgaa acgcaccaca ctggacagcc ctttggggaa gctggagctg tctggttgtg 60 agcagggtc 69 <210> 316 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 316 ccaacgcttg ccaaatcctg acaattcaga accagctctc tgtgacccca atttgagttt 60 tgatgctgtc actaccgt 78 <210> 317 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 317 tgattaccat catggctcag attggctcct atgttcctgc agaagaagcg acaattggga 60 ttgtggatgg cattttcaca ag 82 <210> 318 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 318 ccgccctcac ctgaagagaa acgcgctcct tggcggacac tgggggagga gaggaagaag 60 cgcggctaac ttattcc 77 <210> 319 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 319 ggccaggatc tgtggtggta caattgactc tggccttccg agaaggtacc atcaatgtcc 60 acgacgtgga g 71 <210> 320 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 320 ggctgtggct gaggctgtag catctctgct ggaggtgaga cactctggga actgatttga 60 cctcgaatgc tcc 73 <210> 321 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 321 gcatcaggct gtcattatgg tgtccttacc tgtgggagct gtaaggtctt ctttaagagg 60 gcaatggaag ggcagcacaa ctact 85 <210> 322 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 322 ctgacacttg ccgcagagaa tccctttctc acccacctca tctgcacctt ccagaccaag 60 gaccacct 68 <210> 323 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 323 cgcttgccta actcatactt tcccgttgac acttgatcca cgcagcgtgg cactgggacg 60 taagtggcgc agtctgaatg g 81 <210> 324 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 324 gcgacagctc ctctagttcc accatgtccg cgggcggaga cttcgggaat ccgctgagga 60 aattcaagct ggtgttcc 78 <210> 325 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 325 ggtgtcagat ataaatgtgc aaatgccttc ttgctgtcct gtcggtctca gtacgttcac 60 tttatagctg ctggcaatat cgaa 84 <210> 326 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 326 tgtggatgct ggattgattt caccactggt gcagctgcta aatagcaaag accaggaagt 60 gctgctt 67 <210> 327 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 327 agtgggagac acctgacctt tctcaagctg agattgagca gaagatcaag gagtacaatg 60 cccagatca 69 <210> 328 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 328 cccgttcggt ctgaggaagg ccgggacatg gcgaaccgga tcagtgcctt tggctacctt 60 gagtgctc 68 <210> 329 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 329 aacagagaca ttgccaacca tattggatct gcttgctgtc caaaccagca aacttcctgg 60 gcaaatcac 69 <210> 330 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 330 cgcgagcccc tcattataca ctgggcagcc tccccacagc gcatcgagga atgcgtgctc 60 tcaggcaagg atgtcaacgg cgagtg 86 <210> 331 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 331 cagtggagac cagttgggta gtggtgactg ggtacgctac aagctctgca tgtgtgctga 60 tgggacgctc ttcaagg 77 <210> 332 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 332 gggctcagct ttcagaagtg ctgagttggc agttttcttc tgtcaccaaa agaggtctca 60 atgtggacca gctgaacatg t 81 <210> 333 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 333 tcacatgcca ctttggtgtt tcataatctc ctgggagaga ttgaccagca gtatagccgc 60 ttcctgcaag 70 <210> 334 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 334 gcccgaaacg ccgaatataa tcccaagcgg tttgctgcgg taatcatgag gataagagag 60 ccacg 65 <210> 335 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 335 gccgggaaga ccgtaattgt ggctgcactg gatgggacct tccagaggaa gccatttggg 60 gccatcctga acctggtgcc gctg 84 <210> 336 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 336 tgctgttgct gagtctgttg ccagtcccca gaagaccatg tctgtgttga gctgtatctg 60 tgaagccagg caag 74 <210> 337 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 337 tgtggtgagg aaggagtcag agagctgtga ctgtctccag ggcttccagc tgacccactc 60 tctggg 66 <210> 338 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 338 tggcttcagg agctgaatac cctcccaggc acacacaggt gggacacaaa taagggtttt 60 ggaaccacta ttttctcatc acgacagca 89 <210> 339 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 339 gtatcaggac cacatgcagt acatcggaga aggcgcgaag acaggcatca aagaatgcca 60 gtatcaattc cgaca 75 <210> 340 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Amplicon <400> 340 tttccaaaca tcagcgagtc cacactggag agggagaagc accgtaactt tcaagcgctc 60 ctgtt 65

Claims (70)

Determining the expression level of at least one prognostic RNA transcript or expression product thereof in a biological sample comprising cancer cells obtained from a subject diagnosed with cancer, wherein the prognostic RNA transcript comprises TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A.Catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat G.Catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Hepsin (Hepsin); CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; A transcript of at least one gene selected from the group consisting of TK1, ErbB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, CD68, GSTM1, BCL2, ESR1 (a) ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1; ERBB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2 and CD68; Or if at least one expression unit of the corresponding expression product has increased, the subject is expected to have an increased response to chemotherapy, (b) TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; GSTM1, BCL2, ESR1; Or if at least one expression unit of the corresponding expression product has increased, it is predicted that the subject has the potential to decrease the response to chemotherapy, A method of predicting response to chemotherapy in a subject diagnosed with cancer. The method of claim 1, wherein the prognostic RNA transcript is TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; And a transcript of at least one gene selected from the group consisting of TK1. The method of claim 2, wherein said reaction is a complete pathological reaction. The method of claim 2, wherein the subject is a human patient. The method of claim 2, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer and lung cancer. The method of claim 5, wherein the cancer is breast cancer. The method of claim 6, wherein the cancer is infiltrating breast cancer. 8. The method of claim 7, wherein the cancer is stage II or stage III breast cancer. The method of claim 2, wherein the chemotherapy is adjuvant chemotherapy. The method of claim 2, wherein the chemotherapy is neoadjuvant chemotherapy. The method of claim 10, wherein the neoadjuvant cancer chemotherapy comprises administration of a taxane derivative. The method of claim 11, wherein the taxane is docetaxel or paclitaxel. The method of claim 12, wherein the taxane is docetaxel. The method of claim 11, wherein the chemotherapy further comprises administration of an additional anticancer agent. The method of claim 14, wherein the additional anticancer agent is a member of anthracycline anticancer agent. The method of claim 15, wherein said additional anticancer agent is doxorubicin. The method of claim 15, wherein said additional anticancer agent is a topoisomerase inhibitor. The method of claim 2 comprising determining the expression level of at least two of said prognostic transcripts or expression products thereof. The method of claim 2, comprising determining the expression level of at least five of said prognostic transcripts or expression products thereof. 3. The method of claim 2 comprising determining the expression level of the prognostic transcript or all of their expression products. The method of claim 2, wherein the biological sample is a tissue sample comprising cancer cells. The method of claim 21, wherein the tissue is fixed and embedded in paraffin, fresh, or frozen. The method of claim 21, wherein the tissue is obtained from a microneedle, a core, or other type of biopsy. The method of claim 21, wherein the tissue sample is obtained by microneedle aspiration, bronchial lavage, or coronary biopsy. The method of claim 2, wherein the expression level of said prognostic RNA transcript (s) is determined by RT-PCR or other PCR-based methods. The method of claim 2, wherein the expression level of said expression product (s) is determined by immunohistochemistry. The method of claim 2, wherein the expression level of said expression product (s) is determined by proteomics techniques. The method of claim 2, wherein the assay for measuring the prognostic RNA transcript or expression product thereof is provided in the form of kit (s). The method of claim 1, wherein the prognostic transcript comprises an intron-based sequence and the expression of the intron-based sequence correlates with the expression of a corresponding exon sequence. TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; An array comprising polynucleotides hybridizing to multiple species of TK1, ErbB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, CD68, GSTM1, BCL2, ESR1 genes. 31. The method of claim 30, further comprising: TBP; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; An array comprising polynucleotides that hybridize to multiple species of the TK1 gene. 31. The method of claim 30 further comprising: ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1; An array comprising polynucleotides that hybridize to multiple species of the ERBB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, and CD68 genes. 31. The method of claim 30 further comprising: ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; An array comprising polynucleotides that hybridize to multiple species of the TK1 gene. 31. The method of claim 30, further comprising: TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; An array comprising polynucleotides that hybridize to multiple species of the GSTM1, BCL2, ESR1 genes. 31. The method of claim 30, further comprising: TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; An array comprising polynucleotides that hybridize to multiple species of the RAB6C gene. 31. The array of claim 30, comprising at least five of said polynucleotides. 31. The array of claim 30, comprising at least 10 of said polynucleotides. 31. The array of claim 30, comprising at least 15 of said polynucleotides. The array of claim 30 comprising polynucleotides that hybridize to all of the genes. The array of claim 30 comprising more than one polynucleotide hybridized to the same gene. The array of claim 30, wherein at least one of the polynucleotides comprises an intron-based sequence, wherein expression of the intron-based sequence correlates with expression of the corresponding exon sequence. 42. The array of any one of claims 30-41, wherein said polynucleotide is cDNA. 42. The array of any one of claims 30 to 41 wherein said polynucleotide is an oligonucleotide. (a) TBP in cancer cells obtained from patients to create a personalized genomic profile; ILT.2; ABCC5; CD18; GATA3; DICER1; MSH3; GBP1; IRS1; CD3z; fasl; TUBB; BAD; ERCC1; MCM6; PR; APC; GGPS1; KRT18; ESRRG; E2F1; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNXI; ID2; Rat.catenin; FBXO5; FHIT; MTA1; ERBB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; CDC20; STAT3; ERK1; HLA.DPB1; SGCB; CGA; DHPS; MGMT; CRIP2; MMP12; ErbB3; RAP1GDS1; CDC25B; IL6; CCND1; CYBA; PRKCD; DR4; Heptin; CRABP1; AK055699; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; ZNF38; MCM2; GBP2; SEMA3F; CD31; COL1A1; ER2; BAG1; AKT1; COL1A2; STAT1; Wnt. 5a; PTPD1; RAB6C; Determining normalized expression of an RNA transcript or expression product of a gene or gene set selected from the group consisting of TK1, ErbB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2, CD68, GSTM1, BCL2, ESR1; And (b) generating a report summarizing the data obtained by the gene expression analysis A method of making a personalized genomic profile for a patient, comprising. 45. The method of claim 44, wherein said cancer cell is obtained from a solid tumor. 46. The method of claim 45, wherein the solid tumor is selected from the group consisting of breast cancer, ovarian cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer and lung cancer. 46. The method of claim 45, wherein said cancer cells are obtained from a biopsy sample immobilized in paraffin by immobilizing said tumor. 48. The method of claim 47, wherein said RNA is fragmented. 46. The method of claim 45, wherein said report comprises a recommendation for a treatment technique for said patient. 46. The method of claim 45 further comprising: ILT.2; CD18; GBP1; CD3z; fasl; MCM6; E2F1; ID2; FBXO5; CDC20; HLA.DPB1; CGA; MMP12; CDC25B; IL6; CYBA; DR4; CRABP1; Contig. 51037; VCAM1; FYN; GRB7; AKAP.2; RASSF1; MCP1; MCM2; GBP2; CD31; ER2; STAT1; TK1; ERBB2, CCNB1, BIRC5, STK6, MKI67, MYBL2, MMP11, CTSL2 and CD68; Or if the expression of one or more of the corresponding expression products is determined to be increased, the report includes a prediction that the subject has an increased likelihood of increased response to chemotherapy. 51. The method of claim 50, further comprising treating the patient with a chemotherapeutic agent. The method of claim 51, wherein the patient is receiving adjuvant chemotherapy. The method of claim 51, wherein the patient is receiving neoadjuvant cancer chemotherapy. The method of claim 53, wherein the neoadjuvant cancer chemotherapy comprises administration of a taxane derivative. 55. The method of claim 54, wherein said taxane is docetaxel or paclitaxel. The method of claim 55, wherein the taxane is docetaxel. The method of claim 54, wherein the chemotherapy further comprises administration of an additional anticancer agent. 58. The method of claim 57, wherein said additional anticancer agent is a member of anthracycline anticancer agent. 58. The method of claim 57, wherein said additional anticancer agent is doxorubicin. The method of claim 57, wherein the anticancer agent is a topoisomerase inhibitor. 46. The method of claim 45, further comprising: TBP; ABCC5; GATA3; DICER1; MSH3; IRS1; TUBB; BAD; ERCC1; PR; APC; GGPS1; KRT18; ESRRG; AKT2; A. catenin; CEGP1; NPD009; MAPK14; RUNX1; Rat.catenin; FHIT; MTA1; ErbB4; FUS; BBC3; IGF1R; CD9; TP53BP1; MUC1; IGFBP5; rhoC; RALBP1; STAT3; ERK1; SGCB; DHPS; MGMT; CRIP2; ErbB3; RAP1GDS1; CCND1; PRKCD; Heptin; AK055699; ZNF38; SEMA3F; COL1A1; BAG1; AKT1; COL1A2; Wnt. 5a; PTPD1; RAB6C; GSTM1, BCL2, ESR1; Or when the expression of one or more of the corresponding expression products is determined to be increased, the report comprises a prediction that the subject has a reduced likelihood of a response to chemotherapy. 45. The method of claim 44, wherein said RNA transcript comprises an intron-based sequence and wherein expression of said intron-based sequence correlates with expression of a corresponding exon sequence. (a) ACTB, BAG1, BCL2, CCNB1, CD68, SCUBE2, CTSL2, ESR1, GAPD, GRB7, GSTM1, GUSB, ERBB2, MKI67, MYBL2, PGR, RPLPO, STK6, MMP11, BIRC5, TFRC genes, or expressions thereof Determining the expression level of the RNA transcript of the product, and (b) calculating a recurrence score (RS) Comprising a method of determining the patient's response to chemotherapy. 64. The method of claim 63, wherein the patient with RS> 50 is in the top 50 percentiles of patients who are likely to respond to chemotherapy. The method of claim 63, wherein the patient with RS <35 is in the lower 50 percentile of patients prone to respond to chemotherapy. 66. The method of claim 63, wherein RS is (i) growth factor subsets: GRB7 and HER2; (ii) estrogen receptor subsets: ER, PR, Bcl2, and CEGP1; (iii) proliferation subsets: SURV, Ki.67, MYBL2, CCNB1, and STK15; And (iv) infiltration subsets: CTSL2, and STMY3 Wherein a gene of any of subsets (i) to (iv) can be coexpressed with the gene in the tumor and substituted with a replacement gene having a Pearson correlation coefficient ≥ 0.40 Generate; Subsets (i) to (iv) are determined by weighting the contribution to each breast cancer recurrence to calculate the recurrence score (RS) of the subject. 67. The method of claim 66, wherein the RNA transcripts of CD68, GSTM1 and BAG1 or expression products thereof, or corresponding replacement genes or expression products thereof are measured and the contribution of the gene or replacement gene to breast cancer recurrence is calculated in RS calculation. Further comprising the step of including. 67. The method of claim 66, wherein RS is determined using the formula: RS = (0.23 to 0.70) x GRB7 axis threshold-(0.17 to 0.55) x ER axis + (0.52 to 1.56) x growth axis threshold + (0.07 to 0.21) x infiltration axis + (0.03 to 0.15) x CD68-(0.04 To 0.25) x GSTM1-(0.05 to 0.22) x BAG1 [Wherein, (i) GRB7 axis = (0.45-1.35) × GRB7 + (0.05-0.15) × HER2; (ii) if the GRB7 axis <-2, then the GRB7 axis threshold = -2,      If the GRB7 axis> -2, then the GRB7 axis threshold = GRB7 axis; (iii) the ER axis = (Est1 + PR + Bcl2 + CEGP1) / 4; (iv) axis of proliferation = (SURV + Ki.67 + MYBL2 + CCNB1 + STK15) / 5; (v) if propagation axis <-3.5, propagation axis threshold = -3.5,     If propagation axis ≧ −3.5, then propagation axis threshold = proliferation axis; (vi) infiltration axis = (CTSL2 + STMY3) / 2, Wherein individual contributions of the genes in (iii), (iv) and (vi) are weighted with a coefficient between 0.5 and 1.5, with higher RS indicating an increased likelihood of recurrence of breast cancer. 67. The method of claim 66, wherein RS is determined using the formula: RS (range, 0-100) = + 0.47 × HER2 group score                         -0.34 × ER group score                         + 1.04 × Growth Score                         + 0.10 × infiltration group score                         + 0.05 × CD68                         0.08 × GSTM1                         0.07 x BAG1. 64. The method of claim 63, wherein the RNA transcript comprises an intron-based sequence and the expression of the intron-based sequence correlates with the expression of the corresponding exon sequence.

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