WO2002044423A2 - Method of determining a chemotherapeutic regimen by assaying gene expression in primary tumors - Google Patents

Method of determining a chemotherapeutic regimen by assaying gene expression in primary tumors Download PDF

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Publication number
WO2002044423A2
WO2002044423A2 PCT/US2001/045188 US0145188W WO0244423A2 WO 2002044423 A2 WO2002044423 A2 WO 2002044423A2 US 0145188 W US0145188 W US 0145188W WO 0244423 A2 WO0244423 A2 WO 0244423A2
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Prior art keywords
tumor
gene
expression
determinant
rna
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Application number
PCT/US2001/045188
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French (fr)
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WO2002044423A3 (en
Inventor
Kathleen D. Danenberg
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Response Genetics, Inc.
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Publication date
Priority claimed from US09/877,177 external-priority patent/US6582919B2/en
Priority claimed from US09/877,178 external-priority patent/US7049059B2/en
Priority claimed from US09/879,217 external-priority patent/US7005278B2/en
Priority claimed from US09/988,784 external-priority patent/US6602670B2/en
Application filed by Response Genetics, Inc. filed Critical Response Genetics, Inc.
Priority to AU2002220012A priority Critical patent/AU2002220012A1/en
Publication of WO2002044423A2 publication Critical patent/WO2002044423A2/en
Publication of WO2002044423A3 publication Critical patent/WO2002044423A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to prognostic methods which are useful in
  • the clinical objective is to destroy malignant cells
  • Chemotherapy is based on the use of drugs that are selectively toxic (cytotoxic) to cancer cells.
  • drugs that interfere with cancer cells include drugs that interfere with cytotoxic drugs.
  • nucleic acid synthesis nucleic acid synthesis, protein synthesis, and other vital metabolic processes.
  • 5-fluorouracil As the single most effective agent for the treatment of colon, head and neck, and breast cancers, the primary action of 5-fluorouracil (5-FU) is to inhibit
  • stage colorectal tumors expressing high levels of thymidylate synthase responded poorly when treated with 5-FU/leucovorin. Thus, the patients' survival was low compared to those without elevated TS expression. (Leichman et al., J. Clin Oncol., 15: 3223-3229, 1997).
  • DPD dihydropyrimidine dehydrogenase
  • the "no response" classification included all therapeutic responses with ⁇ 50% tumor shrinkage, progressing growth resulting in a >25% tumor increase
  • DPD is a catabolic enzyme which reduces the 5,6 double bond of 5-FU
  • tumors was relatively narrow (0.6- 2.5 x 10 "3 , 4.2-fold; relative to an internal control)
  • DPD is also an important marker for 5-FU toxicity. It was observed that
  • DPD levels in 5-FU therapy was dramatically illustrated by the occurrence of 19 deaths in Japan from an unfavorable drug interaction between 5-FU and an anti-viral
  • Another class of chemotherapeutic agents specifically inhibits tumor-cell
  • RTKs Receptor tyrosine kinases
  • RTKs are large membrane spanning proteins which possess an extracellular ligand binding domain for growth factors such as epidermal growth factor (EGF) and an intracellular portion which functions as a kinase to phosphorylate tyrosine
  • EGF epidermal growth factor
  • Class I kinases such as the EGFR family of receptor tyrosine kinases include the EGF, HER2-neu, erbB, Xmrk, DER and let23 receptors. These receptors are frequently present in common human cancers such as breast cancer (Sainsbury et al.,
  • gastrointestinal cancer such as colon, rectal or stomach cancer
  • cancers such as thyroid and uterine cancer.
  • cancers such as brain, lung squamous cell, bladder, gastric, breast, head and neck,
  • EGFR disorders are those characterized by EGFR expression by cells normally not expressing EGFR, or increased EGFR activation leading to unwanted cell proliferation, and/or the existence of
  • the EGFR is known to be activated by its ligand EGF
  • TGF-a transforming growth factor-alpha
  • Inhibitors of receptor tyrosine kinases EGFR are employed as selective inhibitors of the growth of mammalian cancer cells (Yaish et al. Science, 1988, 242, 933).
  • erbstatin an EGF receptor tyrosine kinase inhibitor, reduced the
  • Two such styrene derivatives are Class I RTK inhibitors whose effectiveness
  • 4-anilinoquinazoline derivatives are useful as inhibitors of receptor tyrosine kinases.
  • the very tight structure-activity relationships shown by these compounds suggests a
  • Chemotherapy against tumors often requires a combination of agents such as those described above. Accordingly, the identification and quantification of
  • metastases has been intensely pursued. Differential gene expression between a tumor and its metastases not only underlies the mechanism of tumor metastasis, but more importantly to the clinician, it determines the efficacy of chemotherapeutic agents on the primary tumor and matched metastases. Whereas primary tumor
  • specimens are generally available either as pre-treatment paraffin-embedded biopsies
  • metastases are not readily detectable and biopsy specimens of matched tumor
  • FPE paraffin-embedded
  • RNA-based quantitative genetic marker assay when planning any RNA-based quantitative genetic marker assay.
  • the health care professional performing the biopsy must hastily deliver the tissue sample to a
  • tissue sample must remain frozen until it reaches the diagnostic facility, however far
  • routine biopsies generally comprise a heterogenous mix of stromal and tumorous tissue. Unlike with fresh or frozen tissue, FPE biopsy tissue
  • samples are readily microdissected and separated into stromal and tumor tissue and
  • RNA isolation of RNA from fixed tissue, and especially fixed and paraffin embedded tissue results in highly degraded RNA, which is generally not thought to be applicable to gene expression studies.
  • invention to provide a method of quantifying mRNA from primary tumor tissue in
  • the invention relates to a method for determining a chemotherapeutic regimen for an individual, comprising obtaining a mRNA sample from a primary
  • chemotherapeutic regimen comprising a chemotherapeutic agent appropriate for the tumor gene determinant to treat the tumor metastases.
  • the invention further relates to a method of determining whether a chemotherapeutic regimen comprising a chemotherapeutic agent appropriate for a
  • tumor gene determinant in a primary tumor is appropriate for a tumor metastasis
  • tumor gene determinant with a predetermined threshold level and determining the chemotherapeutic regimen for the tumor metastsis.
  • the invention also provides a method of quantifying the amount of tumor
  • FPE paraffin- embedded
  • tyrosine kinase targeted gene expression based chemotherapeutic appropriate for
  • treating the primary tumor is appropriate for treating a tumor metastasis.
  • the invention provides a method of quantifying the amount of DPD, TS
  • FPE embedded
  • tyrosine kinase targeted gene expression based chemotherapeutic appropriate for
  • treating the primary tumor is appropriate for treating a tumor metastasis.
  • the invention also provides a method of quantifying the amount of DPD, TS and/or EGFR mRNA expression in fresh, frozen, fixed or fixed and paraffin-
  • FPE embedded
  • primary tumor is appropriate for treating a tumor metastasis.
  • Figure 1 is a graph showing relative TS gene expression in matching primary
  • Figure 2 is a chart illustrating how to calculate EGFR expression relative to
  • the chart contains data obtained with two test samples, (unknowns 1 and 2), and illustrates how to determine the uncorrected gene
  • UGE expression data
  • the internal control gene in the figure is ⁇ -actin and the calibrator
  • RNA Human Liver Total RNA (Stratagene, Cat #735017).
  • Figure 3 is a chart illustrating how to calculate DPD expression relative to an
  • the chart contains data obtained with two test samples,
  • UGE expression data
  • the internal control gene in the figure is ⁇ -actin and the calibrator RNA is Universal PE RNA; Cat
  • Figure 4 is a chart illustrating how to calculate JS expression relative to an internal control gene.
  • the chart contains data obtained with two test samples,
  • the internal control gene in the figure is ⁇ -actin and the calibrator RNA is Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems.
  • tumor gene determinant refers to a gene whose expression level is indicative of the effectiveness of a specific chemotherapeutic or class of chemotherapeutics. Such tumor gene determinants may include genes whose expression levels prognosticate the effectiveness of anti-metabolite
  • chemotherapeutic agents for example, as shown in pending application 09/877,178
  • JS expression level can prognosticate the effectiveness of a 5-FU based
  • tumor gene determinants may include genes involved in DNA
  • ERCC1 expression level can prognosticate the effectiveness of a genotoxic based chemotherapy.
  • a “predetermined threshold value” is determined by statistically correlating the expression level of a "tumor gene determinant" with the effectiveness of a course
  • a threshold value may be determined by those of skill in the art
  • the Kruskal-Wallis test may be used to test for significant
  • Kaplan-Meier survival curves and the log rank test are used to analyze univariate distributions for survival
  • SPSS Inc. Chicago HI.
  • SPSS Inc. Chicago HI.
  • chemotherapeutic agent specific for the tumor gene determinant refers to any chemotherapeutic agent which is known to target a cancer cell, and has an
  • chemotherapeutic agent specific for the tumor gene determinant is not necessary so long
  • Chemotherapeutic agents specific for a tumor gene there is a correlation between the expression of the tumor gene determinant and the effectiveness of the agent. Chemotherapeutic agents specific for a tumor gene
  • determinant may include, but are not limited to, genotoxic therapies, anti-metabolite therapies and/or receptor tyrosine kinase based therapies.
  • Genes are classes of chemotherapeutic agents that inflict damage on cellular DNA. Examples of genotoxic chemotherapeutic agents
  • Such compounds are meant to be adn inistered alone, or together with other chemotherapies
  • Platinum-based genotoxic chemotherapies comprises heavy metal coordination compounds which form covalent DNA adducts. Generally, these heavy metal compounds bind covalently to DNA to
  • Platinum first agents include analogs or derivatives of any of the
  • Tumors currently manageable by platinum coordination compounds include testicular, endometrial, cervical, gastric, squamous cell,
  • Trans-Diamminedichloroplatinum (II) (trans-DDP) is clinically useless owing, it is thought, to the rapid repair of its DNA adducts.
  • trans-DDP Trans-Diamminedichloroplatinum
  • chemotherapeutic agent herein likely would provide a compound with low toxicity in
  • nonselected cells and high relative toxicity in selected cells.
  • high relative toxicity in selected cells In a preferred
  • the platinum compound is cisplatin.
  • Many compounds are commonly given with platinum-based chemotherapy agents.
  • BEP bleomycin
  • etoposide, cisplatin is used for testicular cancer
  • MVAC memotrexate, vinblastine, doxorabicin, cisplatin
  • MVP mitomycin C, vinblastine, cisplatin
  • genotoxic agents are those that form persistent genomic lesions and are
  • chemotherapeutic agents preferred for use as chemotherapeutic agents in the clinical management of cancer.
  • lesions in a cell's genome can impede DNA replication, impair the replication fidelity of
  • cell death is the resistance of genomic lesions formed therefrom to cellular repair.
  • Genotoxic agents that form persistent genomic lesions e.g., lesions that remain in the genome at least until the cell commits to the cell cycle, generally are more effective
  • DNA alkylating agents affected by levels of DNA repair gene expression are DNA alkylating agents and DNA
  • Psoralens are genotoxic compounds known to be useful in the photochemotherapeutic treatment of cutaneous diseases such as psoriasis, vitiligo, fungal
  • antineoplastic antibiotics which include but are not limited to the following classes of
  • alkylate DNA are those that include the haloethylnitrosoureas, especially the
  • chloroethylnitrosoureas Representative members of this broad class include carmustine,
  • chlorozotocin fotemustine, lomustine, nimustine, ranimustine and streptozotocin.
  • Haloethylnitrosourea first agents can be analogs or derivatives of any of the foregoing
  • Another class of agents, members of which alkylate DNA, include the
  • DNA alkylating agents include the alkyl sulfonates, represented by
  • busulfan the azinidines, represented by benzodepa; and others, represented by, e.g.,
  • metabolite chemotherapeutic agents specific for the tumor gene determinant known to be important in tumor cell metabolism include 5-FU, methotrexate, and ara-C.
  • Receptor tyrosine kinase targeted chemotherapeutic agents are agents that
  • RTKs receptor tyrosine kinases
  • RTKs are over active. Examples of receptor tyrosine kinase targeted chemotherapeutic
  • kinase signaling include 4-anilinoquinazolines such as
  • PCT WO 94/03427 tricyclic polyhydroxyhc compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as
  • tyrosine kinase inhibitors for use in the treatment of cancer.
  • Other agents targeting receptor tyrosine kinase signaling activity include antibodies that
  • Antibodies complexing with the receptor activate serum complement and or mediate antibody-dependent cellular cytotoxicity.
  • Antibodies are selected that greatly inhibit the receptor function by binding the steric vicinity of the ligand binding site of the receptor (blocking the receptor), and/or that bind the growth
  • the antibodies inhibit an autocrine growth cycle (i.e.
  • ligands e.g. TGF-a
  • TGF-a some ligands, e.g. TGF-a, are found lodged in cell membranes, the antibodies serving a
  • the targeting function are directed against the ligand and/or the receptor.
  • the moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment of such a toxin.
  • Enzymatically active toxins and fragments thereof often used are diphtheria, nonbinding
  • abrin abrin, modeccin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
  • the antibodies are conjugated to small molecule
  • Conjugates of the monoclonal antibody and such cytotoxic moieties are made using a variety of bifunctional protein coupling agents. Examples of such reagents are SPDP, IT, bifunctional derivatives of imidoesters such a dimethyl
  • adipimidate HC1 active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine,
  • bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl )-e1-hylenediamine
  • Cytotoxic radiopharmaceuticals for treating cancer may include tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as 1 ,5-difluoro-2,4-dinitrobenzene.
  • the lysing portion of a toxin may be joined to the Fab fragment of the antibodies.
  • Cytotoxic radiopharmaceuticals for treating cancer may include tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as 1 ,5-difluoro-2,4-dinitrobenzene.
  • cytotoxic moiety as used herein is intended to include such isotopes.
  • agents specific for a rumor gene determinant may be chosen by the individual physician
  • the severity of the condition to be treated will vary with the severity of the condition to be treated and to the route of administration.
  • the severity of the condition may, for example, be evaluated, in part, by
  • Suitable routes may include oral, rectal, transdermal,
  • vaginal, transmucosal, or intestinal administration including
  • intramuscular, subcutaneous, intrameduUary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution,
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The invention primarily rests in the observation from archival pathological
  • tumor gene determinants in primary tumors is also appropriate for treating tumor metastases.
  • the present invention allows one to correlate the effectiveness
  • a primary tumor having high level of EGFR mRNA expression is a primary tumor having high level of EGFR mRNA expression.
  • the tumor metastases of patients whose primary tumors are metastases of patients whose primary tumors.
  • rSmRNA are considered likely to be insensitive to TS- targeted chemotherapy.
  • the tumor gene determinant can be any gene whose expression level is indicative of the effectiveness of a specific chemotherapeutic or class of
  • the tumor gene determinants are JS DPD and/or EGFR gene expression in a primary tumor used to treat tumor metastases in the liver.
  • the methods of the invention are applied to solid tumors, most preferably colorectal tumors.
  • Solid or lymphoid primary tumors or portions thereof are surgically resected
  • RNA isolated from frozen or fresh tumor samples is extracted from the cells by any of the melhods typical in die art, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.),
  • Tissue obtained from the patient after biopsy is often fixed, usually by formalin
  • Non-embedded, fixed tissue as well as fixed and embedded tissue may also be used in
  • Solid supports for embedding fixed tissue are envisioned to be
  • RNA is extracted from paraffin-embedded (FPE) tissue cells by any of the
  • FPE tissue means tissue that has been fixed and embedded in a sohd removable support, such
  • RNA may be isolated from an archival pathological sample or biopsy sample which is first deparaffinized.
  • An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent
  • Suitable lower alcohols include, methanol, ethanol,
  • the fixed or fixed and deparaffinized samples can be homogenized using mechanical, sonic or other means of homogenization. Rehydrated
  • samples may be homogenized in a solution comprising a chaotropic agent, such as
  • guanidinium thiocyanate also sold as guanidinium isothiocyanate. Homogenized
  • samples are heated to a temperature in the range of about 50 to about 100 °C in a
  • chaotropic solution which contains an effective amount of a chaotropic agent, such as a
  • guanidinium compound A preferred chaotropic agent is guanidinium thiocyanate.
  • an "effective amount of chaotropic agent” is chosen such that RNA is purified from a paraffin-embedded sample in an amount of greater than about 10-fold that
  • Chaotropic agents include: guanidinium
  • the preferred chaotropic agent for the methods of the invention is a
  • guanidinium compound such as guanidinium isothiocyanate (also sold as guanidinium
  • the guanidinium solution may be of higher concentration such that the final concentration achieved in the sample is in the range of about 1 to about 5M.
  • the guanidinium solution also is preferably buffered to a pH of about 3 to
  • a suitable biochemical buffer such as Tris-Cl.
  • the chaotropic solution may also contain reducing agents, such as dithiothreitol (DTT)
  • the chaotropic solution may also contain RNAse
  • RNA is then recovered from the chaotropic solution by, for example, phenol
  • RNA may then be further purified using the techniques of extraction, electrophoresis,
  • RT-PCR polymerase chain reaction
  • the present invention envisages the quantification of mRNA via use of a PCR-free systems employing, for example
  • quantification of tumor gene determinants and an internal control or house keeping gene is done using a fluorescence based
  • the output of the ABI 7700 (TaqMan® Instrument) is expressed in Ct's or
  • cycle thresholds With the TaqMan® system, a highly expressed gene having a higher number of target molecules in a sample generates a signal with fewer PCR cycles (lower
  • Such an assessment comprises a determination of the overall constitutive
  • House-keeping genes or “internal controls” can include, but are not limited to, the cyclophilin gene, ⁇ -actin gene, the transferrin receptor gene, GAPDH gene, and the like. Most preferably, the internal control gene is ⁇ -actin gene as described by Eads et al, Cancer Research 1999; 59:2302-2306.
  • RNA recovery requires the use of "calibrator RNA.”
  • the "calibrator RNA” is intended to be any available source of accurately pre-quantified
  • primers are used: a forward, and a reverse primer, and a dual labeled fluorogenic oligonucleotide probe that anneals specifically to the cDNA of the gene at issue.
  • fluorogenic probe anneals to the cDNA within the region between where the forward
  • any suitable primers may be used to assess the mRNA expression levels described above. They must provide an accurate assessment of DPD,
  • TS and/or EGFR expression in a fixed paraffin embedded (FPE) tissue are also present.
  • frozen tissue i.e. they have high specificity for their target RNA.
  • Preferred primers for DPD are SEQ ID NO: 4-6.
  • Preferred primers for TS are SEQ ID NO: 7-9.
  • Preferred primers for ⁇ -actin are SEQ ID NO: 10-12.
  • UGE Uncorrected Gene Expression
  • EGFR, TS and DPD, expression is shown in Examples 3, 4, and 5 respectively and illustrated with sample calculations in Figures 2, 3, and 4.
  • Example 6 provides equations for calculating the UGE for any tumor gene determinant, referred to herein as GENEX.
  • a further aspect of this invention provides a method to normalize uncorrected
  • TaqMan® derived tumor gene determinant UGE values (such as but not limited to DPD,
  • TS and or EGFR UGE values from a tissue sample are normalized to samples with
  • TaqMan® derived DPD, TS and/or EGFR values from a tissue sample are known non-TaqMan® derived relative tumor gene determinant: ⁇ actin expression values.
  • ⁇ actin expression values are known non-TaqMan® derived relative tumor gene determinant.
  • TaqMan® derived DPD, TS and/or EGFR values from a tissue sample are known non-TaqMan® derived relative tumor gene determinant.
  • tissue samples are based on the ratio of the RT-PCR signal of a target gene to a
  • tissue samples are formalin fixed and paraffin-embedded (FPE) samples and RNA
  • tissue samples are formalin fixed and paraffin-embedded
  • FPE FPE samples and RNA is extracted from them according to the protocol described in Example 1. Such a determination can be made using standard pre-TaqMan®,
  • cahbrator Human Liver Total RNA (Stratagene, Cat #735017), is 26.95 x 10 "3 .
  • K EGFR may be determined for an internal control gene other than ⁇ -actin and/or a calibrator RNA different than Human Liver Total RNA (Stratagene, Cat #735017).
  • tissue samples are formalin fixed and paraffin-embedded (FPE) samples and RNA
  • K DPD may be dete ⁇ nined for an internal control gene other than ⁇ -actin and/or a
  • tissue samples for which DPD expression levels relative to that particular internal control gene have already been determined are formalin fixed and paraffin-embedded (FPE) samples and RNA is
  • K ra may be determined for an internal control gene other than ⁇ -actin and/or a calibrator RNA different than Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems. To do so, one must calibrate both the internal control gene and the calibrator RNA to -tissue samples for which JS expression levels relative to that
  • tissue samples are formalin
  • the methods of the invention are applicable to a wide range of tissue and tumor
  • diagnostic or prognostic tool for a range of cancers including breast, head and neck,
  • Pre-chemotherapy treatment tumor biopsies are usually available only as fixed paraffin embedded (FPE) tissues, generally containing only a very small amount of FPE
  • tumor gene determinant expression such as DPD, TS and/or EGFR gene
  • RNA is extracted from paraffin-embedded tissue by the following general
  • Steps 2 and 3 are repeated until the majority of paraffin has been dissolved.
  • Steps 5 and 6 are repeated with successively more dilute ethanol solutions:
  • RNA Isolation with Phenol-Chloroform (1) 400 ⁇ L guanidine isothiocyanate solution including 0.5% sarcosine and 8 ⁇ L dithiothreitol is added.
  • tissue homogenizer Ultra-Turrax, KA-Works, Inc., Wilmington, NC
  • speed 1 low speed
  • speed 5 high speed
  • the sample is then heated at about 95 °C for about 5-20 minutes. It is
  • the cap may be affixed with a plastic clamp or
  • RNA is precipitated with about 10 ⁇ L glycogen and with 400
  • RNA is pelleted by centrifugation for about 7 minutes in a benchtop
  • appropriate buffer for further experiments e.g., 50 pi. 5mM Tris chloride, pH 8.0.
  • RNA was isolated from microdissected or non-microdissected formalin fixed paraffin embedded (FPE) tissue as illustrated in Example 1, or from fresh or frozen tissue by a single step guamdinium isocyanate method using the QuickPrepTM Micro
  • RNA pellet was dissolved in 50 ul of 5 mM Tris/Cl at pH 8.0.
  • Transcriptase will extend an oligonucleotide primer hybridized to a single-stranded RNA
  • RNA was reverse transcribed with random hexamers and M-MLV
  • Reverse transcription mix consists of 10 ul 5X buffer (250 mM Tris-HCl, pH 8.
  • gene e.g., ⁇ -actin
  • cDNA was done using a fluorescence based real-time detection method (ABI PRISM 7?00 or 7900 Sequence Detection System [TaqMan ® ], Applied Biosystems, Foster City, CA.) as described by Heid et ah, (Genome Res
  • TS-781 primer TaqMan probe TS-781 (SEQ ID NO: 9) was used.
  • ⁇ -actin TaqMan probe ⁇ -actin -611 (SEQ ID NO: 7) was used.
  • TAMRA 3 'quencher dye
  • the PCR reaction mixture consisted 0.5ml of the reverse transcription reaction
  • oligonucleoride primers 200 nM TaqMan® probe primer, 5 U AmpliTaq Gold
  • x Taqman Buffer A containing a reference dye to a final volume of less than or equal to 25 ml (all reagents Applied Biosystems, Foster City, CA).
  • the forward and reverse primers were respectively EGFR-1753-F
  • TaqMan EGFR-1773 (SEQ ID NO: 3).
  • the forward and reverse primers were respectively DPD 3a-51F (SEQ ID NO:
  • the forward and reverse primers were respectively TS-763F (SEQ ID NO: 1
  • TaqMan ⁇ -actin-611 (SEQ ID NO: 10).
  • EXAMPLE 3 Determining the Uncorrected Gene Expression (UGE) for EGFR
  • amplification reactions are performed on the cahbrator RNA template and are referred to
  • Ct threshold
  • step involves raising the number 2 to the negative DCt, according to the
  • TaqMan® instrument the following calculation is carried out:
  • the normalization calculation entails a multiplication of the UGE with a correction factor (K. EGFR ) specific to EGFR and a particular calibrator RNA.
  • correction factor K EGFR can also be determined for any internal control gene and any accurately pre-quantified calibrator RNA.
  • correction factor K EGFR 1.54.
  • tissue sample on a scale that is consistent with pre-TaqMan® EGFR expression studies
  • TaqMan® apparatus with the K EGFR specific correction factor, given the use of the same internal control gene and calibrator RNA.
  • a K EGFR may be determined using any accurately pre-quantified calibrator RNA
  • Ct cycle threshold
  • DCt calibrator Ct ra - Ct p.actin (From the "calibration" reaction)
  • step involves raising the number 2 to the negative DCt, according to the
  • TaqMan® instrument the following calculation is carried out:
  • the normalization calculation entails a multiplication of the UGE with a
  • correction factor (K rs ) specific to JS and a particular calibrator RNA.
  • factor K ⁇ s can also be determined for any internal control gene and any accurately pre-
  • L121, L150, L220, L164 was divided by its corresponding TaqMan® derived UGE to
  • tissue sample on a scale that is consistent with pre-TaqMan® JS expression studies, one
  • a K ⁇ s may be determined using any accurately pre-quantified calibrator RNA or internal control gene. Future sources of accurately pre-quantified RNA can be cahbrated
  • ⁇ -actin and DPD amplification reactions are the test reactions. Separate ⁇ -actin and DPD amplification reactions are performed on the calibrator RNA and are referred to as the calibration
  • the Taqman instrument will yield four different cycle threshold (Ct) values: Ct ⁇ r ⁇ and Ct p.actin from the test reactions and O ⁇ and Ct p . actin from the calibration
  • step involves raising the number 2 to the negative DCt, according to the
  • the normalization calculation entails a multiplication of the UGE with a correction factor (K ⁇ r ⁇ ) specific to DPD and a particular calibrator RNA.
  • DPD can be determined using any internal control gene and any accurately pre-quantified calibrator RNA: Preferably, the internal control gene ⁇ -actin
  • the internal control gene ⁇ -actin and the calibrator RNA Universal PE RNA
  • a K. DPD may be determined using any accurately pre-quantified calibrator RNA. Future sources of accurately pre-quantified RNA can be calibrated to published samples
  • RNA such as Universal PE RNA; Cat #4307281, lot # 3617812014 described above.
  • control amplification reaction are the test reactions. Separate GENEX and ⁇ -actin amphfication reactions are performed on the cahbrator R ⁇ A template and are referred to as the calibration reactions.
  • the TaqMan® instrument will yield four different cycle
  • Ct threshold (Ct) values: Ct G£r ⁇ : and Ct p . actin from the test reactions and Ct G£NEA - and Ct p.actin
  • DCt calibrator Ct GENEX - Ct p . actin (From the "calibration" reaction)
  • step involves raising the number 2 to the negative DCt, according to the
  • TaqMan® instrument the following calculation is carried out:
  • the normalization calculation entails a multiplication of the UGE with a correction factor (K G£JVE ) specific to GENEX and a particular calibrator RNA.
  • correction factor K EGFR can also be determined for any internal control gene and any
  • the internal control gene ⁇ -actin and the accurately pre-quantified calibrator RNA Human Liver Total RNA (Stratagene,
  • Corrected Relative GENE X ⁇ xpression UG ⁇ x
  • a K GENEX may be determined using any accurately pre-quantified calibrator RNA or internal control gene. Future sources of accurately pre-quantified RNA can be
  • JS gene expressions were measured in 17 sets of tissues from paraffin-embedded
  • the matching metastatic tumor sample is derived from a liver biopsy. Considering the primary tumors and the metastases as separate sets, the mean TS expressions were 5.16 x 10 "3 for primary tumors
  • EXAMPLE 8 Correlation between TS expression in primary tumor and
  • TS mRNA expression relative to expression of the housekeeping gene ⁇ -actin, was measured using a real time fluorescent dye

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Abstract

The invention relates to a method for determining a chemotherapeutic regimen for an individual, comprising obtaining a mRNA sample from a primary tumor specimen; determining a gene expression level for a tumor gene determinant in the specimen; comparing the gene expression level for the tumor gene determinant with a predetermined threshold value for that gene; and providing a chemotherapeutic regimen comprising a chemotherapeutic agent appropriate for the tumor gene determinant to treat the tumor metastases.

Description

METHOD OF DETERMINING A CHEMOTHERAPEUTIC REGIMEN BY
ASSAYING GENE EXPRESSION IN PRIMARY TUMORS
FIELD OF THE INVENTION
The present invention relates to prognostic methods which are useful in
medicine, particularly cancer chemotherapy.
BACKGROUND OF THE INVENTION
Cancer arises when a normal cell undergoes neoplastic transformation and becomes a malignant cell. Transformed (malignant) cells escape normal physiologic
controls specifying cell phenotype and restraining cell proliferation. Transformed cells in an individual's body thus proliferate in the absence of these normal controls,
thus forming a tumor.
When a tumor is found, the clinical objective is to destroy malignant cells
selectively while mitigating any harm caused to normal cells in the individual undergoing treatment. Chemotherapy is based on the use of drugs that are selectively toxic (cytotoxic) to cancer cells. Several general classes of chemotherapeutic drugs have been developed, including drugs that interfere with
nucleic acid synthesis, protein synthesis, and other vital metabolic processes.
Susceptibility of an individual neoplasm to a desired chemotherapeutic drug
or combination of drugs often, however, can be accurately assessed only after a trial period of treatment. The time invested in an unsuccessful trial period poses a
significant risk in the clinical management of aggressive malignancies. Therefore, it is of importance to assess the expression status of genetic determinants targeted by specific chemotherapeutic agents.. For example, if a tumor expresses high levels of
DNA repair genes, it is likely that the tumor will not respond well to low doses of
DNA-damaging genotoxic agents. Thus, the expression status of genetic
determinants of a tumor will help the clinician develop an appropriate
chemotherapeutic regimen specific to the genetic repertoire of the tumor.
As the single most effective agent for the treatment of colon, head and neck, and breast cancers, the primary action of 5-fluorouracil (5-FU) is to inhibit
thymidylate synthase activity (Moertel, C.G. New Engl. J. Med., 330:1136-1142,
1994). For more than 40 years the standard first-line treatment for colorectal cancer was the use of 5-FU alone, but it was supplanted as "standard of care" by the
combination of 5-FU and CPT-11 (Saltz et ah, Irinotecan Study Group. New
England Journal of Medicine. 343:905-14, 2000). Recently, the combination of 5-
FU and oxaliplatin has produced high response rates in colorectal cancers (Raymond
et ah, Semin. Oncol., 25:4-12, 1998). We have previously shown that advanced
stage colorectal tumors expressing high levels of thymidylate synthase (TS) responded poorly when treated with 5-FU/leucovorin. Thus, the patients' survival was low compared to those without elevated TS expression. (Leichman et al., J. Clin Oncol., 15: 3223-3229, 1997).
The mechanism of action and the metabolic pathway of 5-FU have been
intensively studied over the years to identify the most important biochemical
determinants of the drug's anti-tumor activity. The ultimate goal was to improve the
clinical efficacy of 5-FU by a) modulation of its intracellular metabolism and biochemistry and b) measuring response determinants in patients' tumors prior to
therapy to predict which patients are most likely to respond (or not to respond) to the drug. Two major determinants emerged from these studies: 1) the identity of the target enzyme of 5-FU, thymidylate synthase (TS) and 2) the identity of the 5-FU
catabolic enzyme, dihydropyrimidine dehydrogenase (DPD).
The first studies in the area of tumor response prediction to 5-FU based
therapy centered on the target enzyme TS in colorectal cancer. Leichman et al
(Leichman et ah, ,J. Clin Oncol., i5:3223-3229, 1997) carried out a prospective
clinical trial to correlate tumor response to 5-FU with JS gene expression as
determined by RT-PCR in pre-treatment biopsies from colorectal cancers. This
study showed: 1) a large 50-fold range of JS gene expression levels among these
tumors, and 2) strikingly different levels of JS gene expression between responding and non-responding tumors. The range of TS levels of the responding groups (0.5-
4.1 x 10"3, relative to an internal control) was narrower than that of the non-
responding groups (1.6-23.0 x 10"3, relative to an internal control). The investigators
determined a resulting "non-response cutoff threshold level of JS expression above
which there were only non-responders. Thus, patients with JS expression above this "non-response cutoff threshold could be positively identified as non-responders
prior to therapy. The "no response" classification included all therapeutic responses with <50% tumor shrinkage, progressing growth resulting in a >25% tumor increase
and non-progressing tumors with either <50% shrinkage, no change or <25% increase. These tumors had the highest JS levels. Thus, high JS expression
identifies particularly resistant tumors. JS expression levels above a certain
threshold identified a subset of tumors not responding to 5-FU, whereas JS
expression levels below this number predicted gn appreciably higher response rate, yet did not specifically identify responding tumors.
Subsequent studies investigated the usefulness of DPD expression levels as a tumor response determinant to 5-FU treatment in conjunction with JS expression levels. DPD is a catabolic enzyme which reduces the 5,6 double bond of 5-FU,
rendering it inactive as a cytotoxic agent. Previous studies have shown that DPD
levels in normal tissues could influence the bio-availability of 5-FU, thereby
modulating its pharmacokineti.es and anti-tumor activity (Harris et al, Cancer Res.,
SO: 197-201, 1990). Additionally, evidence has been presented that DPD levels in
tumors are associated with sensitivity to 5-FU (Etienne et al, J. Clin. Oncol., 13:
1663-1670, 1995; Beck et al, Eur. J. Cancer, 30: 1517-1522, 1994). Salonga et al, (Clin Cancer Res., 6:1322-1327, 2000, hereby incorporated by reference in its
entirety) investigated gene expression of DPD as a tumor response determinant for
5-FU/leucovorin treatment in a set of tumors in which JS expression had already been determined. As with JS, the range of DPD expression among the responding
tumors was relatively narrow (0.6- 2.5 x 10"3, 4.2-fold; relative to an internal control)
compared with that of the non-responding tumors (0.2-16 x 10"3, 80-fold; relative to
an internal control). There were no responding tumors with a DPD expression
greater than a threshold level of about 2.5 x 10"3. Furthermore, DPD and JS expression levels showed no correlation with one another, indicating that they are independently regulated genes. Among the group of tumors having both JS and
DPD expression levels below their respective "non-response cut-off threshold
levels, 92% responded to 5-FU/leucovorin. Thus, responding tumors could be identified on the basis of low expression levels of DPD and JS.
DPD is also an important marker for 5-FU toxicity. It was observed that
patients with very low DPD levels (such as in DPD Deficiency Syndrome; i.e. thymine uraciluria) undergoing 5-FU based therapy suffered from life-threatening toxicity (Lyss et al, Cancer Invest., 11 : 2390240, 1993). Indeed, the importance of
DPD levels in 5-FU therapy was dramatically illustrated by the occurrence of 19 deaths in Japan from an unfavorable drug interaction between 5-FU and an anti-viral
compound, Sorivudine (Diasio et ah, Br. J. Clin. Pharmacol. 46, 1-4, 1998). It was
subsequently discovered that a metabolite of Sorivudine is a potent inhibitor of
DPD. This treatment resulted in DPD Deficiency Syndrome-like depressed levels of
DPD which increased the toxicity of 5-FU to the patients (Diasio et al. , Br. J. Clin.
Pharmacol. 46, 1-4, 1998).
Thus, because of a) the widespread use of 5-FU protocols in cancer treatment, b) the important role of DPD expression in predicting tumor response to
5-FU and c) the sensitivity of individuals with DPD-Deficiency Syndrome to
common 5-FU based treatments, it is clear that accurate determination of DPD expression levels prior to chemotherapy will provide an important benefit to cancer
patients.
Another class of chemotherapeutic agents specifically inhibits tumor-cell
proliferation by attenuating mitogenic signaling through receptor tyrosine kinases
(RTKs), in cells where RTKs are over active. (Drugs of the Future, 1992, 17, 119). Receptor tyrosine kinases (RTKs) are important in the transduction of mitogenic
signals. RTKs are large membrane spanning proteins which possess an extracellular ligand binding domain for growth factors such as epidermal growth factor (EGF) and an intracellular portion which functions as a kinase to phosphorylate tyrosine
amino acid residues on cytosol proteins, thereby mediating cell proliferation.
Various classes of receptor tyrosine kinases are known based on families of growth
factors which bind to different receptor tyrosine kinases. (Wilks, Advances in Cancer Research, 1993, 60, 43-73)
Class I kinases such as the EGFR family of receptor tyrosine kinases include the EGF, HER2-neu, erbB, Xmrk, DER and let23 receptors. These receptors are frequently present in common human cancers such as breast cancer (Sainsbury et al.,
Brit. J. Cancer, 1988, 58, 458; Guerin et al., Oncogene Res., 1988, 3, 21), squamous
cell cancer of the lung (Hendler et al., Cancer Cells, 1989, 7, 347), bladder cancer
(Neal et al., Lancet, 1985, 366), oesophageal cancer (Mukaida et al, Cancer, 1991,
68, 142), gastrointestinal cancer such as colon, rectal or stomach cancer (Bolen et
al., Oncogene Res., 1987, 1, 149), leukaemia (Konaka et al., Cell, 1984, 37, 1035)
and ovarian, bronchial or pancreatic cancer (European Patent Specification No. 0400586). As further human tumor tissues are tested for the EGF family of receptor
tyrosine kinases it is expected that its widespread prevalence will be established in
other cancers such as thyroid and uterine cancer.
Specifically, EGFR tyrosine kinase activity is rarely detected in normal cells
whereas it is more frequently detectable in malignant cells (Hunter, Cell, 1987, 50,
823). It has been more recently shown that EGFR is overexpressed in many human
cancers such as brain, lung squamous cell, bladder, gastric, breast, head and neck,
oesophageal, gynaecological and thyroid tumours. (W J Gullick, Brit. Med. Bull., 1991, 47, 87). Receptor tyrosine kinases are also important in other
cell-proliferation diseases such as psoriasis. EGFR disorders are those characterized by EGFR expression by cells normally not expressing EGFR, or increased EGFR activation leading to unwanted cell proliferation, and/or the existence of
inappropriate EGFR levels. The EGFR is known to be activated by its ligand EGF
as well as transforming growth factor-alpha (TGF-a).
Inhibitors of receptor tyrosine kinases EGFR are employed as selective inhibitors of the growth of mammalian cancer cells (Yaish et al. Science, 1988, 242, 933). For example, erbstatin, an EGF receptor tyrosine kinase inhibitor, reduced the
growth of EGFR expressing human mammary carcinoma cells injected into athymic nude mice, yet had no effect on the growth of tumors not expressing EGFR. (Toi et
al., Eur. J. Cancer Clin. Oncol., 1990, 26, 722). Various derivatives of styrene are
also stated to possess tyrosine kinase inhibitory properties (European Patent
Application Nos. 0211363, 0304493 and 0322738) and to be of use as anti-tumor
agents. Two such styrene derivatives are Class I RTK inhibitors whose effectiveness
has been demonstrated by attenuating the growth of human squamous cell carcinoma
injected into nude mice (Yoneda et al., Cancer Research, 1991, 51, 4430). It is also known from European Patent Applications Nos. 0520722 and 0566226 that certain
4-anilinoquinazoline derivatives are useful as inhibitors of receptor tyrosine kinases. The very tight structure-activity relationships shown by these compounds suggests a
clearly-defined binding mode, where the quinazoline ring binds in the adenine
pocket and the anilino ring binds in an adjacent, unique lipophilic pocket. Three
4-anilinoquinazoline analogues (two reversible and one irreversible inhibitor) have
been evaluated clinically as anticancer drugs. Denny, Farmaco 2001
Jan-Feb;56(l-2):51-6. Recently, the U.S. FDA approved the use of the monoclonal antibody trastazumab (Herceptin®) for the treatment of HER2-neu overexpressing metastatic breast cancers. Scheurle, et al., Anticancer Res 20:2091-2096, 2000.
Chemotherapy against tumors often requires a combination of agents such as those described above. Accordingly, the identification and quantification of
determinants of resistance or sensitivity to each single drug has become an important
tool to design individual combination chemotherapy.
Moreover, the search for genetic differences between primary tumors and
metastases has been intensely pursued. Differential gene expression between a tumor and its metastases not only underlies the mechanism of tumor metastasis, but more importantly to the clinician, it determines the efficacy of chemotherapeutic agents on the primary tumor and matched metastases. Whereas primary tumor
specimens are generally available either as pre-treatment paraffin-embedded biopsies
or as resection specimens, in many cases, and especially in earlier stages of cancer,
metastases are not readily detectable and biopsy specimens of matched tumor
metastases on which phenotypic analyses could be performed would thus not be
available. Therefore, it is important to determine the degree of variation of gene
expression between primary tumors and metastases. This information is vital in order to determine whether or not a particular chemotherapeutic would be an
effective therapeutic against the both the primary tumor as well as the metastases. To date there has been no reliable way of determining whether a particular
chemotherapy directed toward the expression of a tumor gene determinant
appropriate for a primary tumor is also appropriate for treating a metastsis.
Currently, the only way to reach such a conclusion was to have a fresh or frozen
tissue biopsy of both the primary tumor and its metastasis. This would require a
biopsy of primary tumor and matching tumor metastases. Unfortunately, because tumor metastases are often diffucult to reach by standard surgical procedures and
often only at great risk to the patient, it was previously not possible to determine whether a treatment regiment for the primary tumor would be effective in treating the metastases. Moreover, post-mortem analysis of tumor metastasis samples
immediately frozen or fixed for comparison to similarly fixed matching primary
tumor samples comes too late for the patient.
Previously, there existed no method to accurately and systematically compare the expression of tumor gene determinants in both primary tumor and metastases available in pathological archives. Most patient derived pathological samples are
routinely fixed and paraffin-embedded (FPE) to allow for histological analysis and subsequent archival storage. Thus, most biopsy tissue samples are not useful for
analysis of gene expression because such studies require a high integrity of RNA so
that an accurate measure of gene expression can be made. Currently, gene
expression levels can be only qualitatively monitored in such fixed and embedded
samples by using immunohistochemical staining to monitor protein expression
levels.
The use of frozen tissue by health care professionals as described in Leichman et al, and Reed et al, poses substantial inconveniences. Rapid biopsy
delivery to avoid tissue and subsequent mRNA degradation is the primary concern
when planning any RNA-based quantitative genetic marker assay. The health care professional performing the biopsy, must hastily deliver the tissue sample to a
facility equipped to perform an RNA extraction protocol immediately upon tissue
sample receipt. If no such facility is available, the clinician must promptly freeze the
sample in order to prevent mRNA degradation. In order for the diagnostic facility to
perform a useful RNA extraction protocol prior to tissue and RNA degradation, the tissue sample must remain frozen until it reaches the diagnostic facility, however far
away that may be. Maintaining frozen tissue integrity during transport using specialized couriers equipped with liquid nitrogen and dry ice, comes only at a great expense.
Moreover, routine biopsies generally comprise a heterogenous mix of stromal and tumorous tissue. Unlike with fresh or frozen tissue, FPE biopsy tissue
samples are readily microdissected and separated into stromal and tumor tissue and
therefore, offer an advantage over the use of fresh or frozen tissue. However, isolation of RNA from fixed tissue, and especially fixed and paraffin embedded tissue, results in highly degraded RNA, which is generally not thought to be applicable to gene expression studies.
We report here a significant association between levels of tumor determinant
gene expression in primary tumor with expression of the same tumor determinant
gene in matching metastases in archival samples. Accordingly, it is the object of the
invention to provide a method of quantifying mRNA from primary tumor tissue in
order to provide an early prognosis for genetically targeted chemotherapies to treat tumors throughout the patient's body.
SUMMARY OF THE INVENTION
The invention relates to a method for determining a chemotherapeutic regimen for an individual, comprising obtaining a mRNA sample from a primary
tumor specimen; determining a gene expression level for a tumor gene determinant
in the specimen; comparing the gene expression level for the tumor gene
determinant with a predetermined threshold value for that gene; and providing a
chemotherapeutic regimen comprising a chemotherapeutic agent appropriate for the tumor gene determinant to treat the tumor metastases.
The invention further relates to a method of determining whether a chemotherapeutic regimen comprising a chemotherapeutic agent appropriate for a
tumor gene determinant in a primary tumor is appropriate for a tumor metastasis
comprising, obtaining an mRNA sample from the primary tumor, determining an
expression level of a tumor gene determinant, comparing the expression level of the
tumor gene determinant with a predetermined threshold level and determining the chemotherapeutic regimen for the tumor metastsis.
The invention also provides a method of quantifying the amount of tumor
gene determinant mRNA expression in fresh, frozen, fixed or fixed and paraffin- embedded (FPE) tissue relative to gene expression of an internal control in a primary
tumor in order to determine whether an anti-metabolite, genotoxic, and/or receptor
tyrosine kinase targeted gene expression based chemotherapeutic appropriate for
treating the primary tumor is appropriate for treating a tumor metastasis.
The invention provides a method of quantifying the amount of DPD, TS
and/or EGFR mRNA expression in fresh, frozen, fixed or fixed and paraffin-
embedded (FPE) tissue relative to gene expression of an internal control in a primary tumor in order to determine whether an anti-metabolite, genotoxic, and/or receptor
tyrosine kinase targeted gene expression based chemotherapeutic appropriate for
treating the primary tumor is appropriate for treating a tumor metastasis.
The invention also provides a method of quantifying the amount of DPD, TS and/or EGFR mRNA expression in fresh, frozen, fixed or fixed and paraffin-
embedded (FPE) tissue relative to gene expression of an internal control in a primary
tumor in order to determine whether a 5-FU, platinum, and/or receptor tyrosine kinase targeted gene expression based chemotherapeutic appropriate for treating the
primary tumor is appropriate for treating a tumor metastasis.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing relative TS gene expression in matching primary
and metastatic issue in CRC. All values on the X and Y coordinates are times 103.
Figure 2 is a chart illustrating how to calculate EGFR expression relative to
an internal control gene. The chart contains data obtained with two test samples, (unknowns 1 and 2), and illustrates how to determine the uncorrected gene
expression data (UGE). The chart also illustrates how to normalize UGE generated by the TaqMan® instrument with known relative EGFR values determined by pre- TaqMan® teclmology. This is accomplished by multiplying UGE to a correction
factor KEGFR. The internal control gene in the figure is β-actin and the calibrator
RNA is Human Liver Total RNA (Stratagene, Cat #735017).
Figure 3 is a chart illustrating how to calculate DPD expression relative to an
internal control gene. The chart contains data obtained with two test samples,
(unknowns 1 and 2), and illustrates how to determine the uncorrected gene
expression data (UGE) UCG. The chart also illustrates how to normalize UGE generated by the Taqman instrument with previously published DPD values. This is
accomplished by multiplying UGE to a correction factor KDPD. The internal control gene in the figure is β-actin and the calibrator RNA is Universal PE RNA; Cat
#4307281, lot # 3617812014 from Applied Biosystems.
Figure 4 is a chart illustrating how to calculate JS expression relative to an internal control gene. The chart contains data obtained with two test samples,
(unknowns 1 and 2), and illustrates how to determine the uncorrected gene
expression data (UGE). The chart also illustrates how to normalize UGE generated
by the TaqMan® instrument with previously published JS values. This is accomplished by multiplying UGE to a correction factor Kτs. The internal control gene in the figure is β-actin and the calibrator RNA is Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
A "tumor gene determinant" as used herein refers to a gene whose expression level is indicative of the effectiveness of a specific chemotherapeutic or class of chemotherapeutics. Such tumor gene determinants may include genes whose expression levels prognosticate the effectiveness of anti-metabolite
chemotherapeutic agents. For example, as shown in pending application 09/877,178
and 09/879,217 (both hereby incorporated by reference in their entirety), DPD and
JS expression level can prognosticate the effectiveness of a 5-FU based
chemotherapy. Other tumor gene determinants may include genes involved in DNA
repair, whose expression levels prognosticate the effectiveness of genotoxic chemotherapeutic agents. Alternatively, as shown in pending applications
09/877,178 and applications 09/ (to be assigned)(filed on November 20,
2001)(both hereby incorporated by reference in their entirety) ERCC1 expression level can prognosticate the effectiveness of a genotoxic based chemotherapy. In
another example, EGFR and Her2-neu expression as shown in pending application
09/877,177 (hereby incorporated by reference in its entirety) can prognosticate the
effectiveness of a receptor tyrosine kinase targeted chemotherapy. Furthermore,
increased levels of the tumor determinant gene GST-pi have been found in drug resistant tumors, although the exact mechanism remains unclear.
A "predetermined threshold value" is determined by statistically correlating the expression level of a "tumor gene determinant" with the effectiveness of a course
• of treatment including a "chemotherapeutic agent specific for the tumor gene determinant" in question.
Generally, a threshold value may be determined by those of skill in the art
from tissue samples given a method of determining tumor gene determinant
expression in tissue samples with accompanying information including course of treatment and/or survival time. For example, the Mann- Whitney U test may be used to test for significant associations between the continuous test variable corrected
relative tumor gene determinant expression and dichotomous variables (patient sex, age above and below the median age, presence of weight loss, presence of pleural
effusion, tumor stage). The Kruskal-Wallis test may be used to test for significant
differences in corrected relative tumor gene determinant expression within multiple
groups (ECOG performance status, histopathology). Fisher's exact test may further
be used for the analysis of categorical clinicopathological values including response
and dichotomized corrected relative tumor gene determinant expression values.
Additionally, in order to determine a threshold value, Kaplan-Meier survival curves and the log rank test are used to analyze univariate distributions for survival
and disease-free survival. The maximal chi-square method of Miller and Siegmund (Biometrics 1982; 38:1011-1016) and Halpem (Biometrics 1982; 38:1017-1023) can
be adapted to determine which expression value best segregated patients into poor-
and good prognosis subgroups (in terms of likelihood of surviving), with the log-
rank test as the statistic used to measure the strength of the grouping. To determine a P value that would be interpreted as a measure of the strength of the association
based on the maximal chi-square analysis, 1000 boot-strap-like simulations are used to estimate the distribution of the maximal chi-square statistics under the hypothesis of no association. (Biometrics 1982; 38:1017-1023). Cox's proportional hazards
modeling of factors that are significant in univariate analysis is performed to identify
which factors might have a significant influence on survival. SPSS version 10.0.5
software (SPSS Inc., Chicago HI.) may be used for all statistical analyses.
The methodology for determining a threshold value for the tumor gene
determinant DPD in fresh, frozen, fixed or fixed and paraffin-embedded (FPE) tissue
relative to gene expression of an internal control is found in US applications 09/879,217, filed June 13, 2001 ; 09/842,111, filed April 26, 2001 ; and 09/796,807, filed March 2,
2001, all of which are hereby incorporated by reference in their entirety. The methodology for determining a threshold value for the tumor gene
determinant JS in fresh, frozen, fixed or fixed and paraffin-embedded (FPE) tissue
relative to gene expression of an internal control is found in US application 09/877,178,
filed June 11, 2001, which is hereby incorporated by reference in its entirety.
The methodology for detenr-ining a threshold value for the tumor gene
detenninant EGFR in fresh, frozen, fixed or fixed and paraffin-embedded (FPE) tissue
relative to gene expression of an internal control is found in US application 09/877,177, filed June 11, 2001, which is hereby incorporated by reference in its entirety.
A "chemotherapeutic agent specific for the tumor gene determinant" refers to any chemotherapeutic agent which is known to target a cancer cell, and has an
effectiveness correlating to the expression level of the tumor gene determinant.
Knowledge of the physical interaction between the tumor gene determinant and the
chemotherapeutic agent specific for the tumor gene determinant is not necessary so long
there is a correlation between the expression of the tumor gene determinant and the effectiveness of the agent. Chemotherapeutic agents specific for a tumor gene
determinant may include, but are not limited to, genotoxic therapies, anti-metabolite therapies and/or receptor tyrosine kinase based therapies.
"Genotoxic chemotherapeutic agents" are classes of chemotherapeutic agents that inflict damage on cellular DNA. Examples of genotoxic chemotherapeutic agents
specific for the tumor gene determinant known to be involved in DNA repair are
platinum-based chemotherapies which cause a "bulky adduct" of the DNA, wherein the
primary effect is to distort the three-dimensional conformation of the double helix. Such compounds are meant to be adn inistered alone, or together with other chemotherapies
such as gemcitabine (Gem) or 5-Fluorouracil (5-FU). Platinum-based genotoxic chemotherapies comprises heavy metal coordination compounds which form covalent DNA adducts. Generally, these heavy metal compounds bind covalently to DNA to
form, in pertinent part, cis-l,2-intrastrand dinucleotide adducts. Generally, this class is
represented by cis-diamminedichloroplatinum (IT) (cisplatin), and includes
cis-diammine- (1,1-cyclobutanedicarboxylato) platinum(IT) (carboplatin), cis-diammino
- ( 1 ,2-cyclohexyl) diclιloroplatinum(ir), and cis-( 1 ,2-ethylenedi ammine)
dichloroplatinum(I[). Platinum first agents include analogs or derivatives of any of the
foregoing representative compounds. Tumors currently manageable by platinum coordination compounds include testicular, endometrial, cervical, gastric, squamous cell,
adrenocortical and small cell lung carcinomas along with medulloblastomas and
neuroblastomas. Trans-Diamminedichloroplatinum (II) (trans-DDP) is clinically useless owing, it is thought, to the rapid repair of its DNA adducts. The use of trans-DDP as a
chemotherapeutic agent herein likely would provide a compound with low toxicity in
nonselected cells, and high relative toxicity in selected cells. In a preferred
embodiment, the platinum compound is cisplatin. Many compounds are commonly given with platinum-based chemotherapy agents. For example, BEP (bleomycin,
etoposide, cisplatin) is used for testicular cancer, MVAC (memotrexate, vinblastine, doxorabicin, cisplatin) is used for bladder cancer, MVP (mitomycin C, vinblastine, cisplatin) is used for non-small cell lung cancer treatment. Many studies have
documented interactions between platinum-containing agents. Therapeutic drug synergism, for example, has been reported for many drugs potentially included in a
platinum based chemotherapy. A very short list of recent references for this include the following: Okamoto et al., Urology 2001; 57:188-192.; Tanaka et al., Anticancer
Research 2001; 21:313-315; Slamon et al., Seminars in Oncology 2001; 28:13-19; Lidor
et al., Journal of Clinical Investigation 1993; 92:2440-2447; Leopold et al., NCI Monographs 1987;99-104; Ohta et al., Cancer Letters 2001; 162:39-48; van Moorsel et al., British Journal of Cancer 1999; 80:981-990.
Other genotoxic agents are those that form persistent genomic lesions and are
preferred for use as chemotherapeutic agents in the clinical management of cancer. The
rate of cellular repair of genotoxin-induced DNA damage, as well as the rate of cell
growth via the cell division cycle, affects the outcome of genotoxin therapy. Unrepaired
lesions in a cell's genome can impede DNA replication, impair the replication fidelity of
newly synthesized DNA or hinder the expression of genes needed for cell survival. Thus, one determinant of a genotoxic agent's cytotoxicity (propensity for contributing to
cell death) is the resistance of genomic lesions formed therefrom to cellular repair.
Genotoxic agents that form persistent genomic lesions, e.g., lesions that remain in the genome at least until the cell commits to the cell cycle, generally are more effective
cytotoxins than agents that form transient, easily repaired genomic lesions. A general
class of genotoxiG compounds that are used for treating many cancers and that are
affected by levels of DNA repair gene expression are DNA alkylating agents and DNA
intercalating agents. Psoralens are genotoxic compounds known to be useful in the photochemotherapeutic treatment of cutaneous diseases such as psoriasis, vitiligo, fungal
infections and cutaneous T cell lymphoma. Harrison's Principles of Internal Medicine, Part 2 Cardinal Manifestations of Disease, Ch. 60 (12th ed. 1991). Another general class of genotoxic compounds, members of which can alkylate or intercalate into DNA,
includes synthetically and naturally sourced antibiotics. Of particular interest herein are
antineoplastic antibiotics, which include but are not limited to the following classes of
compounds represented by: amsacrine; actinomycin A, C, D (alternatively known as dactinomycin) or F (alternatively KS4); azaserine; bleomycin; carminomycin (carubicin), daunomycin (daunorubicin), or 14-hydroxydaunomycin (adriamycin or doxorubicin); mitomycin A, B or C; mitoxantrone; plicamycin (mithramycin); and the like. Still another general class of genotoxic agents that are commonly used and that
alkylate DNA, are those that include the haloethylnitrosoureas, especially the
chloroethylnitrosoureas. Representative members of this broad class include carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and streptozotocin.
Haloethylnitrosourea first agents can be analogs or derivatives of any of the foregoing
representative compounds.
Yet another general class of genotoxic agents, members of which alkylate DNA,
includes the sulfur and nitrogen mustards. These compounds damage DNA primarily by forming covalent adducts at the N7 atom of guanine. Representative members of this
broad class include chlorambucil, cyclophosphamide, ifosfamide, melphalan,
mecMoroetharnine, novembicin, trofosfamide and the like. Oligonucleotides or analogs
thereof that interact covalently or noncovalently with specific sequences in the genome
of selected cells can also be used as genotoxic agents, if it is desired to select one or
more predefined genomic targets as the locus of a genomic lesion. Another class of agents, members of which alkylate DNA, include the
ethylenimines and mefcylmelamines. These classes include altretamine
(hexamethylmelamine), triethylenephosphoramide (TEPA), triethylenethiophosphoramide (ThioTEPA) and triethylenemelainine, for example.
Additional classes of DNA alkylating agents include the alkyl sulfonates, represented by
busulfan; the azinidines, represented by benzodepa; and others, represented by, e.g.,
mitoguazone, mitoxantrone and procarbazine. Each of these classes includes analogs
and derivatives of the respective representative compounds.
"Anti-metabolite chemotherapeutic agents" are agents that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes. Examples of anti-
metabolite chemotherapeutic agents specific for the tumor gene determinant known to be important in tumor cell metabolism include 5-FU, methotrexate, and ara-C.
"Receptor tyrosine kinase targeted chemotherapeutic agents" are agents that
specifically inhibit signaling through receptor tyrosine kinases (RTKs) in cells where
RTKs are over active. Examples of receptor tyrosine kinase targeted chemotherapeutic
agents specific for tumor gene determinant known to be involved in receptor tyrosine
kinase signaling include 4-anilinoquinazolines such as
6-acrylamido-4-anilinoquinazoline (Bonvini et al., Cancer Res. 2001 Feb 15;61(4):1671-7) and derivatives, erbstatin (Toi et al., Eur. J. Cancer Clin. Oncol., 1990,
26, 722.), Geldanamycin, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT
WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and
l-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) which have been
described generally as tyrosine kinase inhibitors. Also, Styryl compounds (U.S. Pat. No.
5,217,999), sty l-substituted pyridyl compounds (U.S. Pat. No.5,302,606), certain quinazoline derivatives (EP Application No. 0 566266 Al), seleoindoles and sel'enides
(PCT WO 94/03427), tricyclic polyhydroxyhc compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as
compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer. Other agents targeting receptor tyrosine kinase signaling activity include antibodies that
inhibit growth factor receptor biological function indirectly by mediating cytotoxicity via
a targeting function. Antibodies complexing with the receptor activate serum complement and or mediate antibody-dependent cellular cytotoxicity. The antibodies
that bind the receptor can also be conjugated to a toxin (immunotoxins). Antibodies are selected that greatly inhibit the receptor function by binding the steric vicinity of the ligand binding site of the receptor (blocking the receptor), and/or that bind the growth
factor in such a way as to prevent (block) the Ugand from binding to the receptor. These antibodies are selected using conventional in vitro assays for selecting antibodies which
neutralize receptor function. Antibodies that act as ligand agonists by mimicking the
ligand are discarded by conducting suitable assays as will be apparent to those skilled in
the art. For certain tumor cells, the antibodies inhibit an autocrine growth cycle (i.e.
where a cell secretes a growth factor that then binds to a receptor of the same cell). Since
some ligands, e.g. TGF-a, are found lodged in cell membranes, the antibodies serving a
targeting function are directed against the ligand and/or the receptor. The cytotoxic
moiety of the immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment of such a toxin.
Enzymatically active toxins and fragments thereof often used are diphtheria, nonbinding
active fragments of diphtheria toxin, exotoxin (from Pseudomonas aeruginosa), ricin,
abrin, modeccin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria ofϊicinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin. hi another embodiment, the antibodies are conjugated to small molecule
anticancer drugs. Conjugates of the monoclonal antibody and such cytotoxic moieties are made using a variety of bifunctional protein coupling agents. Examples of such reagents are SPDP, IT, bifunctional derivatives of imidoesters such a dimethyl
adipimidate HC1, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine,
bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl )-e1-hylenediamine,
diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as 1 ,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin may be joined to the Fab fragment of the antibodies. Cytotoxic radiopharmaceuticals for treating cancer may
be made by conjugating radioactive isotopes to the antibodies. The term "cytotoxic moiety" as used herein is intended to include such isotopes.
The exact formulation, route of administration and dosage of chemotherapeutic
agents specific for a rumor gene determinant may be chosen by the individual physician
in view of the patient's condition. (See e.g. Fingl et al., in The Pharmacological Basis of
Therapeutics, 1975, Ch. 1 p. 1). It should be noted that the attending physician would
know how and when to teπninate, interrupt, or adjust administration due to toxicity, or
organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
The magnitude of an administrated dose in the management of the oncogenic disorder of
interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by
standard prognostic evaluation methods. Further, the dose and perhaps dose frequency,
will also vary according to the age, body weight, and response of the individual patient.
Depending on the specific conditions being treated, such agents may be
formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal,
vaginal, transmucosal, or intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intrameduUary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to
name a few. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The invention primarily rests in the observation from archival pathological
samples that expression of tumor gene determinants in primary tumors correlates with
the expression of those tumor gene determinants in matching tumor metastases (a
sample of a metastic cancer tissue derived from the same individual as the primary
tumor sample) Accordingly, a chemotherapeutic regimen designed in view of the
expression of tumor gene determinants in primary tumors is also appropriate for treating tumor metastases. Thus, the present invention allows one to correlate the effectiveness
of a chemotherapeutic regimine treating a primary tumor to also treat the tumor
metastases. For example, a primary tumor having high level of EGFR mRNA expression is
considered likely to be sensitive to receptor tyrosine kinase targeted chemotherapy.
Thus, with the present invention, the tumor metastases of patients whose primary tumors
express high levels, i.e. above a predetermined threshold value, of EGFR mRNA are
considered also likely to be sensitive to receptor tyrosine kinase targeted chemotherapy.
Conversely, the tumor metastases of patients whose primary tumors express low levels, i.e. below a predeteπnined threshold value, of EGFR mRNA are considered likely to be
insensitive to receptor tyrosine kinase targeted chemotherapy.
Similarly, the tumor metastases of patients whose primary tumors express low
levels i.e. below a predetermined threshold value, of JS mRNA are considered likely to
be sensitive to TS targeted chemotherapy. Conversely, the tumor metastases of patients
whose primary tumors express high levels, i.e. above a predetermined threshold value of
rSmRNA are considered likely to be insensitive to TS- targeted chemotherapy.
Providing another example, the tumor metastases of patients whose primary tumors express low levels, i.e. below a predetermined threshold value, of DPD mRNA
are considered likely to be sensitive to 5-FU based chemotherapy. Conversely, the tumor metastases of patients whose primary tumors express high levels, i.e. above a
predetermined threshold value, of DPD mRNA are considered likely to be insensitive to
5-FU- based chemotherapy.
The methodology for determining the expression of a tumor gene determinant in
in fresh, frozen, fixed or fixed and paraffin-embedded (FPE) tissue relative to gene
expression of an internal control is found in US applications 09/879,217, filed June 13,
2001; 09/842,111, filed April 26, 2001; and 09/796,807, filed March 2, 2001, all of which are hereby incorporated by reference in their entirety (describing the methodology
as it relates to DPD expression); in US application 09/877,178 filed June 11, 2001, which is hereby incorporated by reference in its entirety (describing the methodology as
it relates to JS expression), and in US application 09/877,177 filed June 11, 2001, which
is hereby incorporated by reference in its entirety (describing the methodology as it relates to EGFR expression).
This measurement of tumor gene determinant expression in a primary tumor
may then be used for prognosis of a gene targeted chemotherapy to treat metastic tumors throughout the body. The tumor gene determinant can be any gene whose expression level is indicative of the effectiveness of a specific chemotherapeutic or class of
chemotherapeutics. Preferably, the tumor gene determinants are JS DPD and/or EGFR gene expression in a primary tumor used to treat tumor metastases in the liver.
Preferably, the methods of the invention are applied to solid tumors, most preferably colorectal tumors.
Assessment of mRNA expression
Solid or lymphoid primary tumors or portions thereof are surgically resected
from the patient or obtained by routine biopsy. RNA isolated from frozen or fresh tumor samples is extracted from the cells by any of the melhods typical in die art, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.),
Cold Spring Harbor Laboratory Press, New York, (1989). Preferably, care is taken to
avoid degradation of the RNA during the extraction process.
Tissue obtained from the patient after biopsy is often fixed, usually by formalin
(formaldehyde) or gluteraldehyde, for example, or by alcohol immersion. Fixed
biological samples are often dehydrated and embedded in paraffin or other solid supports
known to those of skill in the art. See Plenat et al, Ann Pathol 2001 Jan;21(l):29-47. Non-embedded, fixed tissue as well as fixed and embedded tissue may also be used in
the present methods. Solid supports for embedding fixed tissue are envisioned to be
removable with organic solvents, for example, and allowing for subsequent rehydration of preserved tissue.
RNA is extracted from paraffin-embedded (FPE) tissue cells by any of the
methods as described in US Patent Application No. 09/469,338, filed December 20,
1999, which is hereby incorporated by reference in its entirety. As used herein, FPE tissue means tissue that has been fixed and embedded in a sohd removable support, such
as storable or archival tissue samples. RNA may be isolated from an archival pathological sample or biopsy sample which is first deparaffinized. An exemplary deparaffinization method involves washing the paraffinized sample with an organic
solvent, such as xylene. Deparaffinized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols include, methanol, ethanol,
propanols, and butanols. Deparaffinized samples may be rehydrated with successive
washes with lower alcoholic solutions of decreasing concentration. Alternatively, the sample is simultaneously deparaffinized and rehydrated. RNA is then extracted from the
sample. For RNA extraction, the fixed or fixed and deparaffinized samples can be homogenized using mechanical, sonic or other means of homogenization. Rehydrated
samples may be homogenized in a solution comprising a chaotropic agent, such as
guanidinium thiocyanate (also sold as guanidinium isothiocyanate). Homogenized
samples are heated to a temperature in the range of about 50 to about 100 °C in a
chaotropic solution, which contains an effective amount of a chaotropic agent, such as a
guanidinium compound. A preferred chaotropic agent is guanidinium thiocyanate.
An "effective amount of chaotropic agent" is chosen such that RNA is purified from a paraffin-embedded sample in an amount of greater than about 10-fold that
isolated in the absence of a chaotropic agent. Chaotropic agents include: guanidinium
compounds, urea, formamide, potassium iodiode, potassium thiocyantate and similar compounds. The preferred chaotropic agent for the methods of the invention is a
guanidinium compound, such as guanidinium isothiocyanate (also sold as guanidinium
thiocyanate) and guanidinium hydrochloride. Many anionic counterions are useful, and
one of skill in the art can prepare many guanidinium salts with such appropriate anions.
The effective concentration of guanidinium solution used in the invention generally has a
concentration in the range of about 1 to about 5M with a preferred value of about 4M. If RNA is already in solution, the guanidinium solution may be of higher concentration such that the final concentration achieved in the sample is in the range of about 1 to about 5M. The guanidinium solution also is preferably buffered to a pH of about 3 to
about 6, more preferably about 4, with a suitable biochemical buffer such as Tris-Cl.
The chaotropic solution may also contain reducing agents, such as dithiothreitol (DTT)
and β-mercaptoethanol (BME). The chaotropic solution may also contain RNAse
inhibitors.
RNA is then recovered from the chaotropic solution by, for example, phenol
chloroform extraction, ion exchange chromatography or size-exclusion chromatography. RNA may then be further purified using the techniques of extraction, electrophoresis,
chromatography, precipitation or other suitable techniques known in the art.
The quantification of tumor gene deteπr-inant mRNA from purified total mRNA
from fresh, frozen or fixed is preferably carried out using reverse-transcriptase
polymerase chain reaction (RT-PCR) methods common in the art. Other methods of
quantifying of mRNA include the use of molecular beacons and other labeled probes
useful in multiplex PCR. Additionally, the present invention envisages the quantification of mRNA via use of a PCR-free systems employing, for example
fluorescent labeled probes similar to those of the Invader® Assay (Third Wave
Technologies, Inc.). Preferably, quantification of tumor gene determinants and an internal control or house keeping gene (e.g. β-actin) is done using a fluorescence based
real-time detection method (ABI PRISM 7700 or 7900 Sequence Detection System
[TaqMan®], Applied Biosystems, Foster City, CA.) or similar system as described by
Heid et ah, (Genome Res 1996;6:986-994) and Gibson et a/.(Genome Res 1996;6:995-
1001). The output of the ABI 7700 (TaqMan® Instrument) is expressed in Ct's or
"cycle thresholds." With the TaqMan® system, a highly expressed gene having a higher number of target molecules in a sample generates a signal with fewer PCR cycles (lower
Ct) than a gene of lower relative expression with fewer target molecules (higher Ct). "House keeping" gene or "internal control" is any constitutively or globally
expressed gene whose presence enables an assessment of tumor gene determinant
mRNA levels. Such an assessment comprises a determination of the overall constitutive
level of gene transcription and a control for variations in RNA recovery.
"House-keeping" genes or "internal controls" can include, but are not limited to, the cyclophilin gene, β-actin gene, the transferrin receptor gene, GAPDH gene, and the like. Most preferably, the internal control gene is β-actin gene as described by Eads et al, Cancer Research 1999; 59:2302-2306.
A control for variations in RNA recovery requires the use of "calibrator RNA."
The "calibrator RNA" is intended to be any available source of accurately pre-quantified
control RNA.
As described above, a preferred quantification of gene expression uses a
fluorescence based real time detection method, a preferred TaqMan® system, three
primers are used: a forward, and a reverse primer, and a dual labeled fluorogenic oligonucleotide probe that anneals specifically to the cDNA of the gene at issue. The
fluorogenic probe anneals to the cDNA within the region between where the forward
and the reverse primers anneal. Any suitable primers may used to assess the mRNA expression levels described above. They must provide an accurate assessment of DPD,
TS and/or EGFR expression in a fixed paraffin embedded (FPE) tissue and are also
preferably accurate for determining DPD, TS and/or EGFR expression levels in fresh or
frozen tissue, i.e. they have high specificity for their target RNA. As mRNA derived
from FPE samples is more fragmented relative to that of fresh or frozen tissue and it is therefore, more difficult to quantify.
In the preferred quantification syste Preferred primer for EGFR are SEQ ID
NO: 1-3. Preferred primers for DPD are SEQ ID NO: 4-6. Preferred primers for TS are SEQ ID NO: 7-9. Preferred primers for β-actin are SEQ ID NO: 10-12.
"Uncorrected Gene Expression (UGE)" as used herein refers to the numeric
output of a tumor gene determinant expression relative to an internal control gene
generated by the TaqMan® instrument. The equation used to determine UGE for
EGFR, TS and DPD, expression is shown in Examples 3, 4, and 5 respectively and illustrated with sample calculations in Figures 2, 3, and 4. Example 6 provides equations for calculating the UGE for any tumor gene determinant, referred to herein as GENEX.
A further aspect of this invention provides a method to normalize uncorrected
gene expression (UGE) values acquired from the TaqMan® instrument with "known
relative gene expression" values derived from non-TaqMan® technology. Preferably,
TaqMan® derived tumor gene determinant UGE values (such as but not limited to DPD,
TS and or EGFR UGE values) from a tissue sample are normalized to samples with
known non-TaqMan® derived relative tumor gene determinant: βactin expression values. For example, TaqMan® derived DPD, TS and/or EGFR values from a tissue sample are
normalized to samples with known non TaqMan® derived relative DPD, TS and or
EGER: β-actin expression values.
"Corrected Relative Tumor Gene Determinant Expression" as used herein refers
to normalized tumor gene deteπninant expression whereby UGE is multiplied with a
tumor gene determinant specific correction factor (Kge/!aY), resulting in a value that can be
compared to a known range of tumor gene determinant expression levels relative to an
internal control gene. These numerical values also allow the determination of whether
or not the "Corrected Relative Expression" of a particular tumor sample divided by the "Corrected Relative Expression" of a matching non-tumor sample (i.e., differential expression) falls above or below the "predetermined threshold" level. Example 6
illustrates these calculations in detail.
"Known relative gene expression" values are derived from previously analyzed
tissue samples and are based on the ratio of the RT-PCR signal of a target gene to a
constitutively expressed internal control gene (e.g. β-Actin, GAPDH, etc.). Preferably such tissue samples are formalin fixed and paraffin-embedded (FPE) samples and RNA
is extracted from them according to the protocol described in Example 1. To quantify gene expression relative to an internal control, standard quantitative RT-PCR technology known in the art is used. Pre-TaqMan® technology PCR reactions are run for a fixed
number of cycles (i.e., 30) and endpoint values are reported for each sample. These
values are then reported as a ratio of tumor gene determinant expression to β-actin
expression.
Kge,,ex maY De determined for an internal control gene other than β-actin and/or a calibrator RNA different than Human Liver Total RNA (Stratagene, Cat #735017). To
do so, one must calibrate both the internal control gene and the calibrator RNA to tissue samples for which GeneX tumor gene determinant expression levels relative to that
particular internal control gene have already been deteπrhned (i.e., "known relative gene
expression"). Preferably such tissue samples are formalin fixed and paraffin-embedded
(FPE) samples and RNA is extracted from them according to the protocol described in Example 1. Such a determination can be made using standard pre-TaqMan®,
quantitative RT-PCR techniques well known in the art. Upon such a determination,
such samples have "known relative gene expression" levels of GeneX tumor gene
determinant usefiil in the deteimining a new ~KGeneX specific for the new internal control
and or calibrator RNA as described in Example 3 (regarding KEGFR).
"Corrected Relative EGFR Expression" as used herein refers to normalized
EGFR expression whereby UGE is multiplied with a EGFR specific correction factor
(KEGFR), resulting in a value that can be compared to a known range of EGFR expression levels relative to an internal control gene. Example 3 and Figure 2 illustrate these
calculations in detail. ~K.EGFR specific for EGFR, the internal control β-actin and
cahbrator Human Liver Total RNA (Stratagene, Cat #735017), is 26.95 x 10"3.
KEGFR may be determined for an internal control gene other than β-actin and/or a calibrator RNA different than Human Liver Total RNA (Stratagene, Cat #735017). To
do so, one must calibrate both the internal control gene and the cahbrator RNA to tissue samples for which EGFR expression levels relative to that particular internal control
gene have already been determined (i.e., "known relative gene expression"). Preferably
such tissue samples are formalin fixed and paraffin-embedded (FPE) samples and RNA
is extracted from them according to the protocol described in Example 1. Such a
determination can be made using standard pre-TaqMan®, quantitative RT-PCR
techniques well known in the art. Upon such a determination, such samples have
"known relative gene expression" levels of EGFR useful in the determining a new K.EGFR specific for the new internal control and/or calibrator RNA as described in Example 3.
"Corrected Relative DPD Expression" as used herein refers to normalized DPD
expression whereby UGE is multiplied with a DPD specific correction factor ( DPD), resulting in a value that can be compared to a previously published range of values.
Figure 3 illustrates these calculations in detail.
KDPD may be deteπnined for an internal control gene other than β-actin and/or a
calibrator RNA different than Human Liver Total RNA (Stratagene, Cat #735017). To do so, one must cahbrate both the internal control gene and the calibrator RNA to tissue
samples for which DPD expression levels relative to that particular internal control gene have already been determined (i.e., "known relative gene expression"). Preferably such tissue samples are formalin fixed and paraffin-embedded (FPE) samples and RNA is
extracted from them according to the protocol described in Example 1. Such a determination can be made using standard pre-TaqMan®, quantitative RT-PCR
techniques well known in the art. Upon such a determination, such samples have "known relative gene expression" levels of DPD useful in the deteπnining a new KDPD
specific for the new internal control and/or calibrator RNA as described in Example 5. "Previously published" relative gene expression results are based on the ratio of
the RT-PCR signal of a target gene to a constitutively expressed gene (β-Actin). In pre- TaqMan® technology studies, PCR reactions were run for a fixed number of cycles (i.e.,
30) and endpoint values were reported for each sample. These values were then reported
as a ratio of DPD expression to β-actin expression. Salonga, et al, Clinical Cancer
Research, 6: 1322-1327, 2000, which is hereby incorporated by reference in its entirety.
"Corrected Relative JS Expression" as used herein refers to normalized JS
expression whereby UGE is multiplied with a JS specific correction factor (Kra), resulting in a value that can be compared to a known range of JS expression levels
relative to an internal control gene. Example 4 and Figure 4 illustrate these calculations
in detail. These numerical values allow the determination of whether the "Corrected Relative JS Expression" of a particular sample falls above or below the "predetermined
threshold" level. The predetermined threshold level of Corrected Relative JS
Expression to β-actin level is about 7.5 x 10"3. Kτs specific for JS, the internal control β-
actin and calibrator Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems, is 12.6 x 10"3.
Kra may be determined for an internal control gene other than β-actin and/or a calibrator RNA different than Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems. To do so, one must calibrate both the internal control gene and the calibrator RNA to -tissue samples for which JS expression levels relative to that
particular internal control gene have already been determined (i.e., "known relative gene
expression" or "previously published"). Preferably such tissue samples are formalin
fixed and paraffin-embedded (FPE) samples and RNA is extracted from them according
to the protocol described in Example 1 and in US Patent Application No. 09/469,338, filed December 20, 1999, which is hereby incorporated by reference in its entirety. Such a determination can be made using standard pre-TaqMan®, quantitative RT-PCR
techniques well known in the art. Upon such a determination, such samples have "known relative gene expression" levels of JS useful in the determining a new Kτs
specific for the new internal control and/or calibrator RNA as described in Example 4.
The methods of the invention are applicable to a wide range of tissue and tumor
types and so can be used for assessment of clinical treatment of a patient and as a
diagnostic or prognostic tool for a range of cancers including breast, head and neck,
lung, esophageal, colorectal, and others, h a preferred embodiment, the present methods
are applied to prognosis of colorectal tumors.
Pre-chemotherapy treatment tumor biopsies are usually available only as fixed paraffin embedded (FPE) tissues, generally containing only a very small amount of
heterogeneous tissue. Such FPE samples are readily amenable to microdissection, so
that tumor gene determinant expression, such as DPD, TS and/or EGFR gene
expression, may be determined in tumor tissue uncontaminated with non-malignant
stromal tissue. Additionally, comparisons can be made between non-malignant stromal and tumor tissue within a biopsy tissue sample, since such samples often contain both
types of tissues.
The invention being thus described, practice of the invention is illustrated by the experimental examples provided below. The skilled practitioner will realize that the materials and methods used in the illustrative examples can be modified in various ways.
Such modifications are considered to fall within the scope of the present invention.
EXAMPLES
EXAMPLE 1: RNA Isolation from FPE Tissue
RNA is extracted from paraffin-embedded tissue by the following general
procedure.
A. Deparaffmization and hydration of sections: (1) A portion of an approximately 10 m-M section is placed in a 1.5 mL plastic
centrifuge tube.
(2) 600 μL, of xylene are added and the mixture is shaken vigorously for about
10 minutes at room temperature (roughly 20 to 25 °C).
(3) The sample is centrifuged for about 7 minutes at room temperature at the
maximum speed of the bench top centrifuge (about 10-20,000 x g).
(4) Steps 2 and 3 are repeated until the majority of paraffin has been dissolved.
Two or more times are normally required depending on the amount of paraffin included in the original sample portion.
(5) The xylene solution is removed by vigorously shaking with a lower alcohol,
preferably with 100% ethanol (about 600 μL) for about 3 minutes.
(6) The tube is centrifuged for about 7 minutes as in step (3). The supernatant is
decanted and discarded. The pellet becomes white.
(7) Steps 5 and 6 are repeated with successively more dilute ethanol solutions:
first with about 95% ethanol, then with about 80% and finally with about 70% ethanol.
(8) The sample is centrifuged for 7 minutes at room temperature as in step.
(9) The supernatant is discarded and the pellet is allowed to dry at room temperature for about 5 minutes.
B. RNA Isolation with Phenol-Chloroform (1) 400 μL guanidine isothiocyanate solution including 0.5% sarcosine and 8 μL dithiothreitol is added.
(2) The sample is then homogenized with a tissue homogenizer (Ultra-Turrax, KA-Works, Inc., Wilmington, NC) for about 2 to 3 minutes while gradually increasing the speed from low speed (speed 1) to high speed (speed 5). (3) The sample is then heated at about 95 °C for about 5-20 minutes. It is
preferable to pierce the cap of the tube containing the sample with a fine gauge needle
before heating to 95 °C. Alternatively, the cap may be affixed with a plastic clamp or
with laboratory film. (4) The sample is then extracted with 50 μL 2M sodium acetate at pH 4.0 and
600 μL of phenol/chloroform isoamyl alcohol (10:1.93:0.036), prepared fresh by mixmg
18 mL phenol with 3.6 mL of a 1:49 isoamyl alcohohchloroform solution. The solution is shaken vigorously for about 10 seconds then cooled on ice for about 15 minutes.
(5) The solution is centrifuged for about 7 minutes at maximum speed. The upper (aqueous) phase is transferred to a new tube.
(6) The RNA is precipitated with about 10 μL glycogen and with 400
μL isopropanol for 30 minutes at -20 °C.
(7) The RNA is pelleted by centrifugation for about 7 minutes in a benchtop
centrifuge at maximum speed; the supernatant is decanted and discarded; and the pellet washed with approximately 500 μL of about 70 to 75% ethanol.
(8) The sample is centrifuged again for 7 minutes at maximum speed. The supernatant is decanted and the pellet air dried. The pellet is then dissolved in an
appropriate buffer for further experiments (e.g., 50 pi. 5mM Tris chloride, pH 8.0).
EXAMPLE 2: mRNA Reverse Transcription and PCR Reverse Transcription
RNA was isolated from microdissected or non-microdissected formalin fixed paraffin embedded (FPE) tissue as illustrated in Example 1, or from fresh or frozen tissue by a single step guamdinium isocyanate method using the QuickPrep™ Micro
mRNA purification kit (Amersham Pharmacia Biotech Inc., Piscataway, N. J.) according to the manufacturer's instructions. After precipitation with ethanol and centrifugation,
the RNA pellet was dissolved in 50 ul of 5 mM Tris/Cl at pH 8.0. M-MLV Reverse
Transcriptase will extend an oligonucleotide primer hybridized to a single-stranded RNA
or DNA template in the presence of deoxynucleotides, producing a complementary
strand. The resulting RNA was reverse transcribed with random hexamers and M-MLV
Reverse Transcriptase from Life Technologies. The reverse transcription was
accomplished by mixing 25 ml of the RNA solution with 25.5 ml of "reverse
transcription mix" (see below). The reaction was placed in a thermocycler for 8 min. at 26° C (for binding the random hexamers to RNA), 45 min. at 42° C (for the M-MLV
reverse transcription enzymatic reaction) and 5 min at 95° C (for heat inactivation of
DNAse).
"Reverse transcription mix" consists of 10 ul 5X buffer (250 mM Tris-HCl, pH
8.3, 375 mM KC1, 15 mM MgC12), 0.5 ul random hexamers (50 O.D. dissolved in 550
ul of 10 mM Tris-HCl pH 7.5) 5 ul 10 mM dNTPs (dATP, dGTP, dCTP and dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3mg/ml in 10 mM Tris-HCL, pH 7.5), 1.25 ul RNA Guard 24,800U/ml (RNAse inhibitor) (Porcine #27-0816, Amersham Pharmacia) and 2.5 ul
MMLV 200U (Life Tech Cat #28025-02).
Final concentrations of reaction components are: 50 mM Tris-HCl, pH 8.3, 75
mM KC1, 3 mM MgC12, 1.0 mM dNTP, 1.0 mM DTT, 0.00375. mg/ml BSA, 0.62 U/ul
RNA Guard and 10 U/ ul MMLV.
PCR Quantification of mRNA expression
Quantification of DPD, TS and/or EGFR cDNA and an internal control or house
keeping gene (e.g., β-actin) cDNA was done using a fluorescence based real-time detection method (ABI PRISM 7?00 or 7900 Sequence Detection System [TaqMan®], Applied Biosystems, Foster City, CA.) as described by Heid et ah, (Genome Res
1996;6:986-994); Gibson et al, (Genome Res 1996;6:995-1001). In brief, this method
uses a dual labelled fluorogenic TaqMan® oligonucleotide probe, that anneals
specifically witi in the forward and reverse primers. For EGFR, primer EGFR-1773
(SEQ ID NO: 3), Tm = 70° C was used. For DPD, primerTaqMan probe DPD 3a (SEQ
ID NO: 6) was used. For TS, primer TaqMan probe TS-781 (SEQ ID NO: 9) was used. For β-actin, TaqMan probe β-actin -611 (SEQ ID NO: 7) was used.
Laser stimulation within the capped wells containing the reaction mixture causes emission of a 3 'quencher dye (TAMRA) until the probe is cleaved by the 5' to
3 'nuclease activity of the DNA polymerase during PCR extension, causing release of a
5' reporter dye (6FAM). Production of an amplicon thus causes emission of a fluorescent signal that is detected by the TaqMan® 's CCD (charge-coupled device)
detection camera, and the amount of signal produced at a threshold cycle within the
purely exponential phase of the PCR reaction reflects the starting copy number of the sequence of interest. Comparison of the starting copy number of the sequence of mterest
with the starting copy number of the internal control gene provides a relative gene expression level. TaqMan® analyses yield levels that are expressed as ratios between
two absolute measurements (gene of interest-internal control gene).
The PCR reaction mixture consisted 0.5ml of the reverse transcription reaction
containing the cDNA prepared as described above; 600 nM of each forward and reverse
oligonucleoride primers; 200 nM TaqMan® probe primer, 5 U AmpliTaq Gold
Polymerase, 200 mM each dATP, dCTP, dGTP, 400 mM dTTP, 5.5 mM MgCl2, and 1
x Taqman Buffer A containing a reference dye, to a final volume of less than or equal to 25 ml (all reagents Applied Biosystems, Foster City, CA). For EGFR, the forward and reverse primers were respectively EGFR-1753-F
(SEQ ID NO: 1) and EGFR-R-1823R (SEQ ID NO: 2) and the TaqMan probe was
TaqMan EGFR-1773 (SEQ ID NO: 3).
For DPD, the forward and reverse primers were respectively DPD 3a-51F (SEQ
ID NO: 4) and DPD 3a-13R (SEQ ID NO: 5) and the TaqMan probe was TaqMan DPD
3a (SEQ ID NO: 6).
For TS, the forward and reverse primers were respectively TS-763F (SEQ ID
NO: 7) and TS-82R (SEQ ID NO: 8) and the TaqMan probe was TaqMan TS-781 (SEQ
ID NO: 9). For β-actin, the forward and reverse primers were respectively β-actin-592F
(SEQ ID NO: 11) and β-actin-651R (SEQ ID NO: 12) and the TaqMan probe was
TaqMan β-actin-611 (SEQ ID NO: 10).
Cycling conditions were, 95 °C for 10 min., followed by 45 cycles at 95 °C for
15s and 60 °C for 1 min.
EXAMPLE 3 : Determining the Uncorrected Gene Expression (UGE) for EGFR
Two pairs of parallel reactions are carried out. The "test" reactions and the "calibration" reactions. Figure 2. The EGFR amplification reaction and the β-actin
internal control amplification reaction are the test reactions. Separate EGFR and β-actin
amplification reactions are performed on the cahbrator RNA template and are referred to
as die calibration reactions. The TaqMan® instrument will yield four different cycle
threshold (Ct) values: Ct£G Λ and Ctp.acHn from the test reactions and OLEGFR and Ctp.actin from the calibration reactions. The differences in Ct values for the two reactions are
determined according to the following equation: DCt^t = Ct£G Λ - Ctβ.actin (From the "test" reaction)
DCtcalibrator= Ct£Gra - Ctp.actin (From the "cahbration" reaction)
Next the step involves raising the number 2 to the negative DCt, according to the
following equations.
2-DCt test (From the "test" reaction)
2"DCt Caiibrator (From the "calibration" reaction)
order to then obtain an uncorrected gene expression for EGFR from the
TaqMan® instrument the following calculation is carried out:
Uncorrected gene expression (UGE) for EGFR = 2-DCt test / 2-DCt calibrator
Normalizing UGE with known relative EGFR expression levels
The normalization calculation entails a multiplication of the UGE with a correction factor (K.EGFR) specific to EGFR and a particular calibrator RNA. A
correction factor KEGFR can also be determined for any internal control gene and any accurately pre-quantified calibrator RNA. Preferably, the internal control gene β-actin
and the accurately pre-quantified calibrator RNA, Human Liver Total RNA (Stratagene,
Cat #735017), are used. Given these reagents, correction factor KEGFR equals 1.54.
Normalization is accomplished using a modification of the DCt method described by Applied Biosystems, the TaqMan® manufacturer, in User Bulletin #2 and described above. To carry out this procedure, the UGE of 6 different FPE test tissues
were analyzed for EGFR expression using the TaqMan® methodology described above. The internal control gene β-actin and the calibrator RNA, Human Liver Total RNA
(Stratagene, Cat #735017) was used.
The already known relative EGFR expression level of each sample AG221,
AG222, AG252, Adult Lung, PC3, AdCol was divided by its corresponding TaqMan®
derived UGE to yield an unaveraged correction factor K.
K-maveraged = Known Values / UGE
Next, all of the K values are averaged to determine a single KEGFR correction
factor specific for EGFR, Stratgene Human Liver Total RNA (Stratagene, Cat #735017)
from calibrator RNA, and β-actin.
Therefore, to determine the Corrected Relative EGFR Expression in an unknown
tissue sample on a scale that is consistent with pre-TaqMan® EGFR expression studies,
one merely multiplies the uncorrected gene expression data (UGE) derived from the
TaqMan® apparatus with the KEGFR specific correction factor, given the use of the same internal control gene and calibrator RNA.
Corrected Relative EGFR Expression = UGE x K.EGFR
A KEGFR may be determined using any accurately pre-quantified calibrator RNA
or internal control gene. Future sources of accurately pre-quantified RNA can be
calibrated to samples with known relative EGFR expression levels as described in the method above or may now be cahbrated against a previously calibrated cahbrator RNA such as Human Liver Total RNA (Stratagene, Cat #735017) described above. For example, if a subsequent K.EGFR is determined for a different internal control
gene and/or a different calibrator RNA, one must calibrate both the internal control gene
and the calibrator RNA to tissue samples for which EGFR expression levels relative to
that particular internal control gene have aheady been determined. Such a determination
can be made using standard pre-TaqMan®, quantitative RT-PCR techniques well known
in the art. The known expression levels for these samples will be divided by tiieir corresponding UGE levels to determine a K for that sample. K values are then averaged
depending on the number of known samples to determine a new K.EGFR specific to the
different internal control gene and/or cahbrator RNA.
EXAMPLE 4: Determining the Uncorrected Gene Expression (UGE) for TS
Two pairs of parallel reactions are carried out. The "test" reactions and the
"calibration" reactions. See Figure 4. The JS amplification reaction and the β-actin
internal control amplification reaction are the test reactions. Separate JS and β-actin amplification reactions are performed on the calibrator RNA template and are referred to
as the calibration reactions. The TaqMan® instrument will yield four different cycle threshold (Ct) values: Ctr5 and Ctp_actin from the test reactions and Ctre and Ctp.actin from
the calibration reactions. The differences in Ct values for the two reactions are determined according to the following equation:
DCt^t = Ctj5 - Ctp.actin (From the "test" reaction)
DCtcalibrator= Ctra - Ctp.actin (From the "calibration" reaction)
Next the step involves raising the number 2 to the negative DCt, according to the
following equations. 2"DCt test (From the "test" reaction)
2"DCt caiibrator (From the "calibration" reaction)
hi order to then obtain an uncorrected gene expression for JS from the
TaqMan® instrument the following calculation is carried out:
Uncorrected gene expression (UGE) for JS= 2-uu test / 2"DLt calibrator
Normalizing UGE with known relative TS expression levels
The normalization calculation entails a multiplication of the UGE with a
correction factor (Krs) specific to JS and a particular calibrator RNA. A correction
factor Kτs can also be determined for any internal control gene and any accurately pre-
quantified calibrator RNA. Preferably, the internal control gene β-actin and the
accurately pre-quantified cahbrator RNA, Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems are used. Given these reagents correction factor
Kτs equals 12.6 x 10"3. Normalization is accomplished using a modification of the DCt method
described by Applied Biosystems, the TaqMan® manufacturer, in User Bulletin #2 and described above. To carry out this procedure, the UGE of 6 different previously
published test tissues were analyzed for JS expression using the TaqMan® methodology
described above. These tissue samples are described in Salonga, et al, Clinical Cancer
Research, 6:1322-1327, 2000, which is hereby incorporated by reference in its entirety.
The internal control gene β-actin and the calibrator RNA, Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems was used. The previously published relative JS expression level of each sample L7, L91,
L121, L150, L220, L164 was divided by its corresponding TaqMan® derived UGE to
yield an unaveraged correction factor K. Salonga, et al, Clinical Cancer Research,
6:1322-1327, 2000, incorporated herein by reference in its entirety.
I-™-. ed = Known Values / UGE
Next, all of the K values are averaged to determine a single K.ERCC1 correction
factor specific for JS, Applied Biosystems Universal PE RNA; Cat #4307281, lot #
3617812014 calibrator RNA, and β-actin.
Therefore, to detemiine the Corrected Relative JS Expression in an unknown
tissue sample on a scale that is consistent with pre-TaqMan® JS expression studies, one
merely multiplies the uncorrected gene expression data (UGE) derived from the
TaqMan® apparatus with the Kra specific correction factor, given the use of the same
internal control gene and calibrator RNA.
Corrected Relative JS Expression = UGE x Kτs
A Kτs may be determined using any accurately pre-quantified calibrator RNA or internal control gene. Future sources of accurately pre-quantified RNA can be cahbrated
to samples with known relative ERCC1 expression levels as described in the metiiod
above or may now be calibrated against a previously cahbrated cahbrator RNA such as
Universal PE RNA; Cat #4307281, lot # 3617812014 from Applied Biosystems
described above. For example, if a subsequent Kτs is determined for a different internal control
gene and/or a different calibrator RNA, one must calibrate both the internal control gene
and the calibrator RNA to tissue samples for which JS expression levels relative to that
particular internal control gene have already been determined or published. Such a
determination can be made using standard pre-TaqMan®, quantitative RT-PCR
techniques well known in the art. The known expression levels for these samples will be
divided by their corresponding UGE levels to determine a K for that sample. K values
are then averaged depending on the number of known samples to determine a new Kr5 specific to the different internal control gene and or calibrator RNA.
EXAMPLE 5: Determining the Uncorrected Gene Expression (UGE) for DPD
Two pairs of parallel reactions are carried out. The "test" reactions and the
"calibration" reactions. The DPD amplification reaction and the β-actin internal control
amplification reaction are the test reactions. Separate β-actin and DPD amplification reactions are performed on the calibrator RNA and are referred to as the calibration
reactions. The Taqman instrument will yield four different cycle threshold (Ct) values: Ct^rø and Ctp.actin from the test reactions and O^ and Ctp.actinfrom the calibration
reactions.
The differences in Ct values for the two reactions are determined according to
the. following equation:
DCt^t = Ctβp£ - Ctp.actin (From the "test" reaction)
DCtca]ibrator= Ctorø - Ctp.actin (From the "calibration" reaction)
Next the step involves raising the number 2 to the negative DCt, according to the
following equations. 2"DCt test (From the "test" reaction)
-2"DC'cai-brator (From die "calibration" reaction)
hi order to then obtain an uncorrected gene expression for DPD from the
Taqman instrument the following calculation is carried out:
Uncorrected gene expression (UGE) for DPD = TOC est 12-DCt caIibrator
Normalizing UGE with previously published values
The normalization calculation entails a multiplication of the UGE with a correction factor (Kørø) specific to DPD and a particular calibrator RNA. The
correction factor K.DPD can be determined using any internal control gene and any accurately pre-quantified calibrator RNA: Preferably, the internal control gene β-actin
and the accurately pre-quantified calibrator RNA, Universal PE RNA; Cat #4307281,
lot # 3617812014 from Applied Biosystems, are used.
Normalization is accomplished using modification of the DCt method described
by Applied Biosystems, the Taqman manufacturer, in User Bulletin #2 and described above. To carry out this procedure, the UGE of 6 different previously published test
tissues was analyzed for DPD expression using the Taqman methodology described
above. The internal control gene β-actin and the calibrator RNA, Universal PE RNA;
Cat #4307281, lot # 3617812014 from Applied Biosystems was used.
The relative DPD expression level (PV) of each sample previously described in
Salonga et al, which is hereby incorporated by reference in its entirety, L7, L91, L121,
LI 50, L220 and LI 64, was divided by its corresponding Taqman derived UGE to yield an unaveraged correction factor K. Kuπaveraged = PN / UGE
Next, all of the K values are averaged to determine a single KDPD correction
factor specific for DPD, Universal PE RNA; Cat #4307281, lot # 3617812014 calibrator RNA and β-actin.
Therefore, to determine the Corrected Relative DPD Expression in an uriknown tissue sample on a scale tiiat is consistent with previously published pre-Taqman DPD
expression studies, one merely multiplies the uncorrected gene expression data (UGE) derived from the Taqman apparatus with the K.DPD specific correction factor, given the
use of the same internal control gene and calibrator RNA.
Corrected Relative DPD Expression = UGE x K -D, PD
A K.DPD may be determined using any accurately pre-quantified calibrator RNA. Future sources of accurately pre-quantified RNA can be calibrated to published samples
as described in the method above or may now be calibrated against a previously calibrated calibrator RNA such as Universal PE RNA; Cat #4307281, lot # 3617812014 described above.
EXAMPLE 6: Determining the Uncorrected Gene Expression (UGE) for GENEX tumor gene determinant
Two pairs of parallel reactions are carried out. The "test" reactions and the "calibration" reactions. The GENE amphfication reaction and the β-actin internal
control amplification reaction are the test reactions. Separate GENEX and β-actin amphfication reactions are performed on the cahbrator RΝA template and are referred to as the calibration reactions. The TaqMan® instrument will yield four different cycle
threshold (Ct) values: CtG£rø: and Ctp.actin from the test reactions and CtG£NEA- and Ctp.actin
from the calibration reactions. The differences in Ct values for the two reactions are
determined according to the following equation:
DCttest= C - Ctp.actin (From the "test" reaction)
DCtcalibrator= CtGENEX - Ctp.actin (From the "calibration" reaction)
Next the step involves raising the number 2 to the negative DCt, according to the
following equations.
2"DC test (From the "test" reaction)
2"DCt calibrator (From the "calibration" reaction)
In order to then obtain an uncorrected gene expression for GENEX from the
TaqMan® instrument the following calculation is carried out:
Uncorrected gene expression (UGE) for GENEX= TOC est 1 2-DCt calibrator
Normalizing UGE with known relative GENE X expression levels
The normalization calculation entails a multiplication of the UGE with a correction factor (KG£JVE ) specific to GENEX and a particular calibrator RNA. A
correction factor KEGFR can also be determined for any internal control gene and any
accurately pre-quantified calibrator RNA. Preferably, the internal control gene β-actin and the accurately pre-quantified calibrator RNA,Human Liver Total RNA (Stratagene,
Cat #735017), are used. The correction factor KGENEX is calculated.
Normalization is accomplished using a modification of the DCt method
described by Applied Biosystems, the TaqMan® manufacturer, in User Bulletin #2 and
described above. To carry out this procedure, the UGE of 6 different FPE test tissues are
analyzed for GENE expression using the TaqMan® methodology described above.
The internal control gene β-actin and the cahbrator RΝA, Human Liver Total RΝA
(Stratagene, Cat #735017) is used.
Aheady known relative GENEX expression levels of each sample is divided by
its corresponding TaqMan® derived UGΕ to yield an unaveraged correction factor K.
leveraged = K lOWn Values / UGΕ
Next, all of the K values are averaged to determine a single KG£M: correction
factor specific for GENEX, Stratgene Human Liver Total RNA (Stratagene, Cat
#735017) from calibrator RNA and β-actin. Therefore, to determine the Corrected Relative GENEX Expression in an unknown tissue sample on a scale that is consistent with pre-TaqMan® GENEXtumor
gene determinant expression studies, one merely multiplies the uncorrected gene expression data (UGΕ) derived from the TaqMan® apparatus with the K.GENEX specific
correction factor, given the use of the same internal control gene and calibrator RΝA.
Corrected Relative GENE XΕxpression = UGΕ x A KGENEX may be determined using any accurately pre-quantified calibrator RNA or internal control gene. Future sources of accurately pre-quantified RNA can be
calibrated to samples with known relative GENEX expression levels as described in the
method above or may now be calibrated against a previously calibrated calibrator RNA
such as Human Liver Total RNA (Stratagene, Cat #735017) described above.
For example, if a subsequent KGβV£. is determined for a different internal control
gene and/or a different calibrator RNA, one must calibrate both the internal control gene and the calibrator RNA to tissue samples for which GENEX expression levels relative to
that particular internal control gene have aheady been determined. Such a determination
can be made using standard pre-TaqMan®, quantitative RT-PCR techniques well known in the art. The known expression levels for these samples will be divided by their
corresponding UGΕ levels to determine a K for that sample. K values are then averaged
depending on the number of known samples to determine a new KGENEX -specific to the
different internal control gene and/or calibrator RΝA.
EXAMPLE 7: Correlation between tumor gene determinant expression in
Primary and Metastases
JS gene expressions were measured in 17 sets of tissues from paraffin-embedded
primary colorectal cancers and matched liver metastases using quantitative real-time
PCR (Taqmanό). See Figure 1. A method for mRΝA isolation from such samples is described in US Patent Application No. 09/469,338, filed December 20, 1999, and is
hereby incorporated by reference in its entirety.
Both the matching tumor sample and primary tumor sample have significantly similar expression levels of tumor gene markers. Preferably, the matching metastatic tumor sample is derived from a liver biopsy. Considering the primary tumors and the metastases as separate sets, the mean TS expressions were 5.16 x 10"3 for primary tumors
and 4.5 x 10"3 for metastases. There was no significant difference between the gene
expression values in the primary tumors and the metastases (p=0J3, F test). The
correlation coefficient (R2 value) between TS expression values in the sets of primary and metastatic tissue was 0.95. These data show that TS expression values in primary
tumors accurately reflect those in metastatic tissues and thus, for patients with stage -HI
tumors, therapy can be directed based on TS analyses in primary tumor tissue. Our findings have important practical implications for using TS values as a prognostic
indicator in 5-FU based adjuvant therapy of colorectal cancer.
EXAMPLE 8: Correlation between TS expression in primary tumor and
metastases
RNA was also isolated from 9 matched formalin-fixed, paraffin embedded,
laser microdissected colorectal cancer primary tissues and liver metastases
(total 18 specimens). TS mRNA expression, relative to expression of the housekeeping gene β-actin, was measured using a real time fluorescent dye
quantitative RT-PCR system (TaqmanO). There was a significant linear correlation between TS mRNA expression in the primary and secondary tumors. (Spearman's rho correlation coefficient R=0.683, P=0.042 (two-tailed test)).

Claims

1. A method for deteπnining a chemotherapeutic regimen for an individual having
a primary and metastatic tumor, comprising
a) obtaining a mRNA sample from the indvidual's primary tumor specimen;
b) detemiining a gene expression level for a tumor gene determinant in the
primary tumor specimen; c) comparing the gene expression level for the tumor gene determinant with a
predetermined threshold value for that tumor gene; and d) providing a chemotherapeutic regimen comprising a chemotherapeutic agent
appropriate for the tumor gene determinant to treat the individual having a tumor
metastases.
2. The method of claim 1 wherein the tumor gene deteπr-inant is EGFR.
3. The method of claim 1 wherein the tumor gene determinant is DPD.
4. The method of claim 1 wherein the tumor gene determinant is JS.
5. The method of claim 1 wherein determining gene expression level comprises a
fluorescence based real-time detection method.
6. The metiiod of claim 5 wherein the tumor gene determinant is EGFR.
1. The method of claim 5 wherein the tumor gene deteπriinant is DPD.
8. The method of claim 5 wherein the tumor gene determinant is JS.
9. A method of determining whether a chemotherapeutic regimen comprising a
chemotherapeutic agent appropriate for a tumor gene determinant in a primary tumor is
appropriate for a tumor metastasis comprising,
a) obtaining an mRNA sample from the primary tumor, b) determining an expression level of the tumor gene determinant,
c) comparing the expression level of the tumor gene detemiinant with a
predeteimined threshold level; and d) determ-i-ning the chemotherapeutic regimen for the tumor metastsis.
10. The method of claim 9 wherein the tumor gene deteiminant is EGFR.
11. The method of claim 9 wherein the tumor gene determinant is DPD.
12. The method of claim 9 wherein the tumor gene determinant is JS.
13. A method of determining whether an anti-metabolite, genotoxic, and or receptor tyrosine kinase targeted gene expression based chemotherapeutic appropriate for treating
a primary tumor is appropriate for treating a tumor metastasis comprising quantifying an
amount of tumor gene determinant mRNA expression in fresh, frozen, fixed or fixed and
paraffin-embedded (FPE) tissue relative to gene expression of an internal control in a primary tumor.
14. The mefliod of claim 13 wherein die tumor gene determinant is DPD.
15. The method of claim 13 wherein the tumor gene determinant is JS.
16. The method of claim 13 wherein the tumor gene determinant is EGFR.
PCT/US2001/045188 2000-12-01 2001-12-03 Method of determining a chemotherapeutic regimen by assaying gene expression in primary tumors WO2002044423A2 (en)

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WO2002044413A3 (en) * 2000-12-01 2003-11-06 Response Genetics Inc Method of determining epidermal growth factor receptor and her2-neu gene expression and correlation of levels thereof with survival rates
WO2002070750A2 (en) * 2001-03-02 2002-09-12 Response Genetics, Inc. Method of determining dihydropyrimidine dehydrogenase gene expression
WO2002070750A3 (en) * 2001-03-02 2004-02-19 Response Genetics Inc Method of determining dihydropyrimidine dehydrogenase gene expression
US6905821B2 (en) 2001-03-02 2005-06-14 Response Genetics, Inc. Method of determining Dihydropyrimidine dehydrogenase gene expression
US6956111B2 (en) 2001-03-02 2005-10-18 Response Genetics, Inc. Method of determining dihydropyrimidine dehydrogenase gene expression
US7005278B2 (en) 2001-03-02 2006-02-28 Danenberg Kathleen D Method of determining dihydropyrimidine dehydrogenase gene expression
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