HK1097571B - Method of determining dihydropyrimidine dehydrogenase gene expression - Google Patents

Description

Method for determining dihydropyrimidine dehydrogenase gene expression

The present application is a divisional application of "method for determining dihydropyrimidine dehydrogenase gene expression" in chinese patent application 02809318.6, 3, month and 2, 2001.

Technical Field

More specifically, the invention relates to oligonucleotides and methods, including their use in detecting dihydropyrimidine dehydrogenase (DPD) mRNA expression using RT-PCR.

Background

Transformed (malignant) cells escape normal physiological controls that specify the cell phenotype and inhibit cell proliferation.

Chemotherapeutic regimens are based on the use of drugs that are selectively toxic (cytotoxic) to cancer cells.

5-Fluorouracil (5-FU) is a widely used drug in many different cancer types, including major cancers like the gastrointestinal tract and breast (Moertel, C.G.New Engl. J. Med., 330: 1136-, the combination of 5-FU with CPT-11 or oxaliplatin is particularly toxic to these patients.

5-FU is most typical of most anticancer drugs because only a few patients respond favorably to treatment A number of randomized clinical trials have demonstrated an overall tumor response rate of 15-25% for metastatic colorectal cancer patients to 5-FU as a single agent (Moertel, C.G.New Engl. J. Med., 330: 1136-1142, 1994), in combination with other chemotherapies described above, the tumor response rate to 5-FU based regimens is about 40%. however, most treated patients do not receive significant benefit from receiving 5-FU based chemotherapy, since there is currently no reliable method of predicting the response of an individual tumor to treatment prior to treatment, standard clinical practice is to have all patients receive 5-FU therapy, with the results found to be unsatisfactory in most patients.

The ultimate goal is to improve the clinical efficacy of 5-FU by a) modulating the intracellular metabolism and biochemistry of 5-FU and b) predicting which patients are most likely to react (or not) to the drug by measuring response determinants in the patients prior to treatment. 1) The target enzyme of 5-FU, the properties of Thymidylate Synthase (TS) and 2) the properties of 5-FU metabolizing enzyme, dihydropyrimidine dehydrogenase (DPD).

The first study in the field of tumor response prediction for 5-FU based therapy was performed on its target enzyme, Thymidylate Synthase (TS), in colorectal cancer A prospective clinical trial was performed by Leichman et al (Leichman et al, J.Clin Oncol., 15: 3223-plus 3229, 1997) to establish a link between tumor response to 5-FU and expression of the TS gene, as determined by RT-PCR in a pretreatment biopsy of colorectal cancer, demonstrated that: 1) there is a 50-fold greater range of TS gene expression levels in these tumors; and 2) there was a significant difference between TS gene expression levels in responsive and non-responsive tumors, the range of response group TS levels (0.5-4.1X 10)^-3Relative to internal control) narrower than the non-reacted group (1.6-23.0X 10)^-3Thus, patients with TS expression levels above this "no response cutoff" domain can be positively identified as no responders prior to treatment the "no response" classification includes shrinkage of all tumors by < 50%, progressive growth results in tumor increase by > 25% and non-progressive tumor shrinkage by < 50%, no change or increase by < 25%TS expression levels above a certain threshold are identified as tumor subtypes that are non-responsive to 5-FU, whereas tumors with TS expression below this value are predicted to have a slightly higher response rate.

Subsequent studies investigated the use of DPD expression levels in combination with TS expression levels as determinants of tumor response to 5-FU treatment DPD is a catabolic enzyme that reduces the 5, 6 double bond of 5-FU, rendering it inactive as a cytotoxic agent.previous studies showed that DPD levels in normal tissues can affect 5-FU bioavailability, thereby modulating its pharmacokinetics and antitumor activity (Harris et al, Cancer Res., 50: 197-201, 1990). furthermore, evidence has been proposed that DPD levels in tumors correlate 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). Salong et al (Clin Cancer Res., 6: 1322 Res 1327, 2000, incorporated herein as a reference for a set of tumor response determinants of 5-formyl-FU expression in a set of tumors studied DPD Gene expression, for TS, and non-responsive tumors (0.2-16X 10)^-380 times; equivalent to internal control) the range of DPD expression in the responding tumors was relatively narrow (0.6-2.5 × 10)^-34.2 times; relative to internal control), furthermore, none of the tumors responded had a DPD expression level of more than about 2.5 × 10^-3In a tumor group where both TS and DPD expression levels are below the respective "no response cutoff" threshold level, 92% respond to 5-FU/LV. and thus, responsive tumors can be identified based on low expression levels of DPD and TS.

Patients with very low DPD levels (as in DPD deficiency syndrome, i.e. thymidylate uraemia) undergoing treatment with 5-FU have also been found to be affected by life-threatening toxicity (Lyss et al, Cancer invest, 11: 2390240, 1993). in fact, the importance of DPD levels in 5-FU treatment has been largely explained, since adverse drug interactions between 5-FU and the antiviral compound solivudine cause the death of 19 Japanese (Diasio et al, Br.J.Clin.Pharmacol.46, 1-4, 1998) and subsequently found that metabolites of solivudine are strong inhibitors of DPD. this treatment results in a reduction in DPD deficiency syndrome-like DPD levels, which increases the toxicity of 5-FU to patients (Diasio et al, Br.J.Clin.Pharmacol.46, 1-4, 1998).

Thus, due to a) the broad 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 DPD-deficient syndrome individuals to commonly used 5-FU based treatments, it is clear that accurate determination of DPD prior to chemotherapy will provide important benefits to cancer patients.

Disadvantageously, most pre-treatment tumor biopsies are obtained only as fixed paraffin-embedded (FPE) tissue, particularly formalin-fixed paraffin-embedded tissue that does not contain active enzyme.

The expression of DPD in frozen or fresh tissue can be analyzed using RT-PCR primers and probes however, these primers are not suitable for quantitation of DPD mRNA from fixed tissue by RT-PCR. a) DPD RNA itself is low in level; b) the amount of tissue embedded in paraffin is very small; thus, methods for quantifying DPD mRNA from fixed tissues are needed in order to provide an early prognosis for the intended cancer treatment, since DPD enzyme activity and the corresponding mRNA expression levels have been shown to correlate well (Ishikawa et al, clin. cancer res., 5: 883-889, 1999; johnson et al, Analyt.biochem.278: 175-184, 2000), measuring DPD mRNA expression in FPE samples provides a way to assess the status of DPD expression levels in patients without the need to determine enzymatic activity in fresh tissues furthermore, FPE samples are easy to microdissect and thus DPD gene expression can be determined in tumor tissues not contaminated with stromal tissues.

It is therefore an object of the present invention to provide a method for assessing the level of DPD in a tissue and predicting the likely resistance of a patient's tumor to 5-FU-based therapy by detecting the amount of DPD mRNA in the patient's tumor cells and comparing it to a predetermined threshold expression level.

Disclosure of Invention

One aspect of the present invention provides oligonucleotide primers having the sequence of DPD3A-51F (SEQ ID NO: 1) or DPD3A-134R (SEQ ID NO: 2), and oligonucleotide primers DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8) and sequences substantially identical thereto the present invention also provides oligonucleotide primers having a sequence that hybridizes under stringent conditions to DPD3A-51F (SEQ ID NO: 1), DPD3A-134R (SEQ ID NO: 2), DPD3b-651F (SEQ ID NO: 7), DPD3b-736R (SEQ ID NO: 8) or a complementary sequence thereof.

Furthermore, the present invention relates to a method for determining a chemotherapeutic regimen comprising isolating RNA from a tumor sample; determining the gene expression level of DPD in the sample; comparing the determined level of DPD gene expression to a predetermined threshold level for the gene; determining the chemotherapeutic regimen based on the comparison of the determined DPD gene expression level to a predetermined threshold level.

The present invention also relates to a method for normalizing Uncorrected Gene Expression (UGE) of DPD relative to an internal control gene in a tissue sample, wherein said tissue sample is analyzed using TaqMan techniques, said method being performed by analyzing known expression levels of ERCC1 relative to an internal control of the sample.

Drawings

FIG. 1 shows the ability to compare four different pairs of oligonucleotide primers to amplify DPD mRNA from 10 different formalin-FPE samples sample #1-5 and #8-10 from colon tumor biopsies, #6 from bronchoalveolar tumor biopsies, #7 from small intestine tumor biopsies, oligonucleotide primer pair DPD1(DPD-70F, (SEQ ID NO: 3) and DPD-201R, (SEQ ID NO: 4)), DPD2(DPD2p-1129F, (SEQ ID NO: 5) and DPD2p-1208R, (SEQ ID NO: 6)) are ineffective for measuring the level of DPD mRNA in these samples, oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 651: 7) and DPD 3-3 b (SEQ ID NO: 736R)) are ineffective for determining the level of DPD mRNA in these samples And (5) effect.

FIG. 2 shows DPD mRNA amplification efficiency in frozen tissue samples comparing oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) and DPD1(DPD-70F, (SEQ ID NO: 3) and DPD-201R, (SEQ ID NO: 4)) showing that oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) is not only effective in measuring DPD expression levels in frozen tissue samples (as well as FPE-derived samples), but also more effective than oligonucleotide primer pair DPD1(DPD-70F, (SEQ ID NO: 3) and DPD-201R, (SEQ ID NO: 4)).

FIG. 3 is a graph illustrating how DPD expression relative to an internal control gene is calculated, the graph including data obtained with two test samples, (unknowns 1 and 2), and illustrating how uncorrected gene expression data (UGE) UCG is determined_DPDThe internal control gene in the figure is β -actin and the calibrator RNA is Universal PERRNA from Applied Biosystems, Cat No. 4307281, batch No. 3617812014.

FIG. 4 is a bar graph showing the relative corrected DPD expression levels in each histological type specimen.A bar graph represents the 25 th and 75 th percentile (between quartiles). The mean value is expressed as a horizontal bar in each bar.

Detailed Description

These oligonucleotide primers, DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2) (also referred to as oligonucleotide primer pair DPD3A) and oligonucleotide primers DPD3B-651F (SEQ ID NO: 7) and DPD3B-736R (SEQ ID NO: 8) (also referred to as oligonucleotide primer pair 3B), are particularly effective for determining DPD gene expression in fixed paraffin-embedded (FPE) tumor specimens.

"substantially identical" as used herein with respect to Nucleic Acids means oligonucleotides that hybridize to a target under stringent conditions, and Nucleic acid fragments or complements thereof that are identical in at least about 60%, typically at least about 70%, more typically at least about 80%, usually at least about 90%, more usually at least about 95-98% of the nucleotides when properly aligned with appropriate nucleotide insertions and deletions. 203-213(1984).

The invention includes substantially identical oligonucleotides that hybridize under stringent conditions (as defined herein) to all or a portion of oligonucleotide primer sequence DPD3A-51F (SEQ ID NO: 1), its complementary sequence, DPD3A-134R (SEQ ID NO: 2), or its complementary sequence, hi addition, the invention includes substantially identical oligonucleotides that hybridize under stringent conditions (as defined herein) to all or a portion of oligonucleotide primer sequence DPD3b-651F (SEQ ID NO: 7), its complementary sequence, DPD3b-736R (SEQ ID NO: 8), or its complementary sequence.

Preferably, such conditions prevent hybridization of nucleic acids having 4 or more mismatches out of 20 contiguous nucleotides, more preferably 2 or more mismatches out of 20 contiguous nucleotides, and most preferably 1 or more mismatches out of 20 contiguous nucleotides.

The hybridizing portion of the nucleic acid is typically at least about 10 (e.g., 15) nucleotides long. the portion of the hybridizing nucleic acid that hybridizes is at least about 80%, preferably at least about 95%, or most preferably at least about 98% identical to all or a portion of oligonucleotide primer sequence DPD3A-51F (SEQ ID NO: 1), its complement, DPD3A-134R (SEQ ID NO: 2), or its complement. furthermore, the portion of the hybridizing nucleic acid that hybridizes is at least about 80%, preferably at least about 95%, or most preferably at least about 98% identical to all or a portion of oligonucleotide primer sequence DPD3b-651F (SEQ ID NO: 7), its complement, DPD3b-736R (SEQ ID NO: 8), or its complement.

The stability of a nucleic acid duplex or hybrid is expressed in terms of the melting temperature (Tm), which is the temperature at which the probe dissociates from the target DNA. melting temperature can be used to define the desired stringency conditions it can be used first to determine the lowest temperature at which homologous hybridization can only be performed using a particular concentration of salt (e.g., SSC or SSPE) if the sequence to be identified is substantially identical to the probe, rather than identical.then, assuming that a 1% mismatch results in a 1 ℃ decrease in Tm, the final wash temperature of the hybridization reaction is therefore decreased (e.g., if a sequence with > 95% identity to the probe is found, the final wash temperature is decreased by 5 ℃), indeed, Tm varies between 0.5 ℃ and 1.5 ℃/1% mismatch.

Other guidance regarding such conditions is readily available in the art, e.g., Sambrook, Fischer and Maniatis, molecular cloning, a laboratory manual, (2) at about 68 deg.C, hybridization in 5XSSC/5 XDenhart's solution/1.0% SDS, washing at room temperature in 0.2 XSSC/0.1% SDS, washing at 3XSSC, varying salt concentration and temperature parameters, and achieving optimal levels of identity between the primers and target nucleic acid^nded.)，Cold Spring Harbor laboratory Press, New York, (1989) and F.M. Ausubel et al, Current protocols in Molecular Biology, John Wiley and Sons (1994).

This aspect of the invention relates to the use of a method for reliably extracting RNA from an FPE sample, and secondly to the determination of the amount of DPD mRNA in the sample by using the oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) or an oligonucleotide substantially identical thereto and DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) or an oligonucleotide substantially identical thereto for performing the reverse transcriptase polymerase chain reaction the RNA is extracted from FPE cells by any method for isolating mRNA from a sample as described in US patent application No.09/469,338 filed 12/20/1999, which is incorporated herein by reference in its entirety.

The oligonucleotide primers disclosed herein can allow accurate assessment of DPD gene expression in fixed paraffin-embedded tissues, as well as frozen or fresh tissues (FIG. 2). this is because mRNA derived from FPE samples is fragmented relative to mRNA in fresh or frozen tissues and is therefore more difficult to quantify.thus, the present invention provides oligonucleotide primers suitable for determining DPD expression levels in FPE tissues without prior suitable methods of determination, see FIG. 1.

Specifically, high levels of DPD mRNA expression are associated with resistance to 5-FU-based chemotherapy.

For the use of some embodiments of the present invention for specific tumor types, it is preferred to demonstrate a relationship between DPD gene expression level and viability by compiling a data set that establishes a link between specific DPD expression and clinical resistance to 5-FU-based chemotherapy.

The invention can be used for any tissue type, e.g. in order to test the resistance of tumor tissue, it is necessary to test the tumor tissue, preferably, it is necessary to test a part of the normal tissue from the patient from whom the tumor was obtained, normal tissue is resistant to 5-FU based chemotherapeutic compounds, but it is expected that patients to which the tumor tissue is sensitive may then receive treatment with higher amounts of chemotherapeutic compositions.

RNA can then be obtained from frozen or fresh tumor samples, RNA isolated from cells by any method typical in the art, such as Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2)^nded.), Coldspring Harbor Laboratory Press, New York, (1989) preferably during extraction, care is taken to avoid RNA degradation.

Alternatively, the patient's tissue may be fixed, e.g., typically with formalin (formaldehyde) or gluteraldehyde.

RNA is extracted from FPE cells by any of the methods described in U.S. patent application US09/469,338 filed on 20.12.1999, which is incorporated herein by reference in its entirety.

In a preferred embodiment of the invention, RNA can be isolated from archived pathological or biopsy samples, which are first deparaffinized, a representative deparaffinization method includes washing the paraffin-embedded samples with an organic solvent, such as xylene.

Once the sample is rehydrated, RNA is extracted from the rehydrated tissue. the deparaffinized sample can be homogenized using mechanical, sonic or other means of homogenization.

A "chaotropic agent in an effective concentration" is selected to mean an amount of RNA purified from paraffin-embedded samples that is about 10 times higher than the amount of RNA isolated without a chaotropic agent, chaotropic agents include, e.g., guanidine compounds, urea, formamide, potassium iodide, potassium thiocyanate and similar compounds. More preferably about 4. the chaotropic solution may also contain reducing agents, such as Dithiothreitol (DTT) and beta-mercaptoethanol (BME).

The homogenized sample is heated to about 50 to about 100 ℃ in a chaotropic solution, which contains an effective amount of a chaotropic agent, such as a guanidine compound.

The RNA in the chaotropic solution is then recovered by phenol-chloroform extraction, ion exchange chromatography or size exclusion chromatography.

The quantification of DPD mRNA is preferably performed using reverse transcriptase polymerase chain reaction (RT-PCR) as is commonly used in the art, wherein the DPD mRNA is derived from purified total mRNA from fresh, frozen, or fixed tissue other methods of quantifying DPD mRNA include, for example, the use of molecular markers and other labeled probes as used in multiplex PCRMost preferably, the quantification of DPD cDNA and internal control or housekeeping genes (e.g., beta-actin) is by fluorescence-based real-time assay (ABIPRISM 7700 or 7900Sequence Detection System)Applied Biosystems, foster city, CA.) or with Heid et al (Genome Res 1996; 6: 986-; 6: 995 and 1001) the similar system proceeds ABI7700(Instrument) is expressed in Ct's or "cycle threshold". UtilityIn the system, a high level of expressed genes with a higher number of target molecules in a sample produces a signal and the PCR cycles (lower Ct) are less than those of genes with a lower level of relative expression of fewer target molecules (higher Ct).

It has been found herein that tumors expressing high levels of DPD mRNA are likely to be resistant to 5-FU. as opposed to tumors expressing low amounts of DPD mRNA which are likely to be sensitive to 5-FU.

"housekeeping" gene or "internal control" as used herein is any constitutively or globally expressed gene, the presence of which can assess the level of DPD mRNA, such assessment including controls to determine the overall constitutive level of gene transcription and variations in RNA recovery. 59: 2302-2306.

Controls for variation in RNA recovery require the use of "calibrator RNA" which can be any of the precise predetermined amounts of control RNA available, Universal PE RNA from applied biosystems is preferably used, catalog No. 4307281, batch No. 3617812014.

As used herein, "Uncorrected Gene Expression (UGE)" means a gene product derived from a gene selected from the group consisting ofThe equation for determining UGE is shown in example 4 and is illustrated by the sample calculations of FIG. 3.

Another aspect of the invention provides a method for normalizing Uncorrected Gene Expression (UGE) values using values derived from non-influenza sourcesValues of "known relative Gene expression" of the extract fromPreferably, the method is performed by Salonga et al, Clinical Cancer Research, 6: 1322-1327, 2000 (incorporated herein by reference in its entirety) previously disclosed source-non-disposableRelative DPD of (a): β -actin expression values were normalized to DPD UGE values derived from tissue samples.

As used herein, "corrected relative DPD expression" refers to normalized DPD expression, UGE multiplied by a DPD-specific correction factor (K)_DPD) A value is obtained that can be compared to the known range of expression levels of DPD relative to the internal control gene, FIG. 3 details these calculations.

"previously published" relative gene expression is based on the ratio of the RT-PCR signal of the target gene to the constitutively expressed gene (. beta. -actin) in Pre-In technical studies, the PCR reaction was run for a fixed number of cycles (i.e., 30) and the endpoint values for each sample were reported. 1322-1327, 2000, which is hereby incorporated by reference in its entirety.

A "predetermined threshold level" of DPD expression, as defined herein, is a level of DPD expression above which a tumor may be resistant to a platinum-based chemotherapeutic regimen^-3-2.5×10^-3(about 4.2-fold range.) relative DPD expression of tumors that do not respond to 5-FU-based chemotherapeutic regimen is about 0.2X 10^-3-16×10^-3(about 4.2 fold range.) if relative DPD expression is higher than about 2.0X 10^-3Preferably higher than about 2.510^-3These tumor values allow for the determination of whether the "corrected relative DPD expression" for a particular sample is above or below a "predetermined threshold level". The threshold for the corrected relative DPD expression level is about 2.0X 10^-3-2.5×10^-3.

The methods of the invention are applicable to a variety of tissues and tumor types, and can be used to evaluate clinical treatment of patients, and as a diagnostic or prognostic tool for a variety of cancers, including breast, head and neck, lung, esophageal, colorectal, etc.

Based on the measurement of the amount of DPD mRNA expressed in a tumor, the practitioner in the art can make a clinical resistance of the tumor to 5-FU based chemotherapy "includes the administration of 5-FU, a derivative thereof, either alone or in combination with other chemotherapeutic agents, such as folinic acid or a DPD inhibitor, such as uracil, 5-ethynyluracil, bromovinyluracil, thymine, benzyloxybenzyluracil (BBU) or 5-chloro-2, 4-dihydroxypyridine. And showed significant improvement in antitumor activity in a human cancer xenograft model.

The practice of the invention has been described above and illustrated by the experimental examples given below.

Examples

Example 1

Isolation of RNA from FPE tissue

RNA was extracted from paraffin-embedded tissues by the following general procedure.

A. Deparaffinization and hydration of sections

(1) A portion of the approximately 10. mu.M sections were placed in a 1.5mL plastic centrifuge tube.

(2) mu.L of xylene was added and the mixture was shaken vigorously at room temperature (about 20-25 ℃) for about 10 minutes.

(3) At room temperature, the samples were centrifuged for about 7 minutes at a maximum speed of a bench top centrifuge (about 10-20,000 Xg).

(4) Steps 2 and 3 are repeated until the majority of the paraffin is dissolved, typically 2 or more times depending on the amount of paraffin contained in the original sample portion.

(5) The xylene solution is removed by vigorous shaking with a lower alcohol, preferably 100% ethanol (about 600. mu.L) for about 3 minutes.

(6) The tubes were centrifuged for about 7 minutes according to step (3), the supernatant decanted and discarded, and the pellet turned white.

(7) With more dilute ethanol solution: steps 5 and 6 were repeated sequentially, first with about 95% ethanol, then with about 80% ethanol, and finally with about 70% ethanol.

(8) The sample was centrifuged at room temperature for 7 minutes according to step (3), the supernatant was discarded, and the pellet was dried at room temperature for about 5 minutes.

B. Isolation of RNA Using phenol-chloroform

(1) 400 μ L of guanidinium isothiocyanate solution containing 0.5% sarcosine and 8 μ L of dithiothreitol were added.

(2) The sample was then homogenized with a tissue homogenizer (Ultra-Turrax, IKA-Works, inc., Wilmington, NC) for about 2-3 minutes, with the speed increasing gradually from low (speed 1) to high (speed 5).

(3) The sample is then heated at about 95 ℃ for about 5-20 minutes, preferably before heating to 95 ℃, the cap of the test tube containing the sample is pierced with a fine needle.

(4) The sample was then extracted with 50. mu.L of 2M sodium acetate pH4.0 and 600. mu.L of phenol/chloroform/isoamyl alcohol (10: 1.93: 0.036) freshly prepared with 18mL of phenol and 3.6mL of a 1: 49 solution of isoamyl alcohol/chloroform.

(5) The solution was centrifuged at maximum speed for about 7 minutes and the upper (aqueous) phase was transferred to a new tube.

(6) RNA30 min was precipitated with approximately 10. mu.L glycogen and 400. mu.L isopropanol at-20 ℃.

(7) Granulating the RNA by centrifugation in a bench top centrifuge at maximum speed for about 7 minutes; decanting and discarding the supernatant; the pellet is washed with about 500. mu.L of about 70-75% ethanol.

(8) The sample is centrifuged again at maximum speed for 7 minutes, the supernatant is discarded, and the pellet is air dried, and the pellet is then dissolved in an appropriate buffer for further experiments (e.g., 50pL 5mM Tris chloride, pH8.0).

Example 2

Reverse transcription and PCR of mRNA

Reverse transcription: RNA was isolated from formalin-fixed paraffin-embedded (FPE) tissue, either microdissected or non-microdissected, as exemplified in example 1 and described in U.S. patent application No. US09/469,338, filed 12/20 1999, which is incorporated herein by reference in its entirety, after precipitation with ethanol and centrifugation, RNA pellets were dissolved in 50. mu.L 5mM Tris/Cl at pH8.0, the RNA obtained was random hexamers and reverse transcribed from Life technologies M-MLV (Cat. No. 28025-02) reverse transcription was performed by mixing 25. mu.L of RNA solution with 25.5. mu.L of "reverse transcription mix" (see below). 8 min at 26 ℃ for random hexamer binding to RNA, 45 min at 42 ℃ for M-MLV reverse transcriptase reaction, and 5 min at 95 ℃ for heat inactivation of DNase.

The "reverse transcription mixture" consisted of the following components: 10 μ l of 5 Xbuffer (250mM Tris-HCl, pH8.3, 375mM KCl, 15mM MgCl)₂) 0.5. mu.l of random hexamer (500.D. dissolved in 550. mu.l of 10mM Tris-HCl, pH 7.5), 5. mu.l of 10mM dNTPs (dATP, dGTP, dCTP and dTTP), 5. mu.l of 0.1M DTT, 1.25. mu.l BSA (3 mg/ml in 10mM Tris-HCL, pH 7.5), 1.25. mu.l RNA Guard24, 800U/ml RNase inhibitor (Cat. No. 27-0816, Amersham Pharmacia) and 2.5. mu.l MMLV 200U/μ l (Life Tech Cat. No. 28025-02).

The final concentrations of the reaction components were: 50mM Tris-HCl, pH8.3, 75mM KCl, 3mM MgCl₂1.0mM dNTP, 1.0mM DTT, 0.00375mg/ml BSA, 0.62U/. mu.l RNA Cuard and 10U/. mu.l MMLV.

Quantification of DPD cDNA and internal control or housekeeping gene (e.g.,. beta. -actin, e.g., Eads et al, Cancer Research 1999; 59: 2302-; real-time fluorescence-based detection method (ABIPRISM 7700 or 7900sequence detection System) as described by Gibson et al (Genome Res 1996; 6: 995-1001)Applied Biosystems, Foster City, Calif.) briefly, the method uses a dual labeled fluorogenicOligonucleotide probes, (DPD-530Tc (SEQ ID NO: 3), Tm ═ 70 ℃), which anneal specifically within the forward and reverse primers laser stimulation within the capped wells containing the reaction mixture causes the emission of a3 'quencher dye (TAMRA) until the probe is cleaved by the 5' -3 'nuclease activity of the DNA polymerase during PCR extension, causing the release of a 5' reporter dye (6FAM)sCCD (Charge coupled device) detection the amount of signal generated by the threshold cycle in the pure exponential phase of the PCR reaction, detected by the camera, reflects the initial copy number of the target sequence oligonucleotide primer pairs DPD1(DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)))The probe was DPD-108Tc (SEQ ID NO: 9). the oligonucleotide primer pair DPD2(DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)))The probe was DPD-2p-1154Tc (SEQ ID NO: 10). of oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2))The probe was DPD3A-71Tc (SEQ ID NO: 11). the oligonucleotide primer set of DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)))The probe was DPD3b-685Tc (SEQ ID NO: 12).

The PCR reaction mixture contained the primers from primer pair DPD1(DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)); DPD2(DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)); DPD3B (DPD3b-651F (SEQ ID NO: 7), Tm 58 ℃ and DPD3b-736R (SEQ ID NO: 8), Tm 60 ℃); or oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1), Tm 59 ℃ and DPD3a-134R (SEQ ID NO: 2), Tm 59 ℃.) each PCR primer mixture consists of: mu.l of a reverse transcription reaction containing cDNA and 600nM of each oligonucleotide primer from only one pair (DPD1, DPD2, DPD3 or DPD3A) of primers, 200nM of the corresponding oligonucleotide primerProbes (for DPD1, DPD2, DPD3 or DPD3A), 5U AmpliTaq Gold polymerase, 200. mu.M prepared dATP, dCTP, dGTP, 400. mu.M dTTP, 5.5mM MgCl₂And 1xTaqman buffer A containing a reference dye, the final volume being less than or equal to 25. mu.l (all reagents, Applied Biosystems, Foster City, Calif.) cycling conditions were 45 cycles at 95 ℃ for 10 minutes, followed by 95 ℃ for 15 seconds and 60 ℃ for 1 minute.

Example 3

Oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) allow for strong, reproducible quantification of DPD gene expression using RNA extracted from paraffin-embedded tissues by RT-PCR FIG. 1. oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) also significantly increase the sensitivity of analysis of DPD gene expression in fresh frozen tissues by RT-PCR FIG. 2. the ABI Prism 7700 sequence detection system was used as described in example 2 aboveThe process is carried out.

The product length of amplification with oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) is 84 base pairs.the amplification product corresponds to a region of DPD cDNA spanning a portion of the 5' untranslated region (UTR) and entering exon 1.the product length of amplification with oligonucleotide primer pair DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) is 86 base pairs.the amplification product corresponds to a region of DPD cDNA corresponding to exon 6.

The ability of the oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) to amplify DPD mRNA from 10 different FPE tissue samples was compared to other existing primer sets.samples #1-5 and #8-10 were from a colon tumor biopsy, #6 was from a bronchoalveolar tumor biopsy, #7 was from a small intestine tumor biopsy.the other oligonucleotide primer pairs used were DPD1(DPD-70F, (SEQ ID NO: 3) and DPD-201R, (SEQ ID NO: 4)) and DPD2(DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 1208)).

The oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) accurately determined the most effective of the DPD levels in various samples the oligonucleotide primer pair DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) was also effective but did not produce strong signals the results are shown in FIG. 1.

Example 4

Determining Uncorrected Gene Expression (UGE) for DPD

Two pairs of parallel reactions are performed, the "test" reaction and the "calibration" reaction fig. 7. the DPD amplification reaction and the β -actin internal control amplification reaction are test reactions.The instrument yielded 4 different cycle threshold (Ct) values: ct of test reaction_DPDAnd Ct_Beta-actinAnd Ct of calibration reaction_DPDAnd Ct_Beta-actinThe Ct difference for both reactions was determined according to the following equation:

ΔCt_{test of}＝Ct_DPD-Ct_Beta-actin("test" reaction)

ΔCt_Calibration＝Ct_DPD-Ct_Beta-actin("calibration" reaction)

The next step is to calculate the negative Δ Ct power of 2 according to the following equation.

2^{-Delta Ct test}("test" reaction)

2^{- Δ Ct calibration}("calibration" reaction)

To get fromUncorrected gene expression of DPD was obtained by the instrument and the following calculations were performed:

uncorrected Gene Expression (UGE) ═ 2 for DPD^{-Delta Ct test}/2^{- Δ Ct calibration}

Normalizing UGE with known relative DPD expression levels

The calculation of normalization requires multiplying UGE by a correction factor (K) specific for DPD and a particular calibration RNA_DPD) The correction factor K for any internal control gene and any precisely predetermined amount of calibrator RNA can also be determined_DPDPreferably, an internal control gene β -actin and a precisely predetermined amount of calibrator RNA, Universal PERRNA from Applied Biosystems, Cat 4307281, batch 3617812014 are used.

Normalization was performed using a modification of the Δ Ct method, which was performed by applied biosystems,manufacturer, in User Bulletin #2 and described above, to perform the process, the above is usedMethods, DPD expression of UGE was analyzed for 6 different FPE test tissues using the internal control gene β -actin and calibrator RNA, Universal PE RNA from Applied Biosystems, cat # 4307281, lot # 3617812014.

The relative DPD expression levels (PV) of each of samples L7, L91, L121, L150, L220 and L164, previously described in Salonga et al (incorporated herein by reference in its entirety), divided by their corresponding sourcesUGE, resulting in an uneven correction factor K.

K_{Is not uniform}＝PV/UGE

Next, the total K values were averaged to determine the unique K specific for DPD, calibrator RNA, i.e., Universal PE RNA, Cat No. 4307281, batch No. 3617812014, and beta-actin_DPDA correction factor.

Therefore, in order to measure and predict on a large scaleConsistent with studies of DPD expression, correction in unknown tissue samples relative to DPD expression, one need only use a source fromUncorrected gene expression data (UGE) for an instrument multiplied by K_DPDSpecific correction factors, provided the same internal control gene and calibrator RNA were used.

Corrected relative DPD expression UGE × K_DPD

K_DPDThe precise predetermined amount of RNA from the future source may be calibrated against the sample using known relative DPD expression or may be calibrated against a previously calibrated calibrator RNA, such as Universal PE RNA, Cat No. 4307281, batch No. 3617812014, as described above.

For example, if the K of a different internal control gene and/or a different calibrator RNA is subsequently determined_DPDOne must calibrate the internal control gene and the calibrator RNA relative to the tissue sample in which the level of DPD expression relative to the particular internal control gene has been determinedQuantitative RT-The known expression levels of these samples are divided by their corresponding UGE levels to determine the K values for the samples_DPD.

Example 5

DPD expression in FPE colorectal tumor samples

The method described above was used to analyze 34 tumor samples from 34 patients with advanced colorectal cancer all patients were treated with an intravenous 5-FU/LV combination regimen as part of a prospective multicenter European 5-FU/CPT11 crossover trial V239 all were treated with intravenous 5-FU425mg/m²The treated patients were infused for 15 minutes over 5 consecutive days, and also with 20mg/m over 5 consecutive days²The regimen is used as a first or second line relief therapy.

Patients with progressive or stable disease were classified as non-responders (25 patients, 73.5%). Total mRNA was isolated from microdissected FPE pre-treated tumor samples and the relative mRNA expression levels of DPD/β -actin were measured by quantitative PCR as described above.

The mean corrected DPD/β -actin levels in the response and non-response patient groups were 0.87X 10, respectively^-3And 2.04X 10^-3Relative DPD levels in the non-responder group were significantly lower compared to non-responders (P ═ 0.02), the association between DPD mRNA expression and response to 5-FU/LV in these patients is shown in figure 4.

Sequence listing

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Claims (13)

1, SEQ ID NO: 7 or the complement thereof.

SEQ ID NO: 8 or the complement thereof.

3. A kit for detecting the expression of a dihydropyrimidine dehydrogenase gene, comprising a pair of oligonucleotides consisting of SEQ ID NOs: 7 or the complement thereof and the oligonucleotide of SEQ ID NO: 8 or the complement thereof.

4. A method of determining the relative level of dihydropyrimidine dehydrogenase gene expression in a tumor sample comprising:

(a) isolating mRNA from a tumor sample;

(b) using SEQ ID NO: 7 or the complement thereof and the oligonucleotide primer set forth in SEQ ID NO: 8 or the complement thereof, and amplifying the mRNA; and

(c) comparing the amount of mRNA of step (b) with the amount of mRNA of an internal control gene.

5. The method of claim 4, wherein the tumor sample is frozen.

6. The method of claim 4, wherein the tumor sample is embedded in paraffin after fixation.

7. The method of claim 6, wherein the step of isolating mRNA from the tumor sample is performed in the presence of an effective amount of a chaotropic agent.

8. The method of claim 7, wherein the tumor sample comprises non-tumor tissue and tumor tissue.

9. A method of determining the sensitivity of a tumor sample to 5-fluorouracil, comprising:

(a) isolating mRNA from a tumor sample;

(b) amplifying the mRNA using a pair of oligonucleotide primers to obtain an amplified sample, wherein one primer of the primer pair consists of SEQ ID NO: 7 or the complement thereof, and the other primer of the primer pair consists of SEQ ID NO: 8 or the complement thereof;

(c) determining the amount of dihydropyrimidine dehydrogenase mRNA in the amplified sample;

(d) comparing the amount of dihydropyrimidine dehydrogenase mRNA in the amplified sample to a predetermined threshold level of dihydropyrimidine dehydrogenase expression; and

(e) determining the sensitivity of the tumor sample to 5-fluorouracil based on the difference between the amount of dihydropyrimidine dehydrogenase mRNA in the amplified sample and a threshold level of dihydropyrimidine dehydrogenase gene expression.

10. The method of claim 9, wherein the predetermined threshold level of dihydropyrimidine dehydrogenase gene expression is measured relative to an internal control gene expression level.

11. The method of claim 10, wherein the internal control gene is β -actin.

12. The method of claim 11, wherein the predetermined threshold level of dihydropyrimidine dehydrogenase gene expression is 2.0-2.5 times the level of β -actin gene expression.

13. The method of claim 4 or 9, wherein the tumor sample comprises bronchoalveolar tumor tissue, small intestine tumor tissue or colon tumor tissue.