KR100909116B1 - Method for measuring dihydropyrimidine dehydrogenase gene expression - Google Patents

Abstract

The present invention relates to prognostic methods useful in medicine, in particular for the treatment of cancer. SUMMARY OF THE INVENTION An object of the present invention is to provide a method for evaluating the level of dihydropyrimidine dehydrogenase (DPD) expression in a tissue, to investigate the amount of DPD mRNA in tumor cells of a patient, By comparison the prognosis of possible resistance of patient tumors to treatment with 5-FU based therapies. More specifically, the present invention provides oligonucleotide primer pairs DPD3A and DPD3B, and provides a method comprising their use to detect the level of dihydropyrimidine dehydrogenase (DPD) mRNA.

Description

Method for measuring dihydropyrimidine dehydrogenase gene expression {METHOD OF DETERMINING DIHYDROPYRIMIDINE DEHYDROGENASE GENE EXPRESSION}

The present invention relates to prognostic methods useful for medicine, in particular for cancer chemotherapy. The invention also relates to the evaluation of gene expression of tumor cells of a patient. More specifically, the present invention relates to methods comprising oligonucleotides and their use to detect levels of dihydropyrimidine dehydrogenase (DPD) mRNA expression using RT-PCR.

Cancer occurs when normal cells undergo neoplastic transformation and become malignant cells. The converted (malignant) cells do not undergo normal physiological control to characterize the cell phenotype and maintain cell proliferation. Thus, the converted cells in the subject proliferate in the absence of these normal controls and consequently form tumors.

If tumors are produced, the clinical goal is to selectively destroy tumor cells while mitigating any deleterious effects on normal cells in the subject being treated. Chemotherapy is based on the use of drugs that are selectively toxic to cancer cells. Several general classes of chemotherapeutic agents have been developed, including those that interfere with nucleic acid synthesis, protein synthesis, and other important metabolic processes.                 

5-Fluorouracil (5-FU) is a drug that is very widely used to treat many different types of cancers, such as most cancers such as gastrointestinal and breast cancers. See Moertel, C.G. New Engl. J. Med., 330: 1136-1142 (1994). For more than 40 years, the best standard of care for colorectal cancer has been to use only 5-FU, but this has been replaced by a "standard of care" in combination with 5-FU and CPT-11. Saltz Et al., Irinotecan Study Group. New England Journal of Medicine. 343: 905-14 (2000). Recently, the combination of 5-FU and oxaliplatin has shown a high response rate in colorectal cancer [Raymond et al., Semin. Oncol., 25: 4-12 (1998). Thus, 5-FU is expected to be used for the treatment of cancer for many years, because 5-FU is at the heart of currently used chemotherapy. In addition, single agent 5-FU therapy will continue to be used for patients whose combination therapy with CPT-11 or oxaliplatin is likely to be excessively toxic.

5-FU is typically used for most anticancer drugs, with only a small number of patients responding favorably to the therapy. Large randomized clinical trials have shown that the overall response rate of tumors to 5-FU as a single agent for patients with metastatic colorectal cancer ranges from 15-20%. See Moertel, C.G. New Engl. J. Med., 330: 1136-1142 (1994). When used in combination with other chemotherapeutic agents described above, the tumor response rate for 5-FU based therapies increased to nearly 40%. Nevertheless, most of the patients treated did not show any measurable advantages over patients who underwent 5-FU based chemotherapy, resulting in significant risks, inconveniences, and costs. Since there is no reliable means to predict the responsiveness of an individual's tumor prior to treatment, standard clinical practice performs 5-FU based treatment on all patients, and the majority of patients suffer from unsatisfactory results. It is fully recognized that there is.

The mechanism of action and metabolic pathways of 5-FU have been extensively studied over the years to identify the most important biochemical determinants of the antitumor activity of these agents. The ultimate goal is to anticipate whether the patient will (or will not) respond to the drug by (a) controlling its intracellular metabolism and biochemistry and (b) measuring response determinants in the patient's tumor prior to treatment. Thereby improving the clinical efficacy of 5-FU. Two main determinants were derived from these studies: 1) identification of thymidylate synthase (TS), a target enzyme of 5-FU, and 2) dihydropyrimidine dehydrogenase (DPD), a 5-FU catabolic enzyme. Confirmation of).

The first study in the field of predicting tumor response to 5-FU based therapies focused on target enzyme TS in colorectal cancer. Leichman et al., Leichman et al., J. Clin. Oncol. 15: 3233-3229 (1997) conducted a prospective clinical trial to study the correlation of tumor response to 5-FU and TS gene expression as determined by RT-PCR in pretreatment biopsies from colorectal cancer. This study identified 1) a large 50-fold range of TS gene expression levels in these tumors and 2) significantly different levels in TS gene expression between reactive and nonreactive tumors. TS level range of responders (0.5 to 4.1 compared to the internal control x 10 ^-3) is (1.6-23.0 x 10 compared to inner control ^{- 3)} TS level range of the non-responder group atda narrower than. The researchers determined the resulting "non-responsive cutoff" threshold level of TS expression, with only non-responders present. Thus, patients with TS expression levels above the "non-responsive cutoff" can be reliably identified as non-responders prior to treatment. The "non-responsive" classification includes all therapeutic responses with progressive growth resulting in <50% tumor contraction,> 25% tumor increase and non-progressive tumors with <50% contraction, no change or <25% increase. These tumors have the highest TS levels. Thus, high TS expression confirms tumors that are particularly resistant. TS expression levels above a certain threshold identify subsets of tumors that do not respond to 5-FU, whereas TS expression levels below these levels predict significantly higher response rates, but do not specifically identify reactive tumors. .

Subsequent studies investigated the usefulness of DPD expression levels as a tumor response determinant for 5-FU treatment along with TS expression levels. DPD is a catabolic enzyme that reduces the 5,6 double bond of 5-FU and, by this action, inactivates 5-FU as a cytotoxic agent. Previous studies have shown that DPD levels in normal tissues influence the bioavailability of 5-FU to modulate its pharmacokinetic and antitumor activity. Harris, et al., Cancer Res., 50: 197-201 (1990) ]. Evidence has also been shown 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), incorporated herein by reference) are the tumor response determinants for 5-FU / Lucovorin treatment in a set of tumors in which TS expression has already been measured. The gene expression of DPD was examined. As with TS, the range of DPD expression between tumor response in the range of non-responder tumors. - it was very narrow as compared to (0.2 16 x 10 ^-3, 80 times higher than that of the internal control) (0.6-2.5 x 10 ^-3 to internal control 4.2 times). There was no reactive tumor with DPD expression above the threshold level of about 2.5 × 10 ⁻³ . In addition, DPD and TS expression levels were found to be uncorrelated, indicating that they are independently regulated genes. Between tumor groups that had TS and DPD expression levels below their respective "unresponsive cutoff", 92% responded to 5-FU / LV. Thus, reactive tumors can be identified based on low expression levels of DPD and TS.

In addition, DPD is an important marker for 5-FU toxicity. Patients with very low DPD levels receiving 5-FU based treatment (eg DPD deficiency syndrome; ie, thymine uraciluria) have been observed to suffer from life-threatening toxicity. Lyss et al., Cancer Invest , 11: 2390240 (1993). Indeed, the importance of DPD levels in 5-FU therapy has been dramatically exemplified by the death of 19 people in Japan due to poor drug interactions between 5-FU and the antiviral compound sorivudine (Diasio et al., Br. J. Clin. Pharmacol. 46, 1-4 (1998). Metabolite of sorivudine was later found to be a potent inhibitor of DPD. This treatment resulted in a similarly reduced level of DPD deficiency syndrome of DPD, which increased the toxicity of 5-FU for patients. See Diasio et al., Br. J. Clin. Pharmacol. 46, 1-4 (1998).

Thus, (a) widespread use of the 5-FU protocol in cancer treatment, (b) an important role of DPD expression in predicting tumor response to 5-FU, and (c) DPD for conventional 5-FU-based treatments Because of the sensitivity of patients with deficiency syndrome, it is clear that accurate measurement of DPD expression levels prior to chemotherapy will provide significant benefits for cancer patients.

Measurement of DPD enzyme activity requires a significant amount of new tissue containing the active enzyme. Unfortunately, most pretreatment tumor biopsies are available only in fixed paraffin embedded (FPE) tissues, especially formalin-fixed paraffin embedded tissues, which do not contain active enzymes. Moreover, biopsies generally contain only very small amounts of heterologous tissue.

RT-PCR primer and probe sequences can be used to analyze DPD expression in frozen tissue or new tissue. However, these primers are not suitable for quantification of DPD mRNA from tissues immobilized by RT-PCR. To date, existing primers have shown no or inconsistent results. This includes (a) originally low levels of DPD RNA; (b) very small amounts of tissue embedded in paraffin; And (c) degradation of <100 bp of RNA in paraffins into small manipulations. As a result, other researchers cooperated, but with no success in obtaining an oligonucleotide primer set that enables the quantification of DPD expression in paraffin treated tissues. Therefore, there is a need for a method of quantifying DPD mRNA from fixed tissues to provide an early prognosis for the proposed cancer therapy. As it has been found that DPD enzyme activity and the corresponding mRNA expression levels are closely related [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 specimens provides a method for assessing the state of DPD expression levels in a patient without having to measure enzyme activity in new tissue. In addition, FPE specimens can be easily adapted for microanalysis so that DPD gene expression can be measured in tumor tissue that is not contaminated with stromal tissue.

Accordingly, it is an object of the present invention to provide a method for assessing DPD levels in tissues, and also to provide a method for prognosing the probable resistance of a patient's tumor to treatment using 5-FU based therapy. This is done by measuring the amount of DPD mRNA in tumor cells and comparing it to a predetermined limit expression level.

Summary of the Invention

In one aspect of the invention, oligonucleotide primers DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (as well as oligonucleotide primers having sequences of DPD3A-51F (SEQ ID NO: 1) and DPD3A-134R (SEQ ID NO: 2)) SEQ ID NO: 8) and sequences substantially identical thereto are provided. In addition, the present invention forms hybrids with 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 complements thereof. An oligonucleotide primer having a sequence to be provided is provided.

The invention also relates to a method of determining a chemotherapy comprising the steps of obtaining an mRNA sample from a tumor specimen; Measuring DPD gene expression levels in the specimen; Comparing the measured DPD gene expression level with a predetermined threshold level for that gene; And determining a chemotherapy based on a result of the comparison of the measured gene expression level with a predetermined threshold level.

In addition, the present invention provides for the uncontrolled gene expression (UDE) of DPD for internal control in tissue samples analyzed using the Taqman® technique. To a method of normalizing to DPD expression levels.

1 is a graph showing a comparison of four different oligonucleotide primer pairs for the ability to amplify DPD mRNA derived from ten different formalin-FPE tissue samples. Samples # 1-5 and # 8-10 are from a colon tumor biopsy, sample # 6 is from a bronchoalveolar tumor biopsy, and sample # 7 is from a small intestine tumor biopsy. Oligonucleotide primer pairs 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)] It was not effective for measuring DPD mRNA levels. 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)] were found in various samples. It was effective to check DPD level at.

FIG. 2 shows oligonucleotide primer pairs 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: 2) in frozen tissue samples. 4)] is a graph showing a comparison of the DPD mRNA amplification efficiency, as can be seen through the graph of Fig. 2. As shown in the graph of Fig. ] Was effective in measuring DPD expression levels in frozen tissue samples (as well as FPE derived samples), which was described as oligonucleotide primer pairs DPD1 [DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)]. It was more efficient than

3 is a chart showing how DPD expression is calculated for internal control genes. This chart contains data obtained using two test samples (Unknown 1 and 2) and illustrates how to determine uncorrected gene expression data (UGE) UCG. This chart also illustrates how to standardize the UGE obtained by the Takman instrument using known DPD values. This is done by multiplying the correction factor K _DPD by UGE. The internal control gene in the figure is β-actin and the calibrator RNA is universal PE RNA; Applied Biosystems' Cat # 4307281, lot # 3617812014.

4 shows a boxplot of relative corrected DPD expression levels for each histological type of specimen. The boxes in this figure represent 25 ^th and 75 ^th percentiles (quartile range). The mean value is represented by a horizontal bar in each box. Whiskers represent levels outside the 25 ^th and 75 ^th percentiles, but exclude the external values indicated at the top of the boxes.

Detailed description of the invention

The present invention discloses oligonucleotide primers that enable DPD expression in tissues and oligonucleotide primers that are substantially identical thereto. These oligonucleotide primers, namely DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2) (also referred to herein as oligonucleotide primer pair DPD3A) and oligonucleotide primer DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8) (also referred to herein as oligonucleotide primer pair DPD3B) is particularly effective when used to measure DPD gene expression in fixed paraffin embedded (FPE) tumor specimens.

As used herein, "substantially the same" within the context of a nucleic acid means that oligonucleotides hybridize to a target under stringent conditions and, when comparing nucleic acid fragments or their complementary strands, when they are appropriately aligned with appropriate nucleotide insertions and deletions. Typically at least about 70%, more typically at least about 80%, generally at least about 90% and more generally about 95% to 98% identical. If hybridization is more specific than the overall deficiency of specificity, there is selective hybridization (Kanehisa, Nucleic Acids Res., 12: 203-213 (1984)).

The present invention hybridizes to all or a portion of DPD3A-51F (SEQ ID NO: 1), its complement, DPD3A-134R (SEQ ID NO: 2), or oligonucleotides of its complement under stringent conditions (as described herein). And substantially identical oligonucleotides. Furthermore, the present invention also hybridizes to oligonucleotide primer sequence DPD3b-651F (SEQ ID NO: 7) or its complement, DPD3b-736R (SEQ ID NO: 8) or all or a portion thereof, under stringent conditions (as described herein). And substantially identical oligonucleotides.

Under stringent hybridization conditions, only very complementary, ie substantially identical, nucleic acid sequences hybridize. Preferably, such conditions are nucleic acids having four or more mismatches out of twenty consecutive nucleotides, more preferably nucleic acids having two or more mismatches among twenty consecutive nucleotides, most preferably twenty consecutive Prevents hybridization of nucleic acids bearing one or more mismatches among the nucleotides.

The hybridization portion of the nucleic acid is typically at least 10 (eg 15) nucleotides in length. The hybridization portion of the nucleic acid to hybridize is at least about 80%, preferably at least in sequence to the oligonucleotide primers DPD3A-51F (SEQ ID NO: 1), its complement, DPD3A-134R (SEQ ID NO: 2), or all or a portion of its complement At least about 95%, or most preferably at least about 98%. In addition, the hybridization portion of the nucleic acid to hybridize is at least about 80% to the sequence of the oligonucleotide primer DPD3b-651F (SEQ ID NO: 7) or its complement, DPD3b-736R (SEQ ID NO: 8) or all or a portion of its complement, preferably Preferably at least about 95%, or more preferably at least about 98%.                 

Hybridization of oligonucleotide primers to nucleic acid samples under stringent conditions is defined as follows. Nucleic acid duplex or hybrid stability is expressed as melting temperature (T _m ), which is the temperature at which the prop is separated from the target DNA. This melting temperature is used to define the stringent conditions required. If the sequence is found to be substantially identical rather than identical to the probe, it is first useful to establish the lowest temperature at which homologous hybridization occurs only at the concentration of a particular salt (eg, SSC or SSPE). Subsequently, 1% mismatching results in a 1 ° C. decrease in T _m , thus the final wash temperature in the hybridization reaction decreases (eg, when the sequence is> 95% identical to the probe, the final wash temperature decreases by 5 ° C.). do). In practice, the change in T _m may be between 0.5 ° C. and 1.5 ° C. per 1% mismatch.

Stringent conditions include hybridization at 68 ° C. in 5 × SSC / 5 × Denhardt solution / 1.0% SDS and washing in 0.2 × SSC / 0.1% SDS at room temperature. Moderately stringent conditions include washing in 3 × SSC at 42 ° C. Salt concentration and temperature parameters can be altered to obtain optimal levels of identity between primers and target nucleic acids. Additional instructions related to such conditions are well known in the art, for example, Sambrook, Fisher and Maniatis, Molecular Cloning, a laboratory Press, New York (1989) and FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons (1994).

This aspect of the invention provides an oligonucleotide primer oligonucleotide primer pair DPD3A [DPD3a-51F (SEQ ID NO: 1) and DPD3a to perform reliable extraction methods of RNA from FPE specimens and to perform RT-PCR (reverse transcriptase polymerase chain reaction). -134R (SEQ ID NO: 2)] or an oligonucleotide substantially identical thereto or a specimen by use of DPD3B [DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)] or an oligonucleotide substantially identical thereto The use of a second measure of the content of DPD mRNA in the cell. RNA is extracted from FPE cells by any method for mRNA isolation from such samples as described in US Patent Application 09 / 469,338 (December 20, 1999), which is incorporated herein by reference.

Oligonucleotide primers of the invention allow for the accurate assessment of DPD expression in fixed paraffin embedded (FPE) tissue (FIG. 1). In addition, the oligonucleotide primers of the invention are suitable for measuring DPD expression levels in new or frozen tissue. That is, they possess high specificity for their target RNA. Thus, the method of the present invention is not limited to the use of paraffin embedded tissues. The oligonucleotide primers disclosed herein allow for the accurate assessment of DPD gene expression in fixed paraffin embedded tissues as well as frozen or new tissues (FIG. 2). This is due to the fact that mRNA derived from FPE samples is more fragmented than mRNA from new or frozen tissue and is therefore more difficult to quantify. Accordingly, the present invention provides oligonucleotide primers suitable for use in analyzing DPD expression levels in FPE tissues where no suitable assay previously existed (see FIG. 1).                 

Expression of DPD mRNA is associated with clinical resistance to 5-FU based chemotherapy. In particular, high levels of expression of DPD mRNA are associated with 5-FU based chemotherapy.

The method of the present invention is applied to a wide range of tumor types. This allows for the manufacture of an “tumor expression profile” of an individual so that DPD expression levels can be measured in individual patient samples and predict response to various chemotherapeutic therapies. More preferably, the method of the present invention is applied to bronchoalveolar tumors, small intestine tumors or colon tumors. In the application of some embodiments of the present invention to a particular tumor type, the measurement of clinical tolerance is determined by compiling a data set of correlations between specific DPD expression parameters measured and clinical resistance to 5-FU based chemotherapy. It is desirable to confirm the relationship.

The method of the present invention can be applied to any type of tissue. For example, for the investigation of the resistance of tumor tissue, it is preferable to examine the tumor tissue. Preferably, it is desirable to irradiate a portion of normal tissue from a patient who has acquired a tumor. Although normal tissue is resistant to 5-FU based chemotherapeutic compounds, patients whose tumors are expected to be sensitive to the compounds as described above can be treated with higher amounts of chemotherapeutic compositions.

The method of the present invention comprises obtaining a cell sample derived from a tumor of a patient. Solid tumors or lymphoid tumors, or portions thereof, are surgically dissected from the patient. After its incision, if it is possible to extract RNA from the tissue sample, the sample can be fixed or frozen. It can then be used to obtain RNA. RNA extracted and isolated from frozen or fresh samples of dissected tissue can be prepared by any method known in the art, for example Sambrook, Fisher and Maniatis, Molecular Cloning, a laboratory Press, New York (1989). Extract in the manner described in. It is desirable to avoid degradation of RNA during the extraction process.

Alternatively, the tissue obtained from the patient can be fixed, preferably by formalin (formaldehyde) or glutaraldehyde treatment. In addition, samples immobilized by alcohol soaking may also be considered in the present invention. Immobilized biological samples are often dehydrated and embedded in paraffin or embedded in other solid supports known to those skilled in the art. This solid support can be removed using an organic solvent and is configured to allow subsequent rehydration of the preserved tissue. The fixed and paraffin embedded (FPE) tissue specimens as described above are referred to as storeable tissue samples or storage tissue samples.

RNA is extracted from FPE cells by any method as described in US Patent Application 09 / 469,338 (December 20, 1999), which is incorporated herein by reference. More preferably, the RNA is extracted from tumor cells derived from formalin fixed and paraffin embedded tissue specimens.

In one embodiment of the invention, the RNA is isolated from a storage pathological sample or first deparaffinized biopsy. Exemplary deparaffinization methods include washing paraffinized samples with an organic solvent, such as xylene. Deparaffinized samples can be rehydrated with an aqueous lower alcohol solution. Examples of suitable lower alcohols include methanol, ethanol, propanol and butanol. Deparaffinized samples can be rehydrated by continuous washing with lower alcohol solution at reduced concentration. Alternatively, the sample is deparaffinized and rehydrated simultaneously.

Once the sample is rehydrated, RNA is extracted from the rehydrated tissue. Deparaffinized samples can be homogenized using mechanical homogenization means, ultrasonic homogenization means or other homogenization means. In one embodiment, the rehydrated sample is homogenized in a solution comprising a chaotropic agent, for example guanidinium thiocyanate (also marketed as guanidinium isothiocyanethyr).

"Effective concentration of chaotropic agent" is selected such that RNA is purified from paraffin embedded samples in an amount of at least 10 times that of separating without chaotropic agent. Examples of chaotropic agents include, but are not limited to, guanidinium compounds, urea, formamide, potassium iodide, potassium thiocyanate and similar compounds. Preferred chaotropic agents for the process of the present invention are guanidinium compounds, for example guanidinium isothiocyanate (also available as guanidinium thiocyanate) and guanidinium hydrochloride. Many anionic counterions are useful, and one of ordinary skill in the art can prepare multiple guanidinium salts with suitable anions as described above. The effective concentration of the guanidinium solution used in the present invention is generally in the range of about 1 M to about 5 M, with about 4 M being the preferred concentration. If the RNA is already in solution, the guanidinium solution may be at a higher concentration such that the final concentration obtained in the sample ranges from about 1 M to about 5 M. In addition, the guanidinium solution is preferably buffered to a pH of about 3 to about 6, more preferably about 4, using a suitable biochemical buffer such as Tris-HCl. In addition, the chaotropic solution may be a reducing agent such as dithiothreitol (DTT), (β-mercaptoethanol; BME); And combinations thereof. The chaotropic solution may also contain an RNAse inhibitor.

The homogenized sample may be heated to a temperature in the range of about 50 ° C. to about 100 ° C. in a chaotropic solution containing an effective amount of a chaotropic agent, for example a guanidinium compound. Preferred chaotropic formulation is guinadinium thiocyanate.

RNA can then be recovered from solution by, for example, phenol chloroform extraction, ion exchange chromatography or size exclusion chromatography. The RNA can then be further purified using extraction, electrophoresis, chromatography, precipitation or any suitable technique.

Quantification of DPD mRNA from purified whole mRNA derived from fresh tissue, frozen tissue or fixed tissue is preferably performed using, for example, reverse transcriptase polymerase chain reaction (RT-PCR) known in the art. . Examples of other methods for quantifying DPD mRNA include the use of molecular beacons and other labeled probes useful in multiplex PCR. In the present invention, DPD mRNA can also be quantified using a PCR-free system using a fluorescently labeled probe similar to, for example, a probe from an Invader® assay (Third Wave Technologies Inc.). Most preferably, quantification of DPD cDNA and internal control genes or housekeeping genes (eg β-actin) is performed by fluorescence based real-time detection [ABI PRISM 7700 or 7900 sequence detection system [Tagman®], USA Applied Biosystems, Foster City, Calif. Or similar systems as described in Heid et al., Genome Res 6: 986-994 (1996) and Gibson et al., Genome Res 6: 995-1001 (1996). Perform using The results of the ABI 7700 (Takman Instrument) are expressed in cycle thresholds (Ct). Using the Taqman system, highly expressed genes having a larger number of target molecules in a sample may have lower PCR cycles (relative to lower relative expression genes with higher number of target molecules (higher Ct). Produces a signal with a lower Ct).

The present invention is based in part on the discovery that the relative amount of DPD mRNA is associated with resistance to the chemotherapeutic agent 5-FU. As found herein, tumors expressing high levels of DPD mRNA are considered to be resistant to 5-FU. Conversely, tumors expressing small amounts of DPD mRNA are thought to be sensitive to 5-FU. The expression of tumor DPD mRNA in a patient is determined by comparing it with a predetermined limit expression level of DPD expression.

As used herein, the term “housekeeping” gene or “internal control” gene is meant to include any constitutively expressed gene or fully expressed gene whose presence enables the evaluation of DPD mRNA. Evaluation as described above includes control over changes in the overall constitutive level of gene transcription and RNA recovery. Examples of “housekeeping” genes or “internal control” genes include, but are not limited to, cyclophilin gene, β-actin gene, transferrin receptor gene, GAPDH gene, and the like. Most preferably, the internal control gene is a β-actin gene as described in Eads et al., Cancer Research 59: 2302-2306 (1999).

Control of changes in RNA recovery requires the use of "calibraotr RNA". By "calibrator RNA" is meant any available source of control RNA that has been accurately quantified. Universal PE RNA; Preference is given to using Cat # 4307281, lot # 3617812014 (Applied Biosystems).

As used herein, the term “uncorrected gene expression (UGE)” is a numerical output of DPD expression for internal control genes generated by the Taqman instrument. The equation used to determine the UGE is shown in Example 4, which is illustrated by sample calculation in FIG. 3.

Another aspect of the invention provides a method of normalizing uncorrected gene expression (UGE) values obtained from the Taqman apparatus to already known relative gene expression values derived from non-Takman techniques. The non-tag only induced relative DPD: β-actin expression values (which are already known by Salonga et al., Clinical Cancer Research, 6: 1322-1327, 2000, incorporated herein by reference) are obtained from tissue samples. It is preferred to standardize on the derived DPD UGE.

As used herein, the term "corrected relative DPD expression" refers to normalized DPD expression, where UGE is multiplied by the DPD specificity correction factor (K _DPD ) to yield a comparable value to a range of values already known. 3 shows these calculations in detail.

As used herein, the term "previously published" relative gene expression results are based on the ratio of the RT-PCR signal of the target gene to the constitutively expressed gene (β-actin). In the pre-tagman technique study, PCR reactions are run for a fixed number of cycles (ie, 30 cycles) and endpoint values are recorded for each sample. These values are then reported as the ratio of DPD expression to β-actin expression (Salonga et al., Clinical Cancer Research 6: 1322-1327 (2000), incorporated herein by reference).

As used herein, the term “preset limit” of relative DPD expression is a level of DPD expression above which the tumor has been found to exhibit resistance to 5-FU. Expression levels below this limit are believed to be identified in tumors sensitive to 5-FU. Relative DPD expression ranges from about 0.6 × 10 ⁻³ to less than about 2.5 × 10 ⁻³ (about 4.2-fold range) among tumors responding to 5-FU based chemotherapy in response to tumors. Relative DPD expression of tumors that do not respond to 5-FU based therapy is about 0.2 × 10 ⁻³ to about 16 × 10 ⁻³ (range about 80-fold). If the relative DPD expression is at least about 2.0 × 10 ⁻³ , preferably at least about 2.5 × 10 ⁻³ , the tumor generally does not respond to 5-FU treatment. These values allow to determine whether the "uncalibrated relative DPD expression" of a particular sample falls above or below the "preset threshold" level. The threshold level of the corrected relative DPD expression level is about 2.0 × 10 ⁻³ to about 2.5 × 10 ⁻³ .

The methods of the invention can be applied to a wide range of tissues and tumor types, can be used for the evaluation of treatment in patients, and in a range of cancers including breast cancer, head and neck cancer, lung cancer, esophageal cancer, colorectal cancer and other cancers. It can be used as a diagnostic or prognostic tool. Preferably, the present invention is applied to the prognosis of bronchoalveolar cancer, small bowel cancer or colon cancer.

From measuring the amount of DPD mRNA expressed in the tumor, one skilled in the art can determine the prognosis regarding the clinical resistance of the tumor to 5-FU based chemotherapy. "5-FU-based chemotherapy" refers to 5-FU, its derivatives alone or other chemotherapeutic agents, such as leuboline or DPD inhibitors such as uracil, 5-ethynyluracil, bromovinyluracil, thymine, benzyloxy Administration with benzyluracil (BBU) or 5-chloro-2,4-dihydroxypyridine. In addition, simultaneous administration of the 5′-deoxy-cytidine derivative of Formula I with 5-FU or a derivative thereof is selectively directed to tumor tissue as compared to the combination of 5-FU or a derivative thereof and 5-Ethynyluracil, a DPD inhibitor. It has been shown to significantly improve the delivery of chemotherapeutic agents and also to exhibit significantly improved antitumor activity in human cancer xenograft models.

Having described the present invention as described above, the practice of the present invention will be illustrated by the following examples. Those skilled in the art will recognize that the materials and methods used in the illustrative embodiments may be modified in many ways. It is noted that such modifications are included within the scope of the present invention.

Example 1

Isolation of RNA from FPE Tissue

RNA was extracted from paraffin embedded tissue according to the following general procedure:

A. Deparaffinization and Hydration of Flakes

(1) A portion of about 10 μM flakes was placed in a 1.5 ml plastic centrifuge tube.

(2) 600 μl xylene was added and the mixture was vigorously shaken for about 10 minutes at room temperature (approximately 20 ° C. to 25 ° C.).

(3) Samples were centrifuged using a bench top centrifuge at maximum speed (about 10-200,000 xg) for about 7 minutes at room temperature.

(4) Steps (2) and (3) were repeated until most paraffins were dissolved. Depending on the amount of paraffin contained in the original sample portion, it will usually need to be repeated two or more times.

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

(6) The tube was centrifuged for about 7 minutes as in step (3). The supernatant was decanted and discarded. The pellet turned white.

(7) Steps (5) and (6) were repeated successively using a more diluted ethanol solution: first about 95% ethanol, then about 80% ethanol, finally about 70% ethanol.

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

B. RNA Separation Using Phenol-Chloroform

(1) 400 μl guanidine isothiocyanate containing 8 μl 0.5% sarcosine and dithiothritol was added.

(2) The sample was then homogenized using a tissue homogenizer (Ultra-Turax; Ica-Works, Wilmington, NC) for about 2 to 3 minutes, whereby the homogenization slowed down. It was carried out gradually increasing from (speed 1) to high speed (speed 5).

(3) The sample was then heated at about 95 ° C. for about 5-20 minutes. Preferably, the cap of the tube containing the sample is drilled with fine gauge needles and then heated. Alternatively, the cap may be secured with a plastic clamp or laboratory film.

(4) The sample was then prepared by mixing 50 μl of 2 M sodium acetate and 18 ml of phenol and 3.6 ml of a 1:49 isoamyl alcohol: chloroform solution at pH 4.0 to phenol / chloroform / isoamyl alcohol (10: 1.93: 0.036) was extracted using 600 μl. The solution was vigorously shaken for about 10 seconds and cooled on ice for about 15 minutes.

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

(6) RNA was precipitated at −20 ° C. for 30 minutes using about 10 μl glycogen and 400 μl isopropanol.

(7) the RNA was pelleted for about 7 minutes at maximum speed in a benchtop centrifuge; The supernatant is decanted and discarded; The pellet was washed with about 500 μl of about 70% to 75% ethanol.

(8) The sample was centrifuged again for 7 minutes at maximum speed. The supernatant was decanted and the pellets were air dried. The pellet was then dissolved in a suitable buffer (eg 50 p. 5 mM tris chloride, pH 8.0) for later experiments.

Example 2

mRNA reverse transcription and PCR

Reverse transcription:

RNA was isolated from microincised or unmicroinformed formalin fixed paraffin embedded (FPE) tissue as described in Example 1 and US Application 09 / 469,338 (December 20, 1999; incorporated herein by reference). After precipitation and centrifugation with ethanol, the RNA pellet was dissolved in 50 μl of 5 mM Tris / Cl at pH 8.0. The resulting RNA was reverse transcribed using random hexamers and Life Technologies' M-MLV (CAT # 28025-02). The reverse transcription was performed by mixing 25 μl RNA solution with 25.5 μl “reverse transcription mixture” (see below). The reaction was 8 min at 26 ° C. (to bind random hexamers to RNA), 45 min at 42 ° C. (for M-MLV reverse transcription enzyme reaction) and 5 min at 95 ° C. (for thermal inactivation of DNAse). While placed in a thermal cycler.

"Reverse transcription mixture" is 10 μl of 5 × buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl ₂ ), random hexamer (50 OD, dissolved in 550 μl of 10 mM Tris-HCl pH 7.5). 0.5 μl, 10 mM dNTP (dATP, dGTP, dCTP and dTTP) 5 μl, 0.1 M DTT 5 μl, BSA (3 mg / ml in 10 mM Tris-HCl pH 7.5) 1.25 μl, RNA guard 24,800 U / ml (RNAse Inhibitor) (Amersham Pharmacia Pig # 27-0816) and 2.5 μl of MMLV 200 U / μl (Life Tech Cat # 28025-02).

Final concentrations of reaction components are as follows: 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 1.0 mM dNTP, 1.0 mM DTT, 0.00375 mg / ml BSA, 0.62 U / μL RNA Guard and 10 U / Μl MMLV.

PCR Quantification of mRNA Expression:

Quantification of DPD cDNA and internal control genes or housekeeping genes (ie β-actin as described in Eads et al., Cancer Research 59: 2302-2306 (1999)) is described in Heid et al., Fluorescence-based real-time detection method [ABI PRISM 7700 or 7900 sequence detection system [Tagman®] as described in Genome Res 6: 986-994 (1996) and Gibson et al., Genome Res 6: 995-1001 (1996)]. ], Applied Biosystems, Foster City, Calif., USA. Schematically, this method used a double labeled fluorescence oligonucleotide probe (Takman probe) that specifically annealed in template amplicons spanning the forward and reverse primers. Laser stimulation in the capped wells containing the reaction mixture can be cleaved by the 5 'to 3' nuclease activity of DNA polymerase during PCR elongation, which causes the prop to cause release of the 5 'reporter dye (6FAM). Until, triggered the release of the 3 'quencher dye (TAMRA). Thus, the generation of amplicons is detected by a tackman CCD (charge-coupled device) detection camera and the amount of signal generated in the limit cycle in the pure logarithm of the PCR reaction reflecting the starting copy number of a given sequence. Causes the emission of fluorescent signals. Tagman probes for the oligonucleotide primer pair DPD1 (DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)) are DPD-108Tc (SEQ ID NO: 9). Tagman probes for oligonucleotide primer pairs DPD2 (DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)) are DPD-2p-1154Tc (SEQ ID NO: 10). Tagman probes for oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) are DPD3A-71Tc (SEQ ID NO: 11). Tagman probes for oligonucleotide primer pairs DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) are DPD3b-685Tc (SEQ ID NO: 12).

PCR reactions were described in the 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), T _m = 58 ° C. and DPD3b-736R (SEQ ID NO: 8), T _m = 60 ° C.]; Or oligonucleotide primer DPD3A (DPD3a-51F (SEQ ID NO: 1), T _m = 59 ° C and DPD3a-134R (SEQ ID NO: 2), T _m = 59 ° C). Each PCR reaction mixture contains not only 0.5 μl of reverse transcription reaction containing cDNA, but also oligonucleotide primers from only one pair (DPD1, DPD2, DPD3B or DPD3A) of 600 nM each, 200 nM of the corresponding tackman probe ( For DPD1, DPD2, DPD3B or DPD3A), 1 U Tagman Buffer A containing 5 U Amplicon Gold Polymerase, 200 μM of each dATP, dCTP, dGTP, 400 μM TTP, 5.5 mM MgCl ₂ and Reference Dye Containing up to a final volume of 25 μl (all reagents are available from Applied Biosystems, Foster City, CA). Cycling conditions were 45 cycles for 10 minutes at 95 ° C., followed by 15 seconds at 95 ° C. and 1 minute at 60 ° C.

Example 3

PD expression in FPE tumor samples

Oligonucleotide primer pairs DPD3A [DPD3a-51F (SEQ ID NO: 1) and DPD3a-13R (SEQ ID NO: 2)] and DPD3B [DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)] were obtained from paraffin embedded tissue. Strong and reproducible DPD gene expression by RT-PCR using extracted RNA was enabled (see FIG. 1). In addition, oligonucleotide primers DPD3A [DPD3a-51F (SEQ ID NO: 1) and DPD3a-13R (SEQ ID NO: 2)) also significantly increased the sensitivity of DPD gene expression by RT-PCR in new frozen tissues (see Figure 2). . RT-PCR was performed using the ABI Prism 7700 Sequence Detection System (Tagman) as described in Example 2 above.

30 cycles were used in the PCR reaction. Each cycle consists of denaturation at 96 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and elongation at 72 ° C. for 2 minutes. The amplification products using oligonucleotide primer pairs DPD3A [DPD3a-51F (SEQ ID NO: 1) and DPD3a-13R (SEQ ID NO: 2)] were 84 base pairs in length. Corresponding to the region of DPD cDNA that spans a portion of the 5 'untranslated region (UTR) and develops into exon 1. Amplification products using oligonucleotide primer pairs DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) were 86 base pairs in length. The amplification product corresponded to the region of DPD cDNA corresponding to exon 6.

Oligonucleotide primer pairs DPD3A [DPD3a-51F (SEQ ID NO: 1) and DPD3a-13R (SEQ ID NO: 2)] and DPD3B [DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)] are 10 different FPEs. Their ability to amplify DPD mRNA from tissue samples was compared to other existing primer sets. Samples # 1-5 and # 8-10 are from a colon tumor biopsy, sample # 6 is from a bronchoalveolar tumor biopsy, and sample # 7 is from a small intestine tumor biopsy. Other oligonucleotide primer pairs used were 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)] .

Oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-13R (SEQ ID NO: 2)) are most effective for accurately identifying DPD levels in various samples. The oligonucleotide primer pairs DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) were also effective but did not provide a strong signal. The results are illustrated in FIG.

Example 4

Determination of Uncalibrated Gene Expression (UGE) for DPD

Two pairs of parallel reactions were performed, namely a "test" reaction and a "calibration" reaction. The DPD amplification reaction and the β-actin internal control amplification reaction are the test reactions. Separate β-actin and DPD amplification reactions were performed on calibrator RNA and are called calibration reactions. Tag only mechanism has four different cycle threshold will be calculated (Ct) values: Ct _DPD and Ct _DPD and Ct _{β- actin} Ct from _{β- actin} from the test reactions and the correction response.

The difference in Ct values in the two reactions was determined by the formula:

ΔCt _test = Ct _DPD -Ct _β-actin (from the "test" response)

ΔCt _compensator = Ct _DPD -Ct _β-actin (from the "correction" response)

The next step involves a -ΔCt square of 2 according to the following equation:

2 ^-ΔCt _test (from the "test" response)

2 ^-ΔCt _compensator (from the "calibration" response)

Then, to obtain uncorrected gene expression for DPD from the Taqman instrument, the following calculations were made:

^Uncorrected gene expression (UGE) for DPD = 2 ^-ΔCt _test / 2 ^-ΔCt _corrector

Standardization of UGE using known values

The normalization calculation involves the multiplication of the UGE and DPD and the correction factor (K _DPD ) specific for the particular corrector RNA. The correction factor K _DPD can be determined using any internal control gene and any exactly pre-quantified corrector RNA. Said internal control gene β-actin and correctly pre-quantified corrector RNA, universal PE RNA; Preference is given to using Cat # 4307281, lot # 3617812014 (Applied Biosystems).

Standardization was performed by modifying the method described in User Guide # 2 provided by Applied Biosystems, a manufacturer of Tackman, and the ΔCt method as described above. To carry out this procedure, UGE of six different already known test tissues were analyzed for DPD expression using the Tagman technique as described above. Internal control gene β-actin and corrector RNA, universal PE RNA; Cat # 4307281, lot # 3617812014 (Applied Biosystems) was used.

The relative DPD expression levels (PVs) L7, L91, L121, L150, L220, and L164 of each sample already described in Salonga et al., Incorporated herein by reference, are divided by their corresponding tag only derived UGE and the non-average correction factor K Was calculated.

K _{specific mean} = PV / UGE

Next, all K values were averaged by DPD, Universal PE RNA; Cat # 4307281, lot # 361781204 Calibrator RNA and β-actin specific single K _DPD correction factors were determined.

Thus, uncalibrated gene expression data (UGE) and K _DPD specific correction factors derived from the Taqman device to measure corrected relative DPD expression in unknown tissue samples on a scale consistent with the already known preliminary Taqman DPD expression study. Multiplied by the same internal control gene and calibrator RNA.

Corrected relative DPD expression = UGE x K _DPD

K _DPD can be determined using any correctly pre-quantified calibrator RNA. Future sources of correctly pre-quantified RNA may be calibrated against known samples as described in the above methods, or the aforementioned universal PE RNA; Calibration can also be made for already calibrated calibrator RNA such as Cat # 4307281, lot # 361781204.

Example 5

DPD Expression in FPE Colorectal Tumor Samples

34 tumor samples from 34 patients with advanced colorectal cancer were analyzed using the method described above. All patients were treated with intravenous 5-FU / LV combination therapy as part of the Prospective Multicenter Europian 5-FU / CPT11 Crossover Trial V239. All patients were treated with an intravenous 5-FU 425 mg / m ² infusion over 5 consecutive days of infusion period and also with 20 mg / m ² of leuboline for 5 consecutive days. This therapy is called first or second line palliative therapy.

Nine of the patients (25.5%) responded to 5-FU / LV, with the response defined as any response including complete response, partial response and minimal response. Patients with progressive or stable disease were classified as non-responders (25 patients, 73.5%). Total mRNA was isolated from microanalyzed FPE pretreated tumor samples, and relative mRNA expression levels of DPD / β-actin were measured using quantitative PCR as described.

Average correction DPD:

Β-actin levels for the responded and unresponsive patient groups were 0.87 × 10 ⁻³ and 2.04 × 10 ⁻³ , respectively. The Mann-Whitney U test, which compares the rank of values within two independent sample sets, was used to compare the corrected relative DPD expression levels in the responding and non-responding patient groups. Relative DPD levels were significantly lower in the responding patient group compared to the non-responsive patient population (P = 0.02). The relationship between DPD mRNA expression and 5-FU / LV in these patients is shown in FIG. 4. As can be seen from these data, DPD expression is a prognostic factor of response to 5-FU based chemotherapy.

                                SEQUENCE LISTING <110> Danenberg, K. <120> METHOD OF DETERMINING DIHYDROPYRIMIDINE         DEHYDROGENASE GENE EXPRESSION <130> 11220/155 <140> 09 / 796,807 <141> 2001-03-02 <140> 09 / 842,111 <141> 2001-04-26 <140> 09 / 879,217 <141> 2001-06-13 <160> 12 FastSEQ for Windows Version 4.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 1 aggacgcaag gagggtttg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 2 gtccgccgag tccttactga 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 3 tcactggcag actcgagact gt 22 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 4 tggccgaagt ggaacaca 18 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 5 ctgcctttga ctgtgcaaca tc 22 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 6 attaacaaag ccttttctga agacgat 27 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 7 gaagcctatt ctgcaaagat tgc 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 8 gagtacccca atcgagccaa a 21 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 9 ccgccgagtc cttactgagc acagg 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 10 cacacggcga gctccacaac gtaga 25 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 11 cagtgcctac agtctcgagt ctgccagtg 29 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide Primer <400> 12 aaggaagcac aacttatact tgcaggccca g 31

Claims (32)

Oligonucleotide having the sequence of SEQ ID NO: 1. An oligonucleotide having the sequence of SEQ ID NO: 2. An oligonucleotide having the sequence of SEQ ID NO: 7. An oligonucleotide having the sequence of SEQ ID NO: 8. (i) an oligonucleotide primer pair consisting of an oligonucleotide having the sequence of SEQ ID NO: 1, and an oligonucleotide having the sequence of SEQ ID NO: 2, or (ii) dihydropyrimidine dehydrogenase (DPD) in tissue obtained from a patient comprising an oligonucleotide having a sequence of SEQ ID NO: 7 and an oligonucleotide primer pair consisting of an oligonucleotide having the sequence of SEQ ID NO: 8; ) Kit for detecting expression of a gene. (a) isolating mRNA from an in vitro tumor sample; (b) amplifying the mRNA using an oligonucleotide primer pair consisting of an oligonucleotide having the sequence of SEQ ID NO: 1 and an oligonucleotide having the sequence of SEQ ID NO: 2; And (c) comparing the amount of dihydropyrimidine dehydrogenase (DPD) mRNA obtained in step (b) with the amount of mRNA of the internal control gene; A method for determining the relative level of dihydropyrimidine dehydrogenase (DPD) gene expression in a tumor sample, comprising. The method of claim 6, wherein the ex vivo tumor sample is frozen. The method of claim 6, wherein the ex vivo tumor sample is fixed. The method of claim 8, wherein the ex vivo tumor sample is embedded in paraffin after fixation. The method of claim 8 or 9, wherein the RNA is isolated in the presence of an effective amount of chaotropic agent. 10. The method of any one of claims 6, 8, or 9, wherein the tumor sample comprises non-tumor tissue and tumor tissue. (a) isolating mRNA from an in vitro tumor sample; (b) amplifying the mRNA using an oligonucleotide primer pair consisting of an oligonucleotide having the sequence of SEQ ID NO: 1 and an oligonucleotide having the sequence of SEQ ID NO: 2 to obtain an amplified sample; (c) measuring the amount of dihydropyrimidine dehydrogenase (DPD) mRNA in the amplified sample; (d) comparing the amount of dihydropyrimidine dehydrogenase (DPD) mRNA in the amplified sample with a threshold level for predetermined DPD expression; And (e) predicting the sensitivity of the tumor to 5-fluorouracil-based chemotherapy based on the difference between the amount of DPD mRNA in the amplified sample and the threshold level for DPD gene expression Comprising, a method for predicting the sensitivity of a tumor to 5-fluorouracil-based chemotherapy. The method of claim 12, wherein the threshold level of predetermined DPD gene expression is measured relative to the expression level of an internal control gene. The method of claim 13, wherein said internal control gene is a β-actin gene. The method of claim 14, wherein the predetermined threshold level of DPD gene expression is 2.0-2.5 times the β-actin gene expression level.  The method of claim 12, wherein the ex vivo tumor sample is fixed and embedded. The method of claim 12, wherein said mRNA is isolated in the presence of an effective amount of a chaotropic agent. (a) isolating mRNA from an in vitro tumor sample; (b) amplifying said mRNA using an oligonucleotide primer pair consisting of an oligonucleotide having the sequence of SEQ ID NO: 7 and an oligonucleotide having the sequence of SEQ ID NO: 8; And (c) comparing the amount of mRNA obtained from step (b) with the amount of internal control mRNA A method for determining the relative level of dihydropyrimidine dehydrogenase (DPD) gene expression in a tumor sample, comprising. The method of claim 18, wherein the ex vivo tumor sample is frozen. The method of claim 18, wherein the ex vivo tumor sample is fixed. The method of claim 18, wherein the ex vivo tumor sample is fixed and embedded in paraffin. 22. The method of claim 20 or 21, wherein said mRNA is isolated in the presence of an effective amount of chaotropic agent. The method of claim 18, wherein the ex vivo tumor sample comprises non-tumor tissue and tumor tissue. (a) isolating mRNA from an in vitro tumor sample; (b) amplifying the mRNA using an oligonucleotide primer pair consisting of an oligonucleotide having the sequence of SEQ ID NO: 7 and an oligonucleotide having the sequence of SEQ ID NO: 8 to obtain an amplified sample; (c) measuring the amount of dihydropyrimidine dehydrogenase (DPD) mRNA in the amplified sample; (d) comparing the amount of dihydropyrimidine dehydrogenase (DPD) mRNA in the amplified sample with a threshold level for predetermined DPD expression; And (e) predicting the sensitivity of the tumor to 5-fluorouracil-based chemotherapy based on the difference between the amount of DPD mRNA in the amplified sample and the threshold level for DPD gene expression Comprising, a method for predicting the sensitivity of a tumor to 5-fluorouracil-based chemotherapy. The method of claim 24, wherein the threshold level of predetermined DPD gene expression is measured relative to the expression level of an internal control gene. The method of claim 25, wherein the internal control gene is a β-actin gene. The method of claim 24, wherein the threshold level of predetermined DPD gene expression is 2.0 to 2.5 times the β-actin gene expression level.  The method of claim 24, wherein the ex vivo tumor sample is fixed. The method of claim 24, wherein the ex vivo tumor sample is fixed and embedded in paraffin. The method of claim 24, wherein said mRNA is isolated in the presence of an effective amount of a chaotropic agent. The method of claim 6 or 18, wherein the tumor sample contains bronchoalveolar tumor tissue, small intestine tumor tissue, or colon tumor tissue. The method of claim 12 or 24, wherein the tumor sample contains bronchoalveolar tumor tissue, small intestine tumor tissue, or colon tumor tissue.

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