AU2002305573A1 - Method of determining a chemotherapeutic regimen based on glutathione-s-transferase pi expression - Google Patents

Description

METHOD OF DETERMINING A CHEMOTHERAPEUTIC

REGIMEN BASED ON GLUTATHIONE-S-TRANSFERASE Pi

EXPRESSION

FIELD OF THE INVENTION

[001] The present invention relates to prognostic methods which are useful in

medicine, particularly cancer chemotherapy. More particularly, the invention

relates to assessment of tumor cell gene expression in a patient. The resistance of tumor cells to chemotherapeutic agents that target DNA, especially agents that

damage DNA in the manner of platmating agents is assayed by examining the mRNA expressed from genes involved in DNA repair in humans.

BACKGROUND OF THE INVENTION

[002] Cancer arises when a normal cell undergoes neoplastic transformation and

becomes a malignant cell. Transformed (malignant) cells escape normal physiologic controls specifying cell phenotype and restraining cell proliferation. Transformed

cells in an individual's body thus proliferate, forming a tumor. When a tumor is

found, the clinical objective is to destroy malignant cells selectively while mitigating

any harm caused to normal cells in the individual undergoing treatment.

[003] Chemotherapy is based on the use of drugs that are selectively toxic

(cytotoxic) to cancer cells. Several general classes of chemotherapeutic drugs have

been developed, including drugs that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes. These generally are referred to as antimetabolite drugs. Other classes of chemotherapeutic drugs inflict damage on

cellular DNA. Drugs of these classes generally are referred to as genotoxic.

Susceptibility of an individual neoplasm to a desired chemotherapeutic drug or

'combination of drugs often, however, can be accurately assessed only after a trial

period of treatment. The time invested in an unsuccessful trial period poses a

significant risk in the clinical management of aggressive malignancies.

[004] The repair of damage to cellular DNA is an important biological process

carried out by a cell's enzymatic DNA repair machinery. Unrepaired lesions in a

cell's genome can impede DNA replication, impair the replication fidelity of newly synthesized DNA and/or hinder the expression of genes needed for cell survival.

Thus, genotoxic drugs generally are considered more toxic to actively dividing cells

that engage in DNA synthesis than to quiescent, nondividing cells. Normal cells of many body tissues are quiescent and commit infrequently to re-enter the cell cycle

and divide. Greater time between rounds of cell division generally is afforded for the

repair of DNA damage in normal cells inflicted by chemotherapeutic genotoxins. As a result, some selectivity is achieved for the killing of cancer cells. Many treatment regimens reflect attempts to improve selectivity for cancer cells by coadministering chemotherapeutic drugs belonging to two or more of these general classes.

[005] Because effective chemotherapy in solid tumors usually requires a

combination of agents, the identification and quantification of determinants of

resistance or sensitivity to each single drug has become an important tool to design individual combination chemotherapy. [006] Two widely used genotoxic anticancer drugs that have been shown to

damage cellular DNA are cisplatin (DDP) and carboplatin. Cisplatin and/or

carboplatin currently are used in the treatment of selected, diverse neoplasms of

epithelial and mesenchymal origin, including carcinomas and sarcomas of the

respiratory, gastrointestinal and reproductive tracts, of the central nervous system, and of squamous origin in the head and neck. Cisplatin in combination with other

agents is currently preferred for the management of testicular carcinoma, and in many instances produces a lasting remission. (Loehrer et al., 1984,100 Ann. Int.

Med. 704). Cisplatin (DDP) disrupts DNA structure through formation of intrastrand adducts. Resistance to platinum agents such as DDP has been attributed

to enhanced tolerance to platinum adducts, decreased drug accumulation, or

enhanced DNA repair. Although resistance to DDP is multifactoral, alterations in

DNA repair mechanisms probably play a significant role.

[007] The glutathione-S-transferase (GST) family of proteins is involved in

detoxification of cytotoxic drugs. By catalyzing the conjugation of toxic and

carcinogenic electrophilic molecules with glutathione the GST enzymes protect

cellular macromolecules from damage (Boyer et al., Preparation, characterization and properties of glutathione S-transferases. In: Zakim D, Vessey D (eds.)

Biochemical Pharmacology and Toxicology. New York, NY: John Wiley and Sons,

1985.). A certain isomeric type of these proteins, the glutathione S-transferase Pi

(GST-pi, also to be interchangeably refered to as GSTP1 or GST-π herein) is widely

expressed in human epithelial tissues and has been demonstrated to be over- expressed in several tumors (Terrier et al., Am J Pathol 1990; 137: 845-853;

Moscow et al, Cancer Res 1989; 49: 1422-1428). Increased GST-pi levels have been found in drug resistant tumors, although the exact mechanism remains unclear

(Tsuchida et al., Crit Rev Biochem Mol Biol 1992; 27: 337-384). Previous studies

have suggested that low expression of GST protein (not mRNA) is associated with

response to platinum-based chemotherapy (Nishimura et al., Cancer. Clin Cancer

Res 1996; 2:1859-1865; Tominaga, et al., Am. J. Gastro. 94:1664-1668, 1999; Kase,

et al., Acta Cytologia. 42: 1397-1402, 1998). However, these studies did not measure quantitative gene expression, but used a semi-quantitative

immunohistochemical staining method to measure protein levels. However, quantitative GST-pi gene expression measurements are needed to achieve a very

effective prognostication.

[008] Most pathological samples are routinely fixed and paraffin-embedded (FPE)

to allow for histological analysis and subsequent archival storage. Thus, most

biopsy tissue samples are not useful for analysis of gene expression because such studies require a high integrity of RNA so that an accurate measure of gene

expression can be made. Currently, gene expression levels can be only qualitatively monitored in such fixed and embedded samples by using immunohistochemical

staining to monitor protein expression levels.

[009] Until now, quantitative gene expression studies including those of GST-pi

expression have been limited to reverse transcriptase polymerase chain reaction

(RT-PCR) amplification of RNA from fresh or frozen tissue.

[010] The use of frozen tissue by health care professionals poses substantial

inconveniences. Rapid biopsy delivery to avoid tissue and subsequent mRNA degradation is the primary concern when planning any RNA-based quantitative

genetic marker assay. The health care professional performing the biopsy, must hastily deliver the tissue sample to a facility equipped to perform an RNA extraction

protocol immediately upon tissue sample receipt. If no such facility is available, the

clinician must promptly freeze the sample in order to prevent mRNA degradation.

In order for the diagnostic facility to perform a useful RNA extraction protocol prior

to tissue and RNA degradation, the tissue sample must remain frozen until it reaches

the diagnostic facility, however far away that may be. Maintenance of frozen tissue

integrity during transport using specialized couriers equipped with liquid nitrogen and dry ice, comes only at a great expense.

[011] Routine biopsies generally comprise a heterogenous mix of stromal and

tumorous tissue. Unlike with fresh or frozen tissue, FPE biopsy tissue samples are

readily microdissected and separated into stromal and tumor tissue and therefore,

offer andvantage over the use of fresh or frozen tissue. However, isolation of RNA

from fixed tissue, and especially fixed and paraffin embedded tissue, results in

highly degraded RNA, which is generally not applicable to gene expression studies.

[012] A number of techniques exist for the purification of RNA from biological

samples, but none is reliable for isolation of RNA from FPE samples. For example, Chomczynski (U.S. Pat. No. 5,346,994) describes a method for purifying RNA from tissues based on a liquid phase separation using phenol and guanidine

isothiocyanate. A biological sample is homogenized in an aqueous solution of

phenol and guanidine isothiocyanate and the homogenate thereafter mixed with

chloroform. Following centrifugation, the homogenate separates into an organic

phase, an interphase and an aqueous phase. Proteins are sequestered in the organic

phase, DNA in the interphase, and RNA in the aqueous phase. RNA can be precipitated from the aqueous phase. Unfortunately, this method is not applicable to fixed and paraffin-embedded (FPE) tissue samples.

[013] Other known techniques for isolating RNA typically utilize either guanidine

salts or phenol extraction, as described for example in Sambrook, J. et al, (1989) at

pp. 7.3-7.24, and in Ausubel, F. M. et al, (1994) at pp. 4.0.3-4.4.7. Again, none of

the known methods provides reproducible quantitative results in the isolation of

RNA from paraffin-embedded tissue samples.

[014] Techniques for the isolation of RNA from paraffin-embedded tissues are thus

particularly needed for the study of gene expression in tumor tissues, since

expression levels of certain receptors or enzymes can be used to determine the likelihood of success of a particular treatment.

[015] There is a need for a method of quantifying GST-pi mRNA from paraffinized

tissue in order to provide an early prognosis for proposed genotoxic cancer therapies.

As a result, there has been a concerted yet unsuccessful effort in the art to obtain a quantification of GST-pi expression in fixed and paraffinized (FPE) tissue.

Accordingly, it is the object of the invention to provide a method for assessing GST- pi levels in tissues fixed and paraffin-embedded (FPE) and prognosticate the probable resistance of a patient's tumor to treatment with DNA damaging agents,

creating the type of lesions in DNA that are created by DNA platinating agents, by

examination of the amount of GST-pi mRNA in a patient's tumor cells and comparing it to

a predetermined threshold expression level. SUMMARY OF THE INVENTION [016] In one aspect of the invention there is provided a method for assessing levels

of expression of GST-pi mRNA obtained from fixed and paraffin-embedded (FPE)

fixed and paraffin-embedded (FPE) tumor cells.

[017] In another aspect of the invention there is provided a method of quantifying

the amount of GST-pi mRNA expression relative to an internal control from a fixed and paraffin-embedded (FPE) tissue sample. This method includes isolation of total

mRNA from said sample and determining the quantity of GST-pi mRNA relative to the quantity of an internal control gene's mRNA.

[018] In an embodiment of this aspect of the invention, there are provided

oligonucleotide primers having the sequence of GST-F (SEQ ID NO: 1) or GST-R

(SEQ ID NO:2) and sequences substantially identical thereto. The invention also

provides for oligonucleotide primers having a sequence that hybridizes to SEQ ID

NO: 1 or SEQ ID NO:2 or their complements under stringent conditions.

[019] In yet another aspect of the invention there is provided a method for

determining a chemotherapeutic regimen for a patient, comprising isolating RNA from a fixed and paraffin-embedded (FPE) tumor sample; determining a gene

expression level of GST-pi in the sample; comparing the GST-pi gene expression

levels in the sample with a predeterimined threshold level for the GST-pi gene; and

determining a chemotherapeutic regimen based on results of the comparison of the

GST-pi gene expression level with the predetermined threshold level.

[020] The invention further relates to a method of normalizing the uncorrected

gene expression (UGE) of GST-pi relative to an internal control gene in a tissue sample analyzed using TaqMan® technology to known GST-pi expression levels relative to an internal control from samples analyzed by pre-TaqMan® technology.

BRIEF DESCRIPTION OF THE DRAWING

[021] Figure 1 shows and association between survival and GST-pi corrected

relative mRNA expression in patients with esophagocardiac adenocarcinoma treated

with 5-FU and cisplatin. Patients with GST-pi values above the median/threshold value had a survival advantage compared to those with patients with values below the median/threshold. Censored values are denoted by a tick.

[022] Figure 2 is a graph showing survival analysis confined to patients with TNM

Stage II esophagocardiac adenocarcinoma

[023] Figure 3 is a graph showing survival analysis confined to patients with Stage

IN esophagocardiac adenocarcinoma

[024] Figure 4 is a chart illustrating how to calculate GST-pi expression relative to

an internal control gene. The chart contains data obtained with two test samples, (unknowns 1 and 2), and illustrates how to determine the uncorrected gene

expression data (UGE). The chart also illustrates how to normalize UGE generated

by the TaqMan® instrument with known relative GST-pi values determined by pre- TaqMan® technology. This is accomplished by multiplying UGE to a correction

factor K_GST__pi. The internal control gene in the figure is β-actin and the calibrator

RΝA is Human Liver Total RΝA (Stratagene, Cat. #735017).

[025], Figure 5 shows the oligonucleotide primers used in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[026] The present invention resides in part in the finding that the amount of GST-pi

mRNA is correlated with increased sensitivity to DNA platinating agents. Tumors

expressing high levels of GST-pi mRNA are considered likely to be sensitive to

platinum-based chemotherapy. Conversely, those tumors expressing low amounts of GST-pi mRNA are likely to be insensitive to platinum-based chemotherapy. A

patient's relative expression of tumor GST-pi mRNA is judged by comparing it to a predetermined threshold expression level. Such sensitivity or lack thereof to DNA

platinating agents is determined by a patient's survivability.

[027] The invention relates to a method of quantifying the amount of GST-pi

mRNA expression in fixed and paraffin-embedded (FPE) tissue relative to gene

expression of an internal control. The present inventors have developed

oligonucleotide primers that allow accurate assessment of GST-pi expression in

tissues that have been fixed and embedded. The invention oligonucleotide primers,

GST-F (SEQ ID NO: 1), GST-R (SEQ ID NO: 2), or oligonucleotide primers substantially identical thereto, preferably are used together with RNA extracted from fixed and paraffin embedded (FPE) tumor samples. This measurement of GST-pi

gene expression may then be used for prognosis of platinum-based chemotherapy.

[028] This embodiment of the invention involves first, a method for reliable

extraction of RNA from an FPE sample and second, determination of the content of

GST-pi mRNA in the sample by using a pair of oligonucleotide primers, preferably

oHgionucleotide primer pair GST-F (SEQ ID NO: 1) and GST-R (SEQ ID NO: 2), or oligonucleotides substantially identical thereto, for carrying out reverse transcriptase

polymerase chain reaction. RNA is extracted from the FPE cells by any of the methods for mRNA isolation from such samples as described in US Patent

Application No. 09/469,338, filed December 20, 1999, now U.S. Patent Number

6,248,535, and is hereby incorporated by reference in its entirety.

[029] The present method can be applied to any type of tissue from a patient. For

examination of sensitivity of tumor tissue, it is preferable to examine the tumor

tissue, h a preferred embodiment, a portion of normal tissue from the patient from which the tumor is obtained, is also examined.

[030] The methods of the present invention can be applied over a wide range of

tumor types. This allows for the preparation of individual "tumor expression profiles" whereby expression levels of GST-pi are determined in individual patient

samples and response to various chemotherapeutics is predicted. Preferably, the

methods of the invention are applied to solid tumors, most preferably

esophogocardiac rumors. For application of some embodiments of the invention to

particular tumor types, it is preferable to confirm the relationship of GST-pi gene expression levels to clinical resistance by compiling a data-set that enables

correlation of a particular GST-pi expression and clinical resistance to platinum- based chemotherapy.

[031] A "predetermined threshold level", as defined herein, is a level of GST-pi

expression above which it has been found that tumors are likely to be sensitive to a

platinum-based chemotherapeutic regimen. Expression levels below this threshold

level are likely to be found in tumors insensitive to platinum-based

chemotherapeutic regimen. The range of corrected relative expression of GST-pi,

expressed as a ratio of GST-pi : β-actin, among tumors responding to a platinum-

based chemotherapeutic regimen is more than about 1.0 x 10^"3. Tumors that do not respond to a platinum-based chemotherapeutic regimen have relative expression of

GST-pi : β-actin ratio below about 1.0 x 10^"3. Figure 1. However, the present

invention is not limited to the use of β-actin as an internal control gene.

[032] In performing the method of this embodiment of the present invention, tumor

cells are preferably isolated from the patient. Solid or lymphoid tumors or portions

thereof are surgically resected from the patient or obtained by routine biopsy. RNA

isolated from frozen or fresh samples is extracted from the cells by any of the methods typical in the art, for example, Sambrook, Fischer and Maniatis, Molecular

Cloning, a laboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, New York, (1989). Preferably, care is taken to avoid degradation of the RNA during the

extraction process.

[033] However, tissue obtained from the patient after biopsy is often fixed, usually

by formalin (formaldehyde) or gluteraldehyde, for example, or by alcohol

immersion. Fixed biological samples are often dehydrated and embedded in paraffin or other solid supports known to those of skill in the art. Non-embedded, fixed tissue

may also be used in the present methods. Such solid supports are envisioned to be removable with organic solvents for example, allowing for subsequent rehydration of preserved tissue.

[034] RNA is extracted from the FPE cells by any of the methods as described in

US Patent Application No. 09/469,338, filed December 20, 1999, which is hereby

incorporated by reference in its entirety. Fixed and paraffin-embedded (FPE) tissue

samples as described herein refers to storable or archival tissue samples. RNA may be isolated from an archival pathological sample or biopsy sample which is first deparaffinized. An exemplary deparaffmization method involves washing the

paraffinized sample with an organic solvent, such as xylene, for example.

Deparaffinized samples can be rehydrated with an aqueous solution of a lower

alcohol. Suitable lower alcohols, for example include, methanol, ethanol, propanols,

and butanols. Deparaffinized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example.

Alternatively, the sample is simultaneously deparaffinized and rehydrated. RNA is then extracted from the sample.

[035] For RNA extraction, the fixed or fixed and deparaffinized samples can be

homogenized using mechanical, sonic or other means of homogenization.

Rehydrated samples may be homogenized in a solution comprising a chaotropic

agent, such as guanidinium thiocyanate (also sold as guanidinium isothiocyanate).

Homogenized samples are heated to a temperature in the range of about 50 to about 100 °C in a chaotropic solution, which contains an effective amount of a chaotropic

agent, such as a guanidinium compound. A preferred chaotropic agent is guanidinium thiocyanate.

[036] An "effective concentration of chaotropic agent" is chosen such that at an

RNA is purified from a paraffin-embedded sample in an amount of greater than

about 10-fold that isolated in the absence of a chaotropic agent. Chaotropic agents

include: guanidinium compounds, urea, foπnamide, potassium iodiode, potassium

thiocyantate and similar compounds. The preferred chaotropic agent for the methods

of the invention is a guanidinium compound, such as guanidinium isothiocyanate

(also sold as guanidinium thiocyanate) and guanidinium hydrochloride. Many anionic counterions are useful, and one of skill in the art can prepare many guanidinium salts with such appropriate anions. The effective concentration of

guanidinium solution used in the invention generally has a concentration in the range

of about 1 to about 5M with a preferred value of about 4M. If RNA is already in

solution, the guanidinium solution may be of higher concentration such that the final

concentration achieved in the sample is in the range of about 1 to about 5M. The

guanidinium solution also is preferably buffered to a pH of about 3 to about 6, more

preferably about 4, with a suitable biochemical buffer such as Tris-Cl. The chaotropic solution may also contain reducing agents, such as dithiothreitol (DTT) and β-mercaptoethanol (BME). The chaotropic solution may also contain RNAse

inhibitors.

[037] Homogenized samples may be heated to a temperature in the range of about

50 to about 100 °C in a chaotropic solution, which contains an effective amount of a

chaotropic agent, such as a guanidinium compound. A preferred chaotropic agent is guanidinium thiocyanate.

[038] RNA is then recovered from the solution by, for example, phenol chloroform

extraction, ion exchange chromatography or size-exclusion chromatography. RNA

may then be further purified using the techniques of extraction, electrophoresis, chromatography, precipitation or other suitable techniques.

[039] The quantification of GST-pi mRNA from purified total mRNA from fresh,

frozen or fixed is preferably carried out using reverse-transcriptase polymerase chain

reaction (RT-PCR) methods common in the art, for example. Other methods of

quantifying of GST-pi mRNA include for example, the use of molecular beacons and other labeled probes useful in multiplex PCR. Additionally, the present invention envisages the quantification of GST-pi mRNA via use of PCR-free systems employing, for example fluorescent labeled probes similar to those of the Invader®

Assay (Third Wave Technologies, Inc.). Most preferably, quantification of GST-pi

cDNA and an internal control or house keeping gene (e.g. β-actin) is done using a

fluorescence based real-time detection method (ABI PRISM 7700 or 7900 Sequence

Detection System [TaqMan®], Applied Biosystems, Foster City, CA.) or similar

system as described by Heid et al., (Genome Res 1996;6:986-994) and Gibson et α .(Genome Res 1996;6:995-1001). The output of the ABI 7700 (TaqMan® Instrument) is expressed in Ct's or "cycle thresholds". With the TaqMan® system, a

highly expressed gene having a higher number of target molecules in a sample

generates a signal with fewer PCR cycles (lower Ct) than a gene of lower relative

expression with fewer target molecules (higher Ct).

[040] As used herein, a "house keeping" gene or "internal control" is meant to

include any constitutively or globally expressed gene whose presence enables an

assessment of GST-pi mRNA levels. Such an assessment comprises a determination

of the overall constitutive level of gene transcription and a control for variations in RNA recovery. "House-keeping" genes or "internal controls" can include, but are

not limited to the cyclophilin gene, β-actin gene, the transferrin receptor gene,

GAPDH gene, and the like. Most preferably, the internal control gene is β-actin

gene as described by Eads et al, Cancer Research 1999; 59:2302-2306.

[041] A control for variations in RNA recovery requires the use of "calibrator

RNA." The "calibrator RNA" is intended to be any available source of accurately pre-quantified control RNA. Preferably, Adult Colon, Disease Human Total RNA,

(Cat. No. #735263) from Stratagene, is used. [042] "Uncorrected Gene Expression (UGE)" as used herein refers to the numeric

output of GST-pi expression relative to an internal control gene generated by the

TaqMan® instrument. The equation used to determine UGE is shown in Example 3,

and illustrated with sample calculations in Figure 4.

[043] A further aspect of this invention provides a method to normalize

uncorrected gene expression (UGE) values acquired from the TaqMan® instrument with "known relative gene expression" values derived from non-TaqMan®

technology. Preferably, the known non-TaqMan® derived relative GST-pi : β-actin

expression values are normalized with TaqMan® derived GST-pi UGE values from a tissue sample.

[044] "Corrected Relative GST-pi Expression" as used herein refers to normalized

GST-pi expression whereby UGE is multiplied with a GST-pi specific correction

factor (K_GST__pl), resulting in a value that can be compared to a known range of GST-pi expression levels relative to an internal control gene. Example 3 and Figure 4

illustrate these calculations in detail. These numerical values allow the determination of whether or not the "Corrected Relative GST-pi Expression" of a

particular sample falls above or below the "predetermined threshold" level. The

predetermined threshold level of Corrected Relative GST-pi Expression to β-actin

level is about 1.0 x 10^"3. K_{GST i} specific for GST-pi, the internal control β-actin and

calibrator Adult Colon, Disease Human Total RNA, (Cat. No. #735263) from

Stratagene, is 7.28 x 10^-3.

[045] "Known relative gene expression" values are derived from previously

analyzed tissue samples and are based on the ratio of the RT-PCR signal of a target gene to a constitutively expressed internal control gene (e.g. β-Actin, GAPDH, etc.).

Preferably such tissue samples are formalin fixed and paraffin-embedded (FPE)

samples and RNA is extracted from them according to the protocol described in

Example 1 and in US Patent Application No. 09/469,338, filed December 20, 1999,

now U.S. Patent No. 6,248,535, which is hereby incorporated by reference in its

entirety. To quantify gene expression relative to an internal control standard

quantitative RT-PCR technology known in the art is used. Pre-TaqMan® technology PCR reactions are run for a fixed number of cycles (i.e., 30) and

endpoint values are reported for each sample. These values are then reported as a

ratio of GST-pi expression to β-actin expression. See U.S. Patent No. 5,705,336 to

Reed et al.

[046] K_Gsr • ^maY ^De determined for an internal control gene other than β-actin

and/or a calibrator RNA different than Adult Colon, Disease Human Total RNA,

(Cat. No. #735263) from Stratagene. To do so, one must calibrate both the internal

control gene and the calibrator RNA to tissue samples for which GST-pi expression levels relative to that particular internal control gene have already been determined (i.e., "known relative gene expression"). Preferably such tissue samples are formalin

fixed and paraffin-embedded (FPE) samples and RNA is extracted from them

according to the protocol described in Example 1 and in US Patent Application No.

09/469,338, filed December 20, 1999, which is hereby incorporated by reference in

its entirety. Such a determination can be made using standard pre-TaqMan®,

quantitative RT-PCR techniques well known in the art. Upon such a determination,

such samples have "known relative gene expression" levels of GST-pi useful in the determining a new K_GST__pi specific for the new internal control and/or calibrator RNA

as described in Example 3.

[047] The methods of the invention are applicable to a wide range of tissue and

tumor types and so can be used for assessment of clinical treatment of a patient and

as a diagnostic or prognostic tool for a range of cancers including breast, head and

neck, lung, esophageal, colorectal, and others. In a preferred embodiment, the

present methods are applied to prognosis of esophagocardiac adenocarcinoma.

[048] Pre-chemotherapy treatment tumor biopsies are usually available only as

fixed paraffin embedded (FPE) tissues, generally containing only a very small amount of heterogeneous tissue. Such FPE samples are readily amenable to

microdissection, so that GST-pi gene expression may be determined in tumor tissue

uncontaminated with stromal tissue. Additionally, comparisons can be made

between stromal and tumor tissue within a biopsy tissue sample, since such samples

often contain both types of tissues.

[049] Generally, any oligonucleotide pair that flanks a region of GST-pi gene may

be used to carry out the methods of the invention. Primers hybridizing under stringent conditions to a region of the GST-pi gene for use in the present invention will amplify a product between 20-1000 base pairs, preferably 50-100 base pairs,

most preferably less than 100 base pairs.

[050] The invention provides specific oligonucleotide primers pairs and

oligonucleotide primers substantially identical thereto, that allow particularly accurate assessment of GST-pi expression in FPE tissues. Preferable are

oligonucleotide primers, GST-F (SEQ ID NO: 1) and GST-R (SEQ ID NO: 2), (also referred to herein as the oligonucleotide primer pair GST) and oligonucleotide primers substantially identical thereto. The oliogonucleotide primers GST-F (SEQ

ID NO: 1) and GST-R, (SEQ ID NO: 2) have been shown to be particularly effective

for measuring GST-pi mRNA levels using RNA extracted from the FPE cells by any

of the methods for mRNA isolation, for example as described Example 1 and in US

Patent Application No. 09/469,338, filed December 20, 1999, now U.S. Patent No.

6,248,535, which is hereby incorporated by reference in its entirety.

[051] "Substantially identical" in the nucleic acid context as used herein, means

hybridization to a target under stringent conditions, and also that the nucleic acid

segments, or their complementary strands, when compared, are the same when properly aligned, with the appropriate nucleotide insertions and deletions, in at least

about 60% of the nucleotides, typically, at least about 70%, more typically, at least

about 80%, usually, at least about 90%, and more usually, at least, about 95-98% of

the nucleotides. Selective hybridization exists when the hybridization is more

selective than total lack of specificity. See, Kanehisa, Nucleic Acids Res., 12:203- 213 (1984).

[052] This invention includes substantially identical oligonucleotides that

hybridize under stringent conditions (as defined herein) to all or a portion of the oligonucleotide primer sequence of GST-F (SEQ ID NO: 1), its complement or

GST-R (SEQ ID NO: 2), or its complement.

[053] Under stringent hybridization conditions, only highly complementary, i.e.,

substantially similar nucleic acid sequences hybridize. 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, most preferably one or more mismatch out of 20 contiguous

nucleotides.

[054] The hybridizing portion of the nucleic acids is typically at least 10 (e.g., 15)

nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at

least about 80%, preferably at least about 95%, or most preferably about at least 98%, identical to the sequence of a portion or all of oligonucleotide primer GST-F

(SEQ ID NO: 1), its complement or GST-R (SEQ ID NO: 2), or its complement.

[055] Hybridization of the oligonucleotide primer to a nucleic acid sample under

stringent conditions is defined below. Nucleic acid duplex or hybrid stability is

expressed as a melting temperature (T_m), which is the temperature at which the probe dissociates from the target DNA. This melting temperature is used to define the

required stringency conditions. If sequences are to be identified that are

substantially identical to the probe, rather than identical, then it is useful to first

establish the lowest temperature at which only homologous hybridization occurs

with a particular concentration of salt (e.g. SSC or SSPE). Then assuming that 1% mismatching results in a 1°C decrease in T_m, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having >95% identity with the probe are sought, the final wash temperature is decrease by

5° C). In practice, the change in T_m can be between 0.5°C and 1.5°C per 1% mismatch.

[056] Stringent conditions involve hybridizing at 68° C in 5x SSC/5x Denhart's

solution 1.0% SDS, and washing in 0.2x SSC/0.1% SDS at room temperature.

Moderately stringent conditions include washing in 3x SSC at 42° C. The parameters of salt concentration and temperature be varied to achieve optimal level of identity between the primer and the target nucleic acid. Additional guidance

regarding such conditions is readily available in the art, for example, Sambrook,

Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold

Spring Harbor Laboratory Press, New York, (1989) and F. M. Ausubel et al eds.,

Current Protocols in Molecular Biology, John Wiley and Sons (1994).

[057] Oligonucleotide primers disclosed herein are capable of allowing accurate

assessment of GST-pi gene expression in a fixed or fixed and paraffin embedded tissue, as well as frozen or fresh tissue. This is despite the fact that RNA derived

from FPE samples is more fragmented relative to that of fresh or frozen tissue. Thus, the methods of the invention are suitable for use in assaying GST-pi

expression levels in FPE tissue where previously there existed no way to assay GST-

pi gene expression using fixed tissues.

[058] From the measurement of the amount of GST-pi mRNA that is expressed in

the tumor, the skilled practitioner can make a prognosis concerning clinical resistance of a tumor to a particular genotoxin, preferably a platinum-based

chemotherapy, or to a chemotherapy inducing a similar type of DNA damage. Platinum-based chemotherapies cause a "bulky adduct" of the DNA, wherein the primary effect is to distort the three-dimensional conformation of the double helix.

Such compounds are meant to be administered alone, or together with other

chemotherapies such as gemcitabine (Gem) or 5-Fluorouracil (5-FU).

[059] Many compounds are commonly given with platinum-based chemotherapy

agents. For example, BEP (bleomycin, etoposide, cisplatin) is used for testicular

cancer, MNAC (methotrexate, vinblastine, doxorubicin, cisplatin) is used for bladder cancer, MNP (mitomycin C, vinblastine, cisplatin) is used for non-small cell lung cancer treatment. Many studies have documented interactions between platinum-

containing agents. Therapeutic drug synergism, for example, has been reported for

many drugs potentially included in a platinum based chemotherapy. A very short list

of recent references for this include the following: Okamoto et al., Urology 2001;

57:188-192.; Tanaka et al., Anticancer Research 2001; 21:313-315; Slamon et al.,

Seminars in Oncology 2001; 28:13-19; Lidor et al, Journal of Clinical Investigation 1993; 92:2440-2447; Leopold et al., NCI Monographs 1987;99-104; Ohta et al.,

Cancer Letters 2001; 162:39-48; van Moorsel et al., British Journal of Cancer 1999; 80:981-990.

[060] Platinum-based genotoxic chemotherapies comprises heavy metal

coordination compounds which form covalent DNA adducts. Generally, these heavy

metal compounds bind covalently to DNA to form, in pertinent part,

cis-l,2-intrastrand dinucleotide adducts. Generally, this class is represented by

cis-diamminedichloroplatinum (II) (cisplatin), and includes cis-diammine-

(l,l-cyclobutanedicarboxylato) platinum(π) (carboplatin), cis-diammino - (1,2-cyclohexyl) dichloroplatinum(π), and cis-(l,2-ethylenediammine)

dichloroplatinum(JJ). Platinum first agents include analogs or derivatives of any of the foregoing representative compounds.

[061] Tumors currently manageable by platinum coordination compounds include

testicular, endometrial, cervical, gastric, squamous cell, adrenocortical and small cell

lung carcinomas along with medulloblastomas and neuroblastomas.

Trans-Diamminedichloroplatinum (H) (trans-DDP) is clinically useless owing, it is thought, to the rapid repair of its DNA adducts. The use of trans-DDP as a

chemotherapeutic agent herein likely would provide a compound with low toxicity in nonselected cells, and high relative toxicity in selected cells, hi a preferred

embodiment, the platinum compound is cisplatin.

[062] The invention being thus described, practice of the invention is illustrated by

the experimental examples provided below. The skilled practitioner will realize that

the materials and methods used in the illustrative examples can be modified in

various ways. Such modifications are considered to fall within the scope of the

present invention.

EXAMPLES EXAMPLE 1

RNA Isolation from FPE Tissue

[063] RNA is extracted from paraffin-embedded tissue by the following general

procedure.

A. Deparaffinization and hydration of sections:

[064] (1) A portion of an approximately 10 μM section is placed in a 1.5 mL

plastic centrifuge tube.

[065] (2) 600 μL, of xylene are added and the mixture is shaken vigorously for

about 10 minutes at room temperature (roughly 20 to 25 °C).

[066] (3) The sample is centrifuged for about 7 minutes at room temperature at the

maximum speed of the bench top centrifuge (about 10-20,000 x g).

[067] (4) Steps 2 and 3 are repeated until the majority of paraffin has been

dissolved. Two or more times are normally required depending on the amount of

paraffin included in the original sample portion. [068] (5) The xylene solution is removed by vigorously shaking with a lower

alcohol, preferably with 100% ethanol (about 600 μL) for about 3 minutes.

[069] (6) The tube is centrifuged for about 7 minutes as in step (3). The

supernatant is decanted and discarded. The pellet becomes white.

[070] (7) Steps 5 and 6 are repeated with successively more dilute ethanol

solutions: first with about 95% ethanol, then with about 80% and finally with about 70% ethanol.

[071] (8) The sample is centrifuged for 7 minutes at room temperature as in step

(3). The supernatant is discarded and the pellet is allowed to dry at room

temperature for about 5 minutes.

B. RNA Isolation with Phenol-Chloroform

[072] (1) 400 μL guanidine isothiocyanate solution including 0.5% sarcosine and 8

μL dithiothreitol is added.

[073] (2) The sample is then homogenized with a tissue homogenizer (Ultra-

Turrax, IKA- Works, fric, Wilmington, NC) for about 2 to 3 minutes while gradually increasing the speed from low speed (speed 1) to high speed (speed 5).

[074] (3) The sample is then heated at about 95 °C for about 5-20 minutes. It is

preferable to pierce the cap of the tube containing the sample with a fine gauge

needle before heating to 95 °C. Alternatively, the cap may be affixed with a plastic

clamp or with laboratory film.

[075] (4) The sample is then extracted with 50 μL 2M sodium acetate at pH 4.0

and 600 μL of phenol/chloroform/isoamyl alcohol (10:1.93:0.036), prepared fresh by

mixing 18 mL phenol with 3.6 mL of a 1 :49 isoamyl alcoho chloroform solution. The solution is shaken vigorously for about 10 seconds then cooled on ice for about

15 minutes.

[076] (5) The solution is centrifuged for about 7 minutes at maximum speed. The

upper (aqueous) phase is transferred to a new tube.

[077] (6) The RNA is precipitated with about 10 μL glycogen and with 400

μL isopropanol for 30 minutes at -20 °C.

[078] (7) The RNA is pelleted by centrifugation for about 7 minutes in a benchtop

centrifuge at maximum speed; the supernatant is decanted and discarded; and the

pellet washed with approximately 500 μL of about 70 to 75% ethanol.

[079] (8) The sample is centrifuged again for 7 minutes at maximum speed. The

supernatant is decanted and the pellet air dried. The pellet is then dissolved in an

appropriate buffer for further experiments (e.g., 50 pi. 5mM Tris chloride, pH 8.0).

EXAMPLE 2

mRNA Reverse Transcription and PCR

[080] Reverse Transcription: RNA was isolated from microdissected or non-

microdissected formalin fixed paraffin embedded (FPE) tissue as illustrated in

Example 1 and as previously described in U.S. Application No. 09/469,338 filed

December 20, 1999, now U.S. Patent No. 6,248,535, which is hereby incorporated

by reference in its entirety. After precipitation with ethanol and centrifugation, the

RNA pellet was dissolved in 50 ul of 5 mM Tris/Cl at pH 8.0. M-MLN Reverse

Transcriptase will extend an oligonucleotide primer hybridized to a single-stranded

RΝA or DΝA template in the presence of deoxynucleotides, producing a complementary strand. The resulting RΝA was reverse transcribed with random hexamers and M-MLN Reverse Transcriptase from Life Technologies. The reverse

transcription was accomplished by mixing 25 μl of the RΝA solution with 25.5 μl

of "reverse transcription mix" (see below). The reaction was placed in a

thermocycler for 8 min at 26° C (for binding the random hexamers to RΝA), 45 min

at 42° C (for the M-MLN reverse transcription enzymatic reaction) and 5 min at 95°

C (for heat inactivation of DΝAse).

[081] "Reverse transcription mix" consists of 10 ul 5X buffer (250 mM Tris-HCl,

pH 8.3, 375 mM KC1, 15 mM MgC12), 0.5 ul random hexamers (50 O.D. dissolved

in 550 ul of 10 mM Tris-HCl pH 7.5) 5 ul 10 mM dΝTPs (dATP, dGTP, dCTP and

dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3mg/ml in 10 mM Tris-HCL, pH 7.5), 1.25 ul RΝA Guard 24,800U/ml (RΝAse inhibitor) (Porcine #27-0816, Amersham

Pharmacia) and 2.5 ul MMLV 200U/ul (Life Tech Cat #28025-02).

[082] Final concentrations of reaction components are: 50 mM Tris-HCl, pH 8.3,

75 mM KC1, 3 mM MgC12, 1.0 mM dΝTP, 1.0 mM DTT, 0.00375. mg/ml BSA,

0.62 U/ul RΝA Guard and 10 U/ ul MMLN.

[083] PCR Quantification of mRΝA expression. Quantification of GST-pi cDΝA

and an internal control or house keeping gene (e.g., β-actin) cDΝA was done using a

fluorescence based real-time detection method (ABI PRISM 7700 or 7900 Sequence

Detection System [TaqMan®], Applied Biosystems, Foster City, CA.) as described

by Heid et al, (Genome Res 1996;6:986-994); Gibson et al, (Genome Res

1996;6:995-1001). In brief, this method uses a dual labelled fluorogenic TaqMan®

oligonucleotide probe, (GST-219T (SEQ ID NO: 3), T_m = 69° C), that anneals specifically within the forward and reverse primers. Laser stimulation within the capped wells containing the reaction mixture causes emission of a 3 'quencher dye

(TAMRA) until the probe is cleaved by the 5' to 3'nuclease activity of the DNA

polymerase during PCR extension, causing release of a 5' reporter dye (6FAM).

Production of an an plicon thus causes emission of a fluorescent signal that is

detected by the TaqMan® 's CCD (charge-coupled device) detection camera, and the

amount of signal produced at a threshold cycle within the purely exponential phase

of the PCR reaction reflects the starting copy number of the sequence of interest. Comparison of the starting copy number of the sequence of interest with the starting

copy number of theintemal control gene provides a relative gene expression level. TaqMan® analyses yield values that are expressed as ratios between two absolute

measurements (gene of interest/internal control gene).

[084] The PCR reaction mixture consisted 0.5μl of the reverse transcription

reaction containing the cDNA prepared as described above 600 nM of each oligomicleoride primer (GST-F (SEQ ID NO:l), T_m = 59° C and GST-R (SEQ JD

NO: 2), T_m = 59° C ), 200 nM TaqMan® probe (SEQ ID NO:3), 5 U AmpliTaq Gold

Polymerase, 200 μM each dATP, dCTP, dGTP, 400 μM dTTP, 5.5 mM MgCl₂, and

1 x TaqMan® Buffer A containing a reference dye, to a final volume of less than or

equal to 25 μl (all reagents Applied Biosystems, Foster City, CA). Cycling

conditions were, 95 °C for 10 min, followed by 45 cycles at 95 °C for 15s and 60 °C

for 1 min. Ohgonucleotides used to quantify internal control gene β-Actin were β-

Actin TaqMan® probe (SEQ ID NO: 4), β-Actin-592F (SEQ ID NO: 5) and β-

Actin-651R (SEQ ID NO: 6).

[085] The oligonucleotide primers GST-F (SEQ ID NO: 1) and GST-R (SEQ ID NO: 2), used in the above described reaction will amplify a 72 bp product.

EXAMPLE 3

Determining the Uncorrected Gene Expression (UGE) for GST-pi

[086] Two pairs of parallel reactions are carried out, i.e., "test" reactions and the

"calibration" reactions. The GST-pi amplification reaction and the β-actin internal

control amplification reaction are the test reactions. Separate GST-pi and β-actin

amplification reactions are performed on the calibrator RNA template and are

referred to as the calibration reactions. The TaqMan® instrument will yield four

different cycle threshold (Ct) values: Ct^_j^,- and Ct_p._actin from the test reactions and

Ct_{GST i} and Ct_p._actin from the calibration reactions. The differences in Ct values for

the two reactions are determined according to the following equation:

ΔCt_ter_t = Ct_5ff?|. - Ct_β._actin (From the "test" reaction)

ΔCt_calibrator= Ct_G5r__p(. - Ct_β._actin (From the "calibration" reaction)

[087] Next the step involves raising the number 2 to the negative ΔCt, according to the following equations.

2-^ΔCt _test (From the "test" reaction)

2^"ΔCt _Caii_brator (From the "calibration" reaction)

[088] In order to then obtain an uncorrected gene expression for GST-pi from the

TaqMan® instrument the following calculation is carried out:

Uncorrected gene expression (UGE) for GST-pi = 2^"ΔCt _test / 2^" •ΔCt calibrator

Normalizing UGE with known relative GST-pi expression levels

[089] The normalization calculation entails a multiplication of the UGE with a correction factor (K_GS7^,) specific to GST-pi and a particular calibrator RNA. A

corcection factor K_GST__pi can also be determined for any internal control gene and any

accurately pre-quantified calibrator RNA. Preferably, the internal control gene β-

actin and the accurately pre-quantified calibrator Adult Colon, Disease Human Total

RNA, (Cat. No. #735263) from Stratagene, are used. Given these reagents correction

factor K_{GST i} equals 7.28 x 10^"3.

[090] Normalization is accomplished using a modification of the ΔCt method

described by Applied Biosystems, the TaqMan® manufacturer, in User Bulletin #2 and described above. To carry out this procedure, the UGE of 6 different test

tissues was analyzed for GST-pi expression using the TaqMan® methodology

described above. The internal control gene β-actin and the calibrator RNA,Adult

Colon, Disease Human Total RNA, (Cat. No. #735263) from Stratagene was used.

[091] The known relative GST-pi expression level of each sample 14-1, 14-5, 14-

8, 13-24, 13-25 was divided by its corresponding TaqMan® derived UGE to yield an

unaveraged correction factor K.

Ku_naveraged ⁼ KnOWn Values / UGE

[092] Next, all of the K values are averaged to determine a single K_GST.pi correction

factor specific for GST-pi, Adult Colon, Disease Human Total RNA, (Cat. No.

#735263) from Stratagene from calibrator RNA and β-actin.

[093] Therefore, to determine the Corrected Relative GST-pi Expression in an

unknown tissue sample on a scale that is consistent with pre-TaqMan® GST-pi

expression studies, one merely multiplies the uncorrected gene expression data (UGE) derived from the TaqMan® apparatus with the K^, specific correction

factor, given the use of the same internal control gene and calibrator RNA.

Corrected Relative GST-pi Expression = UGE x K_GST__pi

[094] A K_GST.pi may be determined using any accurately pre-quantified calibrator

RNA or internal control gene. Future sources of accurately pre-quantified RNA can

be calibrated to samples with known relative GST-pi expression levels as described

in the method above or may now be calibrated against a previously calibrated calibrator RNA such as Adult Colon, Disease Human Total RNA, (Cat. No.

#735263) from Stratagene described above.

[095] For example, if a subsequent K_GST__pi is determined for a different internal

control gene and/or a different calibrator RNA, one must calibrate both the internal

control gene and the calibrator RNA to tissue samples for which GST-pi expression

levels relative to that particular internal control gene have already been determined.

Such a determination can be made using standard pre-TaqMan®, quantitative RT- PCR techniques well known in the art. The known expression levels for these

samples will be divided by their corresponding UGE levels to determine a K for that sample. K values are then averaged depending on the number of known samples to

determine a new K^_.^ specific to the different internal control gene and/or calibrator RNA.

EXAMPLE 4

GST-pi Expression Correlates with survivability

[096] Total mRNA was isolated from microdissected FPE pretreatment tumor

samples, and Corrected Relative GST-pi Expression was measured using quantitative RT-PCR as described in Examples 2 and 3. A method for mRNA

isolation from such samples is described in Example 1 and in US Patent Application

No. 09/469,338, filed December 20, 1999, now U.S. Patent No. 6,248,535, and is

hereby incorporated by reference in its entirety.

[097] The values of the gene expressions were correlated with clinical outcome

using appropriate statistical methods. Survival was estimated according to Kaplan

and Meier (Kaplan et al., J Am Stat Assoc 1958; 53: 187-220). Univariate analysis was performed with the log-rank test (Mantel, Chemother Rep 1966; 50: 163-170).

The level of significance was set to P<0.05. All P values reported were based on

two-sided tests.

[098] A total of 31 esophageal or gastroesophageal junction (esophagocardiac)

adenocarcinoma tumor specimens from 31 patients were analysed for GST-pi mRNA

expression analysis. Thirty (97%) of the patients were male, the median age was 64

years (mean 60.9 years, range 36-78 years). The ethnic background of this group included 29 Caucasians, 1 Asian, and 1 African- American. Using TNM clinical

staging criteria, 2 (6.5%) of the patients had Stage I disease, 22 (71%) had Stage π disease, 1 (3.2%) had Stage m disease, and 6 (19.4%) patients had Stage IN disease.

Overall survival was assessable for all patients. The median overall survival was

17.17 months (mean 24.8 months, range 3.8-156.7 months). Twelve (38.7%) of the patients had died and 19 (61.3%) were alive.

[099] The treatment consisted of all patients receiving two cycles of 5-FU given as

800 mg/m² per day for 5 days or 1000 mg/m² per day for 4 days plus 75 mg/m² cisplatin with concurrent 45 Gy radiation, followed by operative resection. For entry

into the study, each patient had to have completed the chemotherapy regimen and the prescribed radiotherapy, undergone a gross complete resection, and lived at least 30

days after surgery.

[100] The influence of tumor stage was accounted for by pooling the data over the

TNM stage strata. Survival curves and log-rank statistics were generated for Stage JJ

and Stage IN disease patients only because of the very small numbers of patients in

the other stages. The median corrected relative GST-pi mRΝA expression level was

1.0 x 10^"3 (mean 0.51 x 10^"3, range 0.0-16.1 x 10^'3, all values GST-pi x 10^'3/β-actin).

An analysis of survival according to GST-pi values showed that patients whose tumors had a relative GST-pi gene expression level higher than the median value had

a statistically significant survival benefit compared to those with levels below the

median value (P=0.0073, log-rank test). Accordingly, the median corrected relative GST-pi mRΝA was assigned to be a threshold value. This association is shown

graphically in Figure 1. The relationship was independent of stage. Figures 2 and 3 show that the association was present if the analysis was confined only to those with

Stage II or Stage IN disease.

[101] GST-pi mRΝA expression is a significant prognostic factor for patients with

esophagocardiac adenocarcinoma who are treated with a cisplatin-containing regimen.

Claims (16)

What is claimed is:

1. A method for determining a platinum-based chemotherapeutic regimen for

treating a tumor in a patient comprising:

(a) obtaining a tissue sample of the tumor and fixing the sample, to obtain a fixed tumor sample;

(b) isolating mRNA from the fixed tumor sample;

(c) subj ecting the mRNA to amplification using a pair of oligonucleotide primers that hybridize under high stringency conditions to a region of the

GST-pi gene, to obtain an amplified sample;

(d) determining the amount of GST-pi mRNA in the amplified sample;

(e) comparing the amount of GST-pi mRNA from step (d) to an amount of mRNA of an internal control gene; and

(f) determining a platinum-based chemotherapeutic regimen based on the

amount of GST-pi mRNA in the amplified sample and a predetermined threshold level for GST-pi gene expression.

2. The method of claim 1 wherein the pair of oligonucleotide primers consist of

SEQ ID NO:l or an oligonucleotide primer substantially identical thereto and

SEQ ID NO:2 or an oligonucleotide primer substantially identical thereto.

3. The method of claim 1 wherein, the tumor is a non-small-cell lung cancer

(NSCLC) tumor.

4. The method of claim 1 wherein, the threshold level of GST-pi gene

expression is about 1.0 x 10^"3 times internal control gene expression level.

5. A method of treating a tumor with a platinum-based chemotherapeutic

regimen comprising:

(a) obtaining a tissue sample of the tumor and fixing the sample,

to obtain a fixed tumor sample;

(b) isolating mRNA from the fixed tumor sample;

(c) subjecting the mRNA to amplification using a pair of oligonucleotide primers that hybridize under stringent

conditions to a region of the GST-pi gene, to obtain an

amplified sample;

(d) determining the amount of GST-pi mRNA in the amplified

sample;

(e) comparing the amount of GST-pi mRNA from step (d) to an

amount of mRNA of an internal control gene;

(f) providing a platinum-based chemotherapeutic regimen comprising a cytotoxic agent when the determined gene

expression level for GST-pi gene is below a predetermined

threshold value.

6. The method of claim 5 wherein, the tumor is an esophagocardiac

adenocarcinoma tumor.

7. The method of claim 5 wherein, the cytotoxic agent is 5-FU or cisplatin or a

combination thereof.

8. A method for determining the level of GST-pi expression in a fixed paraffin

emedded tissue sample comprising;

(a) deparaffinizing the tissue sample; to obtain a deparaffinized sample;

(b) isolating mRNA from the deparaffinized sample in the presence of an

effective amount of a chaotropic agent;

(c) subj ecting the mRNA to amplification using a pair of oligonucleotide

primers that hybridize under stringent conditions to a region of the GST-pi gene, to obtain an amplified sample;

(d) determining the quantity of GST-pi mRNA relative to the quantity of

an internal control gene's mRNA.

9. The method of claim 8 wherein, the pair of oligonucleotide primers consists of the oligonucleotide primer pair GST or a pair of oligonucleotide primers

substantially similar thereto.

10. The method of claim 8 wherein, the internal control gene is β-actin.

11. The method of claim 8 wherein, mRNA isolation is carried out by

(a) heating the tissue sample in a solution comprising an effective

concentration of a chaotropic compound to a temperature in the range of about 75 to about 100 °C for a time period of about 5 to about 120 minutes;

and

(b) recovering said mRNA from said chaotropic solution.

12. An oligonucleotide primer having the sequence of SEQ ID NO: 1 or and an

oligonucleotide substantially identical thereto.

13. An oligonucleotide primer having the sequence of SEQ JD NO: 2 or and an oligonucleotide substantially identical thereto.

14. A kit for detecting expression of a GST-pi gene comprising, oHgionucleotide pair GST or an oligonucleotide pair substantially identical thereto.

15. The method of claim 1 wherein, step (b) comprises the step of heating the fixed tumor sample the in the presence of an effective concentration of a

chaotropic agent wherein the heating occurs at a temperature from about 50°C to about 100°C.

16. The method of claim 1 wherein, the tumor is an esophageal adenocarcinoma

tumor.