AU2002249768A1 - Method of determining a chemotherapeutic regimen based on ERCC1 expression - Google Patents

Description

METHOD OF DETERMINING A CHEMOTHERAPEUTIC

REGIMEN BASED ON ERCCl EXPRESSION

FIELD OF THE INVENTION

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 survival of

patients treated with chemotherapeutic agents that target DNA, especially agents that

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

BACKGROUND OF THE INVENTION

Cancer arises when a normal cell undergoes neoplastic transformation and

becomes a malignant cell. Transformed (malignant) cells escape normal physiologic

controls specifying cell phenotype and restraining cell proliferation. Transformed

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

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

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

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

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

been developed, including drugs that interfere with nucleic acid synthesis, protein

synthesis, and other vital metabolic processes. 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 geno toxic. 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.

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 co administering chemotherapeutic drugs belonging to two or more of these general classes.

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.

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 etal., 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. Excision repair of bulky

DNA adducts, such as those formed by platinum agents, appears to be mediated by

genes involved in DNA damage recognition and excision. Cleaver et al.,

Carcinogenesis 11:875-882 (1990); Hoeijmakers et al., Cancer Cells 2:311-320

(1990); Shivji et al., Cell 69:367-374 (1992). Indeed, cells carrying a genetic defect

in one or more elements of the enzymatic DNA repair machinery are extremely sensitive to cisplatin. Fraval et al. (1978), 51 Mutat. Res. 121, Beck and Brubaker

(1973), 116 J. Bacteriol 1247.

The excision repair cross-complementing (ERCCl) gene is essential in the

repair of DNA adducts. The human ERCCl gene has been cloned. Westerveld et al.,

Nature (London) 310:425-428 (1984); Tanaka et al., Nature 348:73-76 (1990).

Several studies using mutant human and hamster cell lines that are defective in this

gene and studies in human tumor tissues indicate that the product encoded by

ERCCl is involved in the excision repair of platinum-DNA adducts. Dabholkar et

al., J. Natl. Cancer Inst. 84:1512-1517 (1992); Dijt et al, Cancer Res. 48:6058-6062

(1988); Hansson et al, Nucleic Acids Res. 18: 35-40 (1990).

When transfected into DNA-repair deficient CHO cells, ERCCl confers

cellular resistance to cisplatin along with the ability to repair platinum-DNA adducts. Hansson et al., Nucleic Acids Res. 18: 35-40 (1990). Currently accepted

models of excision repair suggest that the damage recognition/excision step is rate-

limiting to the excision repair process.

The relative levels of expression of excision repair genes such as ERCCl in

malignant cells from cancer patients receiving platinum-based therapy has been

examined. Dabholkar et al., J. Natl. Cancer Inst. 84:1512-1517 (1992). ERCCl

overexpression in gastric cancer patients has been reported to have a negative impact

on tumor response and ultimate survival when treated with the chemotherapeutic

regimen of cisplatin (DDP)/fluorouracil (Metzger, et l., J Gin Oncol 16: 309,

1998). Recent evidence indicates that gemcitabine (Gem) may modulate ERCCl

nucleotide excision repair (NER) activity. Thus, intratumoral levels of ERCCl expression may be a major prognostic factor for determining whether or not DDP

and GEM would be an effective therapeutic cancer patients.

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

to allow for histo logical 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.

Until now, quantitative gene expression studies including those of ERCCl

expression have been limited to reverse transcriptase polymerase chain reaction

(RT-PCR) amplification of RNA from fresh or frozen tissue. U.S. Patent No.

5,705,336 to Reed et al., discloses a method of quantifying ERCCl mRNA from

ovarian tumor tissue and determining whether that tissue will be sensitive to platinum-based chemotherapy. Reed et al., quantify ERCCl mRNA from frozen

ovarian tumor biopsies.

The use of frozen tissue by health care professionals as described in Reed et

al., poses substantial inconveniences. Rapid biopsy delivery to avoid tissue and

subsequent mRNA degradation is the primary concern when planning any RNA-

based quantitative genetic marker assay. The health care professional performing

the biopsy, must hastily deliver the tissue sample to a facility equipped to perform an

RNA extraction protocol immediately upon tissue sample receipt. If no such facility

is available, the clinician must promptly freeze the sample in order to prevent

mRNA degradation. In order for the diagnostic facility to perform a useful RNA

extraction protocol prior to tissue and RNA degradation, the tissue sample must

remain frozen until it reaches the diagnostic facility, however far away that may be. Maintenance of frozen tissue integrity during transport using specialized couriers

equipped with liquid nitrogen and dry ice, comes only at a great expense.

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.

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.

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.

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.

There is a need for a method of quantifying ERCCl 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 ERCCl expression in fixed and paraffinized

(FPE) tissue. Accordingly, it is the object of the invention to provide a method for

assessing ERCCl 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 ERCCl mRNA in a patient's

tumor cells and comparing it to a predetermined threshold expression level. SUMMARY OF THE INVENTION

In one aspect of the invention there is provided a method for assessing levels

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

fixed and paraffin-embedded (FPE) tumor cells.

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

the amount of ERCCl 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 ERCCl mRNA relative to

the quantity of an internal control gene's mRNA.

In- an embodiment of this aspect of the invention, there are provided

oligonucleotide primers having the sequence of ERCC1-504F (SEQ ID NO: 1) or

ERCC1-574R (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.

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 ERCCl in the sample; comparing the ERCCl gene expression

levels in the sample with a predeterimined threshold level for the ERCCl gene; and

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

ERCCl gene expression level with the predetermined threshold level.

The invention further relates to a method of normalizing the uncorrected

gene expression (UGE) of ERCCl relative to an internal control gene in a tissue sample analyzed using TaqMan® technology to known ERCCl expression levels

relative to an internal control from samples analyzed by pre-TaqMan® technology.

DESCRIPTION OF THE DRAWING

Figure 1 is a graph showing the overall survival of patients receiving

Cisplatin/Gem treatment vs. Corrected Relative ERCCl Expression in NSCLC.

Patient Corrected Relative ERCCl Expression levels lower than the threshold of 6.7

x 10^"3 correlated with significantly better survival. While patient Corrected Relative

ERCCl Expression levels higher than the threshold of 6.7 x 10^"3 correlated with

significantly worse survival. (P=0.009 Log rank test)

Figure 2 is a chart illustrating how to calculate Corrected Relative ERCCl

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 ERCCl

values determined by pre-TaqMan® technology. This is accomplished by

multiplying UGE to a correction factor K_ERCCJ. The internal control gene in the

figure is β-actin and the calibrator RNA is Human Liver Total RNA (Stratagene,

Cat. #735017).

Figure 3 is a table showing the demographic details of the 56 patients in the

study, tumor stage and cell types. The median number of treatment cycles received

was 3 (range 1-6). Fourteen patients (25%) had previously received chemotherapy,

mostly (9 patients) taxane therapy alone or in combination with DDP or carboplatin.

Three of the 56 patients had received radiotherapy and 5 patients had under gone

surgical resection of the primary tumor. Figure 4 is a table showing patients with Corrected ERCCl expression levels

below the threshold had a significantly longer median survival of 61.6 weeks (95%

CI. 42.4, 80.7 weeks) compared to 20.4 weeks (95% CI. 6.9, 33.9 weeks) for

patients with Corrected ERCCl levels above the threshold. Adjusted for tumor stage,

the log rank statistic for the association between low or high ERCCl expression and

overall survival was 3.97 and the P value was 0.046. The unadjusted log rank results

are shown in this figure. Also shown are factors that were significantly associated

with overall survival on univariable analysis using Kaplan Meier survival curves, and

the log rank test. These were the presence of pretreatment weight loss and the

ECOG performance status. Patient age (P=0.18), sex (P=0.87), tumor stage

(P=0.99), tumor cell type (P=0.63), and presence of pleural effusion (P=0.71) were

not significant prognostic factors for overall survival. Corrected Relative ERCCl Expression level, ECOG performance status, and weight loss remained significant

prognostic factors for survival in the Cox proportional hazards regression model

multivariable analysis. P values for a Cox regression model stratified on tumor stage

were 0.038 for ERCCl, 0.017 for weight loss, and 0.02 for ECOG performance

status (PS 0 versus 1 or 2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention resides in part in the finding that the amount of

ERCCl mRNA in a tumor correlates with survival in patients treated with DNA

platinating agents. Patients with tumors expressing high levels of ERCCl mRNA are

considered likely to be resistant to platinum-based chemotherapy and this have lower

levels of survivability. Conversely, those patients whose tumors expressing low

amounts of ERCCl mRNA are likely to be sensitive to platinum-based

chemotherapy and have greater levels of survivability. A patient's relative

expression of tumor ERCCl mRNA is judged by comparing it to a predetermined

threshold expression level.

The invention relates to a method of quantifying the amount of ERCCl

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 ERCCl expression in

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

ERCC1-504F (SEQ ID NO: 1), ERCC1-574R (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 ERCCl gene expression may then be used for prognosis of

platinum-based chemotherapy.

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

ERCCl mRNA in the sample by using a pair of oligonucleotide primers, preferably

oligionucleotide primer pair ERCC1-504F (SEQ ID NO: 1) and ERCC1-574R (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, and is hereby

incorporated by reference in its entirety.

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

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

tissue. In a preferred embodiment, a portion of normal tissue from the patient from

which the tumor is obtained, is also examined. Patients whose normal tissues are

expected to be resistant to platinum-based chemotherapeutic compounds, i.e., show

a high level of ERCCl gene expression, but whose tumors are expected to be

sensitive to such compounds, i.e., show a low level of ERCCl gene expression, may then be treated with higher amounts of the chemotherapeutic composition.

Patients showing a level of ERCCl gene expression below the threshold

level, may be treated with higher amounts of the chemotherapeutic composition

because they are expected to have greater survivability than patients with tumors

expressing a level of ERCCl gene expression above the threshold level.

Alternatively, the clinician may determine that patients with tumors expressing a

level of ERCCl gene expression above the threshold level may not derive any

significant benefit from chemotherapy given their low expected survivability.

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 ERCCl 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 Non-Small

Cell Lung Cancer (NSCLC) tumors. For application of some embodiments of the

invention to particular tumor types, it is preferable to confirm the relationship of

ERCCl gene expression levels to survivability by compiling a dataset that enables

correlation of a particular ERCCl expression and clinical resistance to platinum-

based chemotherapy.

A "predetermined threshold level", as defined herein, is a corrected relative

level of ERCCl tumor expression above which it has been found that tumors are

likely to be resistant to a platinum-based chemotherapeutic regimen. Tumor

expression levels below this threshold level are likely to be found in tumors sensitive

to platinum-based chemotherapeutic regimen. The range of corrected relative

expression of ERCCl, expressed as a ratio of ERCCl : β-actin, among tumors

responding to a platinum-based chemotherapeutic regimen is less than about 6.7 x

10^"3. Tumors that do not respond to a platinum-based chemotherapeutic regimen

have relative expression of ERCCl : β-actin ratio above about 6.7 x 10^"3. See

Example 4.

A "predetermined threshold level" is further defined as tumor corrected

relative ERCCl expression levels above which patients receiving a platinum-based

chemotherapeutic regimen are likely to have low survivability. Tumor corrected

relative ERCCl expression levels below this threshold level in patients receiving a

platinum-based chemotherapeutic regimen correlate to high patient survivability.

The threshold corrected relative ERCCl expression, expressed as a ratio of ERCCl :

β-actin, is about 6.7 x 10^"3. Figure 1, see Example 4. However, the present invention

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

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.

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 deparaffinization 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.

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

homogenized using mechanical, sonic or other means of homogenization.

Rehydrated samples may be homogenized in a solution comprising a chaotropic

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

Homogenized samples are heated to a temperature in the range of about 50 to about

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

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

guanidinium thiocyanate.

An "effective 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, formamide, potassium iodiode, potassium

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

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

(also sold as guanidinium thiocyanate) and guanidinium hydro chloride. 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.

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.

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.

The quantification of ERCCl 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 ERCCl 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 ERCCl 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

ERCCl 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 ^ .(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).

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 ERCCl mRNA levels. Such an assessment comprises a determination

of the overall constitutive level of gene transcription and a control for variations in

RNA recovery. "House-keeping" genes or "internal controls" can include, but are

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

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

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

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

RNA." The "calibrator RNA" is intended to be any available source of accurately

pre-quantified control RNA. Preferably, Human Liver Total RNA (Stratagene, Cat.

#735017) is used.

"Uncorrected Gene Expression (UGE)" as used herein refers to the numeric

output of ERCCl 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 2.

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 ERCCl : β-

actin expression values are normalized with TaqMan® derived ERCCl UGE values

from a tissue sample.

"Corrected Relative ERCCl Expression" as used herein refers to normalized

ERCCl expression whereby UGE is multiplied with a ERCCl specific correction

factor (K_ERC ), resulting in a value that can be compared to a known range of

ERCCl expression levels relative to an internal control gene. Example 3 and Figure

2 illustrate these calculations in detail. These numerical values allow the

determination of whether or not the "Corrected Relative ERCCl Expression" of a

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

predetermined threshold level of Corrected Relative ERCCl Expression to β-actin

level is about 6.7 x 10^"3. ¥L._mccl specific for ERCCl, the internal control β-actin and

calibrator Human Liver Total RNA (Stratagene, Cat. #735017), is 1.54 x 10^'3.

"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,

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 ERCCl expression to β-actin expression.

See U.S. Patent No. 5,705,336 to Reed et al.

Kg_Rca may be determined for an internal control gene other than β-actin

and/or a calibrator RNA different than Human Liver Total RNA (Stratagene, Cat.

#735017). To do so, one must calibrate both the internal control gene and the

calibrator RNA to tissue samples for which ERCCl 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 ERCCl useful in the determining a new

K_mccJ specific for the new internal control and/or calibrator RNA as described in

Example 3.

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 Non-Small Cell Lung Cancer (NSCLC).

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 ERCCl 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.

Generally, any oligonucleotide pair that flanks a region of ERCCl gene may

be used to carry out the methods of the invention. Primers hybridizing under

stringent conditions to a region of the ERCCl 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.

The invention provides specific oligonucleotide primers pairs and

oligonucleotide primers substantially identical thereto, that allow particularly

accurate assessment of ERCCl expression in FPE tissues. Preferable are

oligonucleotide primers, ERCC1-504F (SEQ ID NO: 1) and ERCCl (SEQ ID NO:

2), (also referred to herein as the oligonucleotide primer pair ERCCl) and

oligonucleotide primers substantially identical thereto. The oliogonucleotide

primers ERCC1-504F (SEQ ID NO: 1) and ERCCl, (SEQ ID NO: 2) hybridize to

the ERCCl gene (SEQ ID NO: 7) under stringent conditions and have been shown to

be particularly effective for measuring ERCCl 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, which is hereby incorporated by reference in its entirety.

"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).

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 ERCC1-504F (SEQ ID NO: 1), its complement

or ERCC1-574R (SEQ ID NO: 2), or its complement.

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.

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 ERCCl -

504F (SEQ ID NO: 1), its complement or ERCC1-574R (SEQ ID NO: 2), or its

complement.

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^, 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.

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).

Oligonucleotide primers disclosed herein are capable of allowing accurate

assessment of ERCCl 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 ERCCl

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

ERCCl gene expression using fixed tissues.

From the measurement of the amount of ERCCl mRNA that is expressed in

the tumor, the skilled practitioner can make a prognosis concerning clinical

resistance of a tumor to a particular genotoxin or the survivability of a patient

receiving a particular genotoxin. A platinum-based chemotherapy or a chemotherapy

inducing a similar type of DNA damage, is the preferable genotoxin.

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).

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(II) (carboplatin), cis-diammino -

(1,2-cyclohexyl) dichloroplatinum(II), and cis-(l,2-ethylenediammine)

dichloroplatinum(II). Platinum first agents include analogs or derivatives of any of

the foregoing representative compounds.

Tumors currently manageable by platinum coordination compounds include

testicular, endometrial, cervical, gastric, squamous cell, adrenocortical and small cell lung carcinomas along with medulloblastomas and neuroblastomas.^"

Trans-Diamminedichloroplatinum (II) (trans-DDP) is clinically useless owing, it is

thought, to the rapid repair of its DNA adducts. The use of trans-DDP as a

chemotherapeutic agent herein likely would provide a compound with low toxicity

in nonselected cells, and high relative toxicity in selected cells. In a preferred

embodiment, the platinum compound is cisplatin.

Many compounds are commonly given with platinum-based chemotherapy

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

cancer, 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., ΝCI Monographs 1987;99-104; Ohta et al.,

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

80:981-990.

Other genotoxic agents are those that form persistent genomic lesions and are

preferred for use as chemotherapeutic agents in the clinical management of cancer.

The rate of cellular repair of genotoxin-induced DΝA damage, as well as the rate of

cell growth via the cell division cycle, affects the outcome of genotoxin therapy.

Unrepaired lesions in a cell's genome can impede DΝA replication, impair the replication fidelity of newly synthesized DNA or hinder the expression of genes

needed for cell survival. Thus, one determinant of a genotoxic agent's cytotoxicity

(propensity for contributing to cell death) is the resistance of genomic lesions

formed therefrom to cellular repair. Genotoxic agents that form persistent genomic

lesions, e.g., lesions that remain in the genome at least until the cell commits to the

cell cycle, generally are more effective cytotoxins than agents that form transient,

easily repaired genomic lesions.

A general class of genotoxic compounds that are used for treating many

cancers and that are affected by levels of ERCCl expression are DNA alkylating

agents and DNA intercalating agents. Psoralens are genotoxic compounds known to be useful in the photochemotherapeutic treatment of cutaneous diseases such as

psoriasis, vitiligo, fungal infections and cutaneous T cell lymphoma. Harrison's Principles of Internal Medicine, Part 2 Cardinal Manifestations of Disease, Ch. 60

(12th ed. 1991). Another general class of genotoxic compounds, members of which

can alkylate or intercalate into DNA, includes synthetically and naturally sourced

antibiotics. Of particular interest herein are antineoplastic antibiotics, which include

but are not limited to the following classes of compounds represented by: amsacrine;

actinomycin A, C, D (alternatively known as dactinomycin) or F (alternatively KS4);

azaserine; bleomycin; carminomycin (carubicin), daunomycin (daunorubicin), or

14-hydroxydaunomycin (adriamycin or doxorubicin); mitomycin A, B or C;

mitoxantrone; plicamycin (mithramycin); and the like.

Still another general class of genotoxic agents that are commonly used and

that alkylate DNA, are those that include the haloethylnitrosoureas, especially the

chloroethylnitrosoureas. Representative members of this broad class include carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and

streptozotocin. Haloethylnitrosourea first agents can be analogs or derivatives of any

of the foregoing representative compounds.

Yet another general class of genotoxic agents, members of which alkylate

DNA, includes the sulfur and nitrogen mustards. These compounds damage DNA

primarily by forming covalent adducts at the N7 atom of guanine. Representative

members of this broad class include chlorambucil, cyclophosphamide, ifosfamide,

melphalan, mechloroethamine, novembicin, trofosfamide and the like.

Oligonucleotides or analogs thereof that interact covalently or noncovalently with

specific sequences in the genome of selected cells can also be used as genotoxic

agents, if it is desired to select one or more predefined genomic targets as the locus

of a genomic lesion.

Another class of agents, members of which alkylate DNA, include the

ethylenimines and methylmelamines. These classes include altretamine

(hexamethylmelamine), triethylenephosphoramide (TEPA),

triethylenethiophosphoramide (ThioTEPA) and triethylenemelamine, for example.

Additional classes of DNA alkylating agents include the alkyl sulfonates,

represented by busulfan; the azinidines, represented by benzodepa; and others,

represented by, e.g., mitoguazone, mitoxantrone and procarbazine. Each of these

classes includes analogs and derivatives of the respective representative compounds.

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

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

the materials and methods used in the illustrative examples can be modified in various ways. Such modifications are considered to fall within the scope of the

present invention.

EXAMPLES

EXAMPLE 1

RNA Isolation from FPE Tissue

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

procedure.

A. Deparaffinization and hydration of sections:

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

plastic centrifuge tube.

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

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

(3) The sample is centrifuged for about 7 minutes at room temperature at the maximum speed of the bench top centrifuge (about 10-20,000 x g).

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

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

paraffin included in the original sample portion.

(5) The xylene solution is removed by vigorously shaking with a lower

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

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

supernatant is decanted and discarded. The pellet becomes white.

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

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

about 70% ethanol.

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

(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

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

μL dithiothreitol is added.

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

Turrax, IKA- Works, Inc., Wilmington, NC) for about 2 to 3 minutes while gradually

increasing the speed from low speed (speed 1) to high speed (speed 5).

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

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

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

clamp or with laboratory film.

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

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

mixing 18 mL phenol with 3.6 mL of a 1:49 isoamyl alcoholxhloroform solution.

The solution is shaken vigorously for about 10 seconds then cooled on ice for about

15 minutes.

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

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

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

μL isopropanol for 30 minutes at -20 °C

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

centrifuge at maximum speed; the supernatant is decanted and discarded; and the pellet washed with approximately 500 μL of about 70 to 75% ethanol.

(8) The sample is centrifuged again for 7 minutes at maximum speed. The supernatant is decanted and the pellet air dried. The pellet is then dissolved in an

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

EXAMPLE 2

mRNA Reverse Transcription and PCR

Reverse Transcription: RNA was isolated from microdissected or non-

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

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

December 20, 1999, 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-MLV Reverse Transcriptase will extend an

oligonucleotide primer hybridized to a single-stranded RNA or DNA template in the

presence of deoxynucleotides, producing a complementary strand. The resulting RNA was reverse transcribed with random hexamers and M-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).

"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 RNA Guard 24,800U/ml (RNAse inhibitor) (Porcine #27-0816, Amersham

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

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

75 mM KC1, 3 mM MgC12, 1.0 mM dNTP, 1.0 mM DTT, 0^*00375. mg/ml BSA,

0.62 U/ul RNA Guard and 10 U/ ul MMLV.

PCR Quantification of mRNA expression. Quantification of ERCCl

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

using a fluorescence based real-time detection method (ABI PRISM 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, (ERCCl -53 OTc (SEQ ID NO: 3), T_m = 70° 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 amplicon thus causes emission of a fluorescent signal that is

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

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

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

Comparison of the starting copy number of the sequence of interest with the starting

copy number of theinternal 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). The PCR reaction mixture consisted 0.5μl of the reverse transcription

reaction containing the cDNA prepared as described above 600 nM of each

oligonucleoride primer (ERCC1-504F (SEQ ID NO. l), T_m = 59° C and ERCC1-

574R (SEQ ID NO: 2), T_m = 58° 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. Oligonucleotides used to quantify internal control gene β-

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

5) and β-Actin-651R (SEQ ID NO: 6).

The oligonucleotide primers ERCC1-504F (SEQ ID NO:l) and ERCC1-

574R (SEQ ID NO: 2), used in the above described reaction will amplify a 71bp

product.

EXAMPLE 3

Determining the Uncorrected Gene Expression (UGE) for ERCCl

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

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

control amplification reaction are the test reactions. Separate ERCCl 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^^ and Ct_p._act;n from the test reactions and

Ct_ERCC1 and Ct_p._oct&lfrom the calibration reactions. The differences in Ct values for the two reactions are determined according to the following equation:

ΔCt_test= t^cc_j - Ct_p._oc(in (From the "test" reaction)

ΔCt_caIibrator= Ct^_Rcc - Ct_p._actin (From the "calibration" reaction)

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

2^"ΔCt _tcst (From the "test" reaction)

2^*ΔC'_ca_i_bra.or (From the "calibration" reaction)

In order to then obtain an uncorrected gene expression for ERCCl from the

TaqMan® instrument the following calculation is carried out:

Uncorrected gene expression (UGE) for ERCCl = 2 -ΔCt _test / / 2 <->-ΔCt calibrator

Normalizing UGE with known relative ERCCl expression levels

The normalization calculation entails a multiplication of the UGE with a correction factor (K^cc_;) specific to ERCCl and a particular calibrator RNA. A

correction factor K^^ can also be determined for any internal control gene and any

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

actin and the accurately pre-quantified calibrator RNA Human Liver Total RNA

(Stratagene, Cat. #735017), are used. Given these reagents correction factor K^c,

equals 1.54 x 10^"3.

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 ERCCl expression using the TaqMan® methodology

described above. The internal control gene β-actin and the calibrator RNA,Human Liver Total RNA (Stratagene, Cat. #735017) was used.

The known relative ERCCl expression level of each sample AG221,

AG222, AG252, Adult Lung, PC3, AdCol was divided by its corresponding

TaqMan® derived UGE to yield an unaveraged correction factor K.

Ku_naverag_ed = KllOWIl Valu^βS / UGE

Next, all of the K values are averaged to determine a single K^^ correction

factor specific for ERCCl, Human Liver Total RNA (Stratagene, Cat. #735017)

from calibrator RNA and β-actin.

Therefore, to determine the Corrected Relative ERCCl Expression in an

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

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

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

Corrected Relative ERCCl Expression = UGE x K^,-^

A K^_JCC; 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 ERCCl expression levels as described

in the method above or may now be calibrated against a previously calibrated

calibrator RNA such as Human Liver Total RNA (Stratagene, Cat. #735017)

described above.

For example, if a subsequent K^c, 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 ERCCl 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 specific to the different internal control gene and/or

calibrator RNA.

EXAMPLE 4

All patients were enrolled in the Cisplatin/Gemcitabine arm of a prospective

multicenter three arm randomized trial (GEPC/98-02, Spanish Lung Cancer Group Phase III trial of Cisplatin/Gemcitabine (CG) versus Cisplatin/ Gemcitabine /

Ninorelbine (CGN) versus sequential doublets of Gemcitabine / Ninorelbine

followed by Ifosfamide / Ninorelbine (GNAN) in advanced ΝSCLC). All patients

received Gem 1250 mg/m² days 1,8 plus CDDP 100mg/m² day 1 every 3 weeks.

Eligibility criteria for GEPC/98-02 were measurable stage IV (with brain metastases

eligible if asymptomatic) or stage IIIB (malignant pleura! and/or pericardial effusion

and/or supraclavicular adenopathy) ΝSCLC and Eastern Cooperative Group (ECOG)

performance score 0-2. All patients had chest x-ray and a computed tomography

(CT) scan of the chest and upper abdomen before entry into the study and underwent

repeat evaluations at least every 6 weeks. Tumor response was assessed according to

WHO criteria as complete response, partial response, stable disease, and progressive disease. Tumors were reassessed during treatment with the same imaging methods

used to establish the baseline tumor measurement.

Total mRNA was isolated from microdissected FPE pretreatment tumor

samples, and Corrected Relative ERCCl 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, and is hereby incorporated by reference in

its entirety.

Statistical Analysis

The Mann- Whitney U test was used to test for significant associations

between the continuous test variable Corrected Relative ERCCl Expression and

dichotomous variables (patient sex, age above and below the median age, presence of weight loss, presence of pleural effusion, tumor stage). The Kruskal-Wallis test

was used to test for significant differences in Corrected Relative ERCCl Expression

within multiple groups (ECOG performance status, histopathology). Fisher's exact

test was used for the analysis of categorical clinicopatho logical values including

response and dichotomized Corrected Relative ERCCl Expression values.

All patients were followed from first study treatment until death or until the

data were censored. Kaplan-Meier survival curves and the log rank test were used to

analyze univariate distributions for survival and disease-free survival. The maximal

chi-square method of Miller and Siegmund (Biometrics 1982; 38:1011-1016 and

Halpera (Biometrics 1982; 38:1017-1023) was adapted to determine which

expression value best segregated patients into poor- and good prognosis subgroups (in terms of likelihood of surviving), with the log-rank test as the statistic used to

measure the strength of the grouping. To determine a P value that would be

interpreted as a measure of the strength of the association based on the maximal chi-

square analysis, 1000 boot-strap-like simulations were used to estimate the

distribution of the maximal chi-square statistics under the hypothesis of no

association.(Biometrics 1982; 38: 1017-1023) Cox's proportional hazards modeling

of factors that were significant in univariate analysis was performed to identify

which factors might have a significant influence on survival. SPSS version 10.0.5

software (SPSS Inc., Chicago 111.) was used for all statistical analyses. All P values were

two-sided.

Corrected Relative ERCCl Expression Levels.

ERCCl mRNA expression was detectable in all 56 samples analyzed. The

median Corrected Relative ERCCl Expression, relative to the expression of the

internal control housekeeping gene β-actin, was 6.7 x 10^"3 (range 0.8 x 10^"3 - 24.6 x

10^"3). There were no significant associations between Corrected Relative ERCCl

Expression levels and any of the factors age (P= 0.66), sex (P=0.18) presence of

weight loss in the six months prior to randomization (P=0.74), tumor stage (IIIB

versus IN, P=0.39), or presence of pleural effusion (P=0.25, all Mann- Whitney U

test). There were also no significant differences between the Corrected Relative

ERCCl Expression levels among patients with different performance status grades

(P=0.48, Kruskal-Wallis test) or different tumor cell types (all four tumor types,

P=0.10, Kruskal-Wallis test), but Corrected Relative ERCCl Expression levels were significantly higher in SCC tumors (median 8.6 x 10^"3) compared to

adenocarcinomas (median 5.2 x 10^"3, P=0.015, Mann- Whitney test).

Response to chemotherapy

The tumor response frequencies for the 47 patients who were evaluable for

response are shown in Figure 3. The overall response rate was 44.7%. The Corrected

Relative ERCCl Expression levels in the complete response and partial response i.e. "responding" tumors (median 4.3 x 10^"3, range 1.2 x 10^"3-24.6 x 10^"3) were not

significantly different from the levels in the stable disease and progressive disease

i.e. "non-responding" tumors (median 7.85 x 10^"3, range 0.8 x 10^"3-24.3 x 10^"3,

P=0.31 Mann- Whitney test). There were also no significant differences between the

proportion of responding and non-responding tumors with Corrected Relative

ERCCl Expression values greater and less than any ERCCl level (all Fisher's exact

test). The response rate in tumors with Corrected Relative ERCCl Expression below

the threshold value ("low" expression, 52% responders) was higher than for tumors

with Corrected Relative ERCCl Expression above the threshold value ("high"

expression, 36.4% responders, Fisher's exact test, P = 0.38).

Association between patient overall survival and Corrected Relative ERCCl

Expression levels

The median overall survival time was 36.6 weeks (range 0-113.4 weeks) and

the median time to progression was 24.4 weeks (range 0-102.9 weeks). Use of the

log rank test and the maximal chi-square statistic to identify threshold Corrected

Relative ERCCl Expression levels that segregated patients into poor- and good- prognosis subgroups showed that the range of discriminatory values included the

median value, which was therefore used as the threshold value for the survival

analysis. Therefore, the threshold Corrected Relative ERCCl Expression value was

determined to be 6.7 x 10^"3 for NSCLC Figure 1 shows the Kaplan-Meier survival

curve for patients with intratumoral Corrected Relative ERCCl Expression levels

above and below the threshold Corrected Relative ERCCl Expression level. As

shown in Figure 4, patients with Corrected Relative ERCCl Expression levels below

the threshold value had a significantly longer median survival of 61.6 weeks (95%

CI. 42.4, 80.7 weeks) compared to 20.4 weeks (95% CI. 6.9, 33.9 weeks) for

patients with Corrected Relative ERCCl Expression levels above the threshold

value. Adjusted for tumor stage, the log rank statistic for the association between

low or high Corrected Relative ERCCl Expression and overall survival was 3.97 and the P value was 0.046. The unadjusted log rank results are shown in Figure 4.

A separate Corrected Relative ERCCl Expression threshold value of 5.8 x

10^"3 was tested because this value was shown in a previous study to be associated

with overall survival for patients with gastric cancer. (Metzger et al., J Clin Oncol

1998; 16:309-316). Overall survival was significantly better for the group of NSCLC

patients in this study with Corrected Relative ERCCl Expression levels less than 5.8

x 10^"3 compared to those with ERCCl levels less than 5.8 x 10^"3 (log rank statistic

6.37, P=0.011), although a higher 6.7 x 10^"3 Corrected Relative ERCCl Expression

threshold level is a more powerful discriminator.

Other factors that were significantly associated with overall survival on

univariable analysis using Kaplan Meier survival curves and the log rank test were the presence of pretreatment weight loss and the ECOG performance status (Table 2). Patient age (P=0.18), sex (P=0.87), tumor stage (P=0.99), tumor cell type

(P=0.63), and presence of pleural effusion (P=0.71) were not significant prognostic

factors for overall survival. Corrected Relative ERCCl Expression level, ECOG

performance status, and weight loss remained significant prognostic factors for

survival in the Cox proportional hazards regression model multivariable analysis

(Figure 4). P values for a Cox regression model stratified on tumor stage were 0.038

for ERCCl, 0.017 for weight loss, and 0.02 for ECOG performance status (PS 0

versus 1 or 2).

This study found an association between lower ERCCl mRNA expression

levels and improved survival after treatment with a platinum-based

chemotherapeutic for patients with cancer.

Claims (14)

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) subjecting the mRNA to amplification using a pair of oligonucleotide

primers that hybridize under stringent conditions to a region of the

ERCCl gene, to obtain an amplified sample; (d) determining the amount of ERCCl mRNA in the amplified sample;

(e) comparing the amount of ERCCl 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 ERCCl mRNA in the amplified sample and a

predetermined threshold level for ERCCl gene expression.

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

oligonucleotide primer pair ERCCl, or pair of oligonucleotide primers

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 ERCCl gene

expression is about 6.7 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 ERCCl gene, to obtain an amplified sample;

(d) determining the amount of ERCCl mRNA in the amplified sample;

(e) comparing the amount of ERCCl mRNA from step (d) to an

amount of mRNA of an internal control gene;

(f) providing a platinum-based chemotherapeutic regimen

comprising a genotoxic agent when the determined gene

expression level for ERCCl gene is below a predetermined threshold value.

6. The method of claim 5 wherein, the tumor is a non-small-cell lung cancer (NSCLC) tumor.

7. The method of claim 5 wherein, the genotoxic agent is gemcitabine, cisplatin

or a combination thereof.

8. A method for determining the level of ERCCl 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) subjecting the mRNA to amplification using a pair of oligonucleotide

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

(d) determining the quantity of ERCCl 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 ERCCl 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 ID NO: 2 or and an

oligonucleotide substantially identical thereto.

14. A kit for detecting expression of an ERCCl gene comprising,

oligionucleotide pair ERCCl or an oligonucleotide pair substantially identical thereto.