AU2002232409A1 - Method of determining epidermal growth factor receptor and Her2-neu gene expression and correlation of levels thereof with survival rates - Google Patents

Description

METHOD OF DETERMINING EPIDERMAL GROWTH FACTOR

RECEPTOR AND HER2-neu GENE EXPRESSION AND

CORRELATION OF LEVELS THEREOF WITH SURVIVAL

RATES

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 surviviability of a patient whose tumor cell gene expression is analyzed. Additionally, the sensitivity of tumor cells to receptor tyrosine kinase targeted chemotherapeutic regimen is assayed by examining the mRNA expression ofthe EGFR and Her2-neu genes in humans.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of cancer-related deaths among both males

and females in western countries. In the United States, approximately 171,000 new

cases of lung cancer are diagnosed and 160,000 individuals die from this disease

each year. Despite improvements in the detection and treatment of lung cancer in

the past two decades, the overall 5-year survival remains less than 15% . Ginsberg,

et al., In: DeVita, et al., Cancer: Principles in Practice of Oncology. Ed. 5, pp. 858-

910. Philadelphia: Lipincott-Raven Publishers, 1997. To further improve the

survival rate in patients with Non-Small Cell Lung Carcinoma (NSCLC), their

prognostic classification will provide more accurate and useful diagnostic tools and, eventually,

more effective therapeutic options.

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 dmgs have been developed, including drugs that interfere with nucleic acid synthesis, protein synthesis, and other vital metabolic processes. These generally are referred to as anti-metabolite drugs. Other classes of chemotherapeutic drugs inflict damage on cellular DNA. Drugs of these classes generally are referred to as genotoxic. Additionally, a class of chemotherapeutic agents specifically inhibit mitogenic

signaling through receptor tyrosine kinases (RTKs), in cells where RTKs are over active. (Drugs ofthe Future, 1992, 17, 119).

Susceptibility of an individual neoplasm to a desired chemotherapeutic drug

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

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

significant risk in the clinical management of aggressive malignancies. Therefore, it

is of importance to assess the expression status of genetic determinants targeted by

specific chemotherapeutic agents. For example, if a tumor expresses high levels of

DNA repair genes, it is likely that the tumor will not respond well to low doses of

DNA-damaging genotoxic agents. Thus, the expression status of genetic determinants of a tumor will help the clinician develop an appropriate

chemotherapeutic regimen specific to the genetic repertoire ofthe tumor.

Receptor tyrosine kinases (RTKs) are important in the transduction of

mitogenic signals. RTKs are large membrane spanning proteins which possess an

extracellular ligand binding domain for growth factors such as epidermal growth

factor (EGF) and an intracellular portion which functions as a kinase to

phosphorylate tyrosine amino acid residues on cytosol proteins thereby mediating

cell proliferation. Various classes of receptor tyrosine kinases are known based on

families of growth factors which bind to different receptor tyrosine kinases. (Wilks, Advances in Cancer Research, 1993, 60, 43-73)

Class I kinases such as the EGF-R family of receptor tyrosine kinases include the EGF, HER2-neu, erbB, Xmrk, DER and let23 receptors. These receptors are frequently present in common human cancers such as breast cancer (Sainsbury et al., Brit. J. Cancer, 1988, 58, 458; Guerin et al., Oncogene Res., 1988, 3, 21), squamous cell cancer ofthe lung (Hendler et al., Cancer Cells, 1989, 7, 347), bladder cancer (Neal et al., Lancet, 1985, 366), oesophageal cancer (Mukaida et al, Cancer, 1991, 68, 142), gastrointestinal cancer such as colon, rectal or stomach

cancer (Bolen et al., Oncogene Res., 1987, 1, 149), leukaemia (Konaka et al., Cell,

1984, 37, 1035) and ovarian, bronchial or pancreatic cancer (European Patent

Specification No. 0400586). As further human tumor tissues are tested for the EGF

family of receptor tyrosine kinases it is expected that its widespread prevalence will

be established in other cancers such as thyroid and uterine cancer.

Specifically, EGFR tyrosine kinase activity is rarely detected in normal cells

whereas it is more frequently detectable in malignant cells (Hunter, Cell, 1987, 50,

823). It has been more recently shown that EGFR is overexpressed in many human cancers such as brain, lung squamous cell, bladder, gastric, breast, head and neck,

oesophageal, gynaecological and thyroid tumours. (W J Gullick, Brit. Med. Bull.,

1991, 47, 87). Receptor tyrosine kinases are also important in other

cell-proliferation diseases such as psoriasis. EGFR disorders are those characterized

by EGFR expression by cells normally not expressing EGFR, or increased EGFR

activation leading to unwanted cell proliferation, and/or the existence of

inappropriate EGFR levels. The EGFR is known to be activated by its ligand EGF

as well as transforming growth factor-alpha (TGF- ).

The Her2-neu protein is also a member ofthe class I receptor tyrosine kinase (RTK) family. Yarden and Ullrich, Annu. Rev. Biochem. 57:443, 1988;

Ullrich and Schlessinger, Cell 61:203, 1990. Her2-neu protein is structurally related to EGFR. Carraway, et al., Cell 78:5, 1994; Carraway, et al., J. Biol. Chem. 269: 14303, 1994. These receptors share a common molecular architecture and contain two cysteine-rich regions within their cytoplasmic domains and structurally related enzymatic regions within their cytoplasmic domains.

Ligand-dependent activation of Her2-neu protein is thought to be mediated by neuactivating factor (NAF) which can directly bind to ρl65(Her2-neu) and

stimulate enzymatic activity. Dougall et al., Oncogene 9:2109, 1994; Samata et al.,

Proc. Natl. Acad. Sci. USA 91 :1711, 1994. Ligand-independent homodimerization

of Her2-neu protein and resulting receptor activation is facilitated by

over-expression of Her2-neu protein. An activated Her2-neu complex acts as a

phosphokinase and phosphorylates different cytoplasmic proteins. HER2-neu

disorders are characterized by inappropriate activity or over-activity of HER2-neu

have increased HER2-neu expression leading to unwanted cell proliferation such as

cancer. Inhibitors of receptor tyrosine kinases EGFR and HER2-neu are employed

as selective inhibitors ofthe growth of mammalian cancer cells (Yaish et al.

Science, 1988, 242, 933). For example, erbstatin, an EGF receptor tyrosine kinase

inhibitor, reduced the growth of EGFR expressing human mammary carcinoma cells

injected into athymic nude mice, yet had no effect on the growth of tumors not

expressing EGFR. (Toi et al., Eur. J. Cancer Clin. Oncol., 1990, 26, 722.) Various

derivatives of styrene are also stated to possess tyrosine kinase inhibitory properties

(European Patent Application Nos. 0211363, 0304493 and 0322738) and to be of

use as anti-tumour agents. Two such styrene derivatives are Class I RTK inhibitors

whose effectiveness has been demonstrated by attenuating the growth of human

squamous cell carcinoma injected into nude mice (Yoneda et al., Cancer Research,

1991, 51, 4430). It is also known from European Patent Applications Nos. 0520722

and 0566226 that certain 4-anilinoquinazoline derivatives are useful as inhibitors of

receptor tyrosine kinases. The very tight structure-activity relationships shown by

these compounds suggests a clearly-defined binding mode, where the quinazoline

ring binds in the adenine pocket and the anilino ring binds in an adjacent, unique

lipophilic pocket. Three 4-anilinoquinazoline analogues (two reversible and one

irreversible inhibitor) have been evaluated clinically as anticancer drugs. Denny,

Far aco 2001 Jan-Feb;56(l-2):51-6. Recently, the U.S. FDA approved the use of

the monoclonal antibody trastazumab (Herceptin®) for the treatment of HER2-neu

overexpressing metastatic breast cancers. Scheurle, et al., Anticancer Res 20:2091-

2096, 2000.

Because effective chemotherapy against tumors often 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. Studies have unsuccessfully attempted to reliably

correlate the relative levels of expression of EGFR and/or HER2-neu in malignant

cells from cancer patients with survivability.

The prognostic importance of EGFR and in NSCLC has heretofore remained

controversial. Studies using binding assays correlated increased EGFR expression

with advanced stage NSCLC and shortened overall survival,, whereas studies using

semi-quantitative techniques for measuring EGFR mRNA or protein expression

failed to show a consistent correlation with clinical outcome. Veale et al., Br. J.

Caner 68:162-165, 1993; Fujino et al., Eur. Cancer 32:2070-2074, 1996; Rusch,et al., Cancer Res 53:2379-2385, 1993; Pfeiffer, et al., Br J Cancer 74:86-91, 1996;

Pastorino, et al.,. J Clin Oncol 75:2858-2865, 1997. Studies of ΕGFR expression in

NSCLC tumors using immunohistochemical methods have shown frequencies for

ΕGFR overexpression between 32% and 47% in NSCLC tumors. Veale et al., Br. J.

Caner 55:513-516, 1987; Veale et al., Br. J. Caner 68:162-165, 1993; Fujino et al., Εur. Cancer 32:2070-2074, 1996; Rusch,et al., Cancer Res 53 :2379-2385, 1993;

Pastorino et al., J.Clin.Onc. 15:2858-2865, 1997; Tateishi, et al., Εur J Cancer

27:1372-75, 1991; Rachwal, et al., Br J Cancer 72:56-64,1995; Rusch, et al., Cancer

Res 75:2379-85,1993; Pfeiffer, et al., Br J Cancer 75:96-9, 1998; Ohsaki, et al.,

Oncol Rep 7:603-7,2000. Moreover, significant differences in EGFR expression

has been reported among histological subtypes, generally with higher EGFR

expression in SCC compared to AC and LC. Fujino et al., Εur. Cancer 32:2070-

2074, 1996; Veale et al., Br. J. Caner 55:513-516, 1987; Pastorino et al., J.Clin.Onc.

15:2858-2865, 1997; Pfeiffer, et al., Br J Cancer 76?:96-9, 1998; Ohsaki et al,

Oncol. Rep. &:603-7, 2000. However, these studies reported no consistent

correlation of EGFR overexpression with lung cancer patient survival. Observations of a purported correlation of EGFR overexpression with a

decrease in patient survival were made in some inconclusive studies. Veale et al,

1987; Ohsaki et al, 2000. However, Veale et al, analyzed a population of only

nineteen NSCLC patients. Ohsaki et al, correlated EGFR protein expression with

poor prognosis in NSCLC patients with p53 overexpression (P=0.024).

As with EGFR, the prognostic importance of HER2-neu and in NSCLC has

heretofore remained controversial. HER2-neu protein overexpression has been

demonstrated in NSCLC, including squamous cell carcinoma, adenocarcinoma, and

large cell carcinoma. Veale et al, 1987; Schneider, et al. Cancer Res 49:4968-

4971 , 1989; Kem et al. Cancer Res. 50:5184-5191, 1990; Weiner, et al. Cancer Res 50:421-425, 1990; Scheurle, et al, Anticancer Res. 20:2091-2096, 2000. Earlier studies, using protein assays, reported an association of HER2-neu protein overexpression and inferior overall survival in pulmonary adenocarcinomas (AC). Kern, et al. Cancer Res 50:5184-5191, 1990; Kern et al, J Clin Invest 93:516-20, 1994. However, contradictory studies reported no correlation of HER2-neu protein

overexpression with inferior overall survival in pulmonary adenocarcinomas (AC). Pfeiffer et al, Br. J. Cancer 74:86-91, 1996.

Another critical question is the evaluation of interrelationships between

HER2-neu and EGFR co-overexpression as prognosticators of cancer. Tateishi et

al, (Eur. J. Cancer 27: 1372-75, 1991), measured EGFR and HER2-neu protein co-

expression, in 13% of AC analysed, and found that co-overexpression of these two

genes correlated with inferior 5 -year survival. However, as with HER2-neu

overexpression alone, association between HER2-neu and EGFR co-expression and

survival in squamous cell carcinoma (SCC) and large cell carcinoma (LCC) ofthe

lung has not been reported. Inconsistent methodologies for the determination of EGFR and HER2-neu

expression levels has been at the root ofthe problem in determining to what extent

expression of these genes may be used to prognosticate cancer patient survivability.

Heretofore investigations oϊHER2-neu and EGFR expression in NSCLC has

resulted in enormous variations in frequencies of NSCLC tumors scored positive for

both EGFR and HER2-neu expression. Overexpression of HER2-neu, defined as

positive protein staining in adenocarcinomas (AC), was reported in 13-80%, in 2-

45% in squamous cell carcinomas (SCC), and in 0-20% in large cell carcinomas

(LC) by using paraffin embedded tissue on light microscope slides and HER2-neu

antisera. Pfeiffer et al, 1996; Kern et al, 1 90; Kern et al, 1994; Tateishi et al,

1991; Shi, et al, Mol Carcing 5:213-8, 1992; Bongiorno, et al, J Thorac

Cardiovasc Surg 707:590-5,1994; Harpole, et al, Clin Cancer Res 7:659-64, 1995;

Volm et al, Anticancer Res 12: 11 -20,1992. Moroever, a recent report illustrates the non-specificity of current protocols designed to assess HER2-neu expression levels. The HercepTest® for measurement of HER2-neu expression in invasive breast cancers was shown to have very high false positivity. Jacobs et al, J Clin Oncol

77:1983-1987, 1999.

If a precise, accurate, and consistent method for determining the expression

levels of EGFR and HER2-neu existed, one could ascertain what expression levels

correlate to patient survivability and whether or not a receptor tyrosine kinase

targeted chemotherapy is appropriate. Consistent demonstration of EGFR and/or

ER2-neu overexpression in NSCLC, using a standardized method, is desirable in

establishing clinical trials for current and future receptor tyrosine kinase targeted

chemotherapies, e.g., chemotherapeutic agents, antibody-based drugs, to treat

cancers overexpressing these receptors. The current protocols for measuring EGFR and/or HER2-neu gene

expression, aside from being insufficiently accurate for tumor prognostication,

suffer from a second limitation in that they require a significant amount of fresh

tissue that contains non-degraded mRNA. Most patient derived pathological

samples are routinely fixed and paraffin-embedded (FPE) to allow for histological

analysis and subsequent archival storage. Thus, most biopsy tissue samples are not

useful for analysis of gene expression because such studies require a high integrity

of RNA so that an accurate measure of gene expression can be made. Currently, gene expression levels can be only qualitatively monitored in such fixed and embedded samples by using immunohistochemical staining to monitor protein expression levels.

The use of frozen tissue by health care professionals poses substantial inconveniences. Rapid biopsy delivery to avoid tissue and subsequent mRNA degradation is the primary concern when planning any RNA-based quantitative

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

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

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

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

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

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

integrity during transport using specialized couriers equipped with liquid nitrogen

and dry ice, comes only at a great expense. 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 thought to be 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. etal, (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 then be used to determine the

likelihood of success or appropriateness of a particular treatment.

We report here a significant association between high levels of the

intratumoral EGFR mRNA and high levels of intratumoral HER2-neu mRNA with

an inferior survivability. Accordingly, it is the object ofthe invention to provide a

method of quantifying EGFR and/or HER2-neu mRNA from tumor tissue in order to provide an early prognosis for receptor tyrosine kinase targeted chemotherapies. It is also the object ofthe invention to provide a method for assessing EGFR and/or HER2-neu levels in tissues fixed and paraffin-embedded (FPE) and predicting the probable sensitivity of a patient's tumor to treatment with receptor tyrosine kinase targeted chemotherapy by examining the ainountEGER and/or HER2-neu mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression

level.

SUMMARY OF THE INVENTION

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

of expression of EGFR mRNA obtained from fresh, frozen, fixed or fixed and

paraffin-embedded (FPE) tumor cells.

In another aspect ofthe invention there is provided a method for assessing

levels of expression of ^'HER2-neu mRNA obtained from fresh, frozen, fixed or fixed

and paraffin-embedded (FPE) tumor cells.

In another aspect ofthe invention there is provided a method of quantifying the amount of ^'EGFR mRNA expression relative to an internal control from a fresh, frozen, fixed or fixed and paraffin-embedded (FPE) tissue sample. This method includes isolation of total mRNA from said sample and determining the quantity of

EGFR mRNA relative to the quantity of an internal control gene's mRNA.

In another aspect of he invention there is provided a method of quantifying the amount of HELR2-neu mRNA expression relative to an internal control from a

fresh, frozen, fixed or fixed and paraffin-embedded (FPE) tissue sample. This method includes isolation of total mRNA from said sample and determining the

quantity of HER2-neu mRNA relative to the quantity of an internal control gene's

mRNA.

In an embodiment of this aspect ofthe invention, there are provided

oligonucleotide primers having the sequence of EGFR-1753F (SEQ ID NO: 1) or

EGFR-1823R (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 another embodiment of this aspect ofthe invention, there are provided

oligonucleotide primers having the sequence of HER2-neu 267 IF (SEQ ID NO: 4)

or HER2-neu 2699R (SEQ ID NO: 5) and sequences substantially identical thereto.

The invention also provides for oligonucleotide primers having a sequence that

hybridizes to SEQ ID NO: 4 or SEQ ID NO: 5 or their complements under stringent

conditions.

In yet another aspect ofthe invention there is provided a method for

determining a receptor tyrosine kinase targeted chemotherapeutic regimen for a patient, comprising isolating RNA from a fresh, frozen, fixed or fixed and paraffin- embedded (FPE) tumor sample; isolating RNA from a fresh, frozen, fixed or fixed and paraffin-embedded (FPE) matching non-malignant tissue sample; determining a gene expression level of EGFR in both samples; dividing the level of EGFR expression in the tumor sample with the EGFR expression level in the matching non-malignant tissue sample to determine a differential expression level; comparing

the differential EGFR gene expression level with a predeterimined threshold level for the EGFR gene; and determining a chemotherapeutic regimen based on results

ofthe comparison ofthe differential EGFR gene expression level with the

predetermined threshold level

In yet another aspect ofthe invention there is provided a method for

determining a receptor tyrosine kinase targeted chemotherapeutic regimen for a

patient, comprising isolating RNA from a fresh, frozen, fixed or fixed and paraffin-

embedded (FPE) tumor sample; isolating RNA from a fresh, frozen, fixed or fixed

and paraffin-embedded (FPE) matching non-malignant tissue sample; determining a

gene expression level of HER2-neu in both samples; dividing the level of HER2-neu expression in the tumor sample with the HER2-neu expression level in the matching

non-malignant tissue sample to determine a differential expression level; comparing

the differential HER2-neu gene expression levels with a predeteri ined threshold

level for the HER2-neu gene; and determining a chemotherapeutic regimen based on

results of the comparison of the differential HER2-neu gene expression level with

the predetermined threshold level

In yet another aspect ofthe invention there is provided a method for

determining a receptor tyrosine kinase targeted chemotherapeutic regimen for a patient, comprising isolating RNA from a fresh, frozen, fixed or fixed and paraffin- embedded (FPE) tumor sample; isolating RNA from a fresh, frozen, fixed or fixed and paraffin-embedded (FPE) matching non-malignant tissue sample; determining gene expression levels of HER2-neu and EGFR in both ofthe samples; dividing the level of EGFR expression in the tumor sample with the EGFR expression level in the matching non-malignant tissue sample to determine a EGFR differential expression level; dividing the level of HEJU-neu expression in the tumor sample with the HEJU-neu expression level in the matching non-malignant tissue sample to

determine a differential HEJU-neu expression level; comparing the differential

HEJU-neu and EGFR gene expression levels with a predeterimined threshold level

for each ofthe HEJU-neu and EGFR genes; and determining a chemotherapeutic

regimen based on results of the comparison of the differential HEJU-neu and EGFR

gene expression levels with the predetermined threshold levels.

In yet another aspect ofthe invention there is provided a method for

determining the survivability of a patient, comprising isolating RNA from a fresh,

frozen, fixed or fixed and paraffin-embedded (FPE) tumor sample; isolating RNA from a fresh, frozen, fixed or fixed and paraffin-embedded (FPE) matching non-

malignant tissue sample; determining a gene expression level of EGFR in both

samples; dividing the level of EGFR expression in the tumor sample with the E FR

expression level in the matching non-malignant tissue sample to determine a

differential expression level; comparing the differential EGFR gene expression level

with a predeterimined threshold level for the EGFR gene; and determining the

survivability of a patient based on results ofthe comparison ofthe differential EGFR gene expression levels with ti

In yet another aspect ention , ,n υ -. , •. .., a method for

determining the survivability of a patient, comprising isolating RNA from a fresh, frozen, fixed or fixed and paraffin-embedded (FPΕ) tumor sample; isolating RNA from a fresh, frozen, fixed or fixed and paraffin-embedded (FPΕ) matching non- malignant tissue sample; determining a gene expression level of HEJU-neu in both samples; dividing the level of HER2-neu expression in the tumor sample with the

EGFR expression level in the matching non-malignant tissue sample ^* ermine a

differential expression level; comparing the differential HE⁷ . >e expression levels with a predeterimined threshold level for the HI gene; and

determining the survivability of a patient based < of the comparison of the

differential HER2-neu gene expression leve' m the predetermined threshold

level

In yet another aspect ofthe invention there is provided a method for

determining the survivability of a patient, comprising isolating RNA from a fresh,

frozen, fixed or fixed and paraffin-embedded (FPΕ) tumor sample; isolating RNA

from a fresh, frozen, fixed or fixed and paraffin-embedded (FPΕ) matching non- malignant tissue sample; determining gene expression levels of HEJU-neu and

EGFR in both of he samples; dividing the level of EGFR expression in the tumor

sample with the EGFR expression level in the matching non-malignant tissue

sample to determine a EGFR differential expression level; dividing the level of

HER2-neu expression in the tumor sample with the HER2-neu expression level in

the matching non-malignant tissue sample to determine a HEJU-neu differential

expression level; comparing the differential HEJU-neu and EGFR gene expression

levels with a predetermined threshold level for each ofthe HER2-neu and EGFR

genes; and determining the survivability of a patient based on results of the comparison of the EGFR and HER2~neu gene expression levels with the predetermined threshold levels.

The invention further relates to a method of normalizing the uncorrected gene expression (UGE) of EGFR and HEJU-neu relative to an internal control gene in a tissue sample analyzed using TaqMan® technology to known EGFR&nd HER2- neu expression levels relative to an internal control from samples analyzed by pre-

TaqMan® technology.

DESCRIPTION OF THE DRAWINGS

Figure 1. Estimated probability of survival of curatively resected non-small

cell lung cancer patients versus the HER2-neu mRNA expression status. The median

survival was not reached in the low HER2-neu expression group compared to 31 J

months (95% CI: 21.96- 40.24) in the highH£R2-new expression group(P=0.004).

Figure 2. Estimated probability of survival of curatively resected non-small cell lung cancer patients versus the EGFR mRNA expression status. A trend towards inferior overall survival was observable for the high EGFR expression group, but

did not reach statistical significance. The median survival was not reached in the

low EGFR expression group compared to 32.37 months (95% CJ: 8.43-56.31) in

the high EGFR expressor group (E=0J76).

Figure 3. Estimated probability of survival of curatively resected non-small

cell lung cancer patients versus combined patterns of EGFR and HER2-neu co-

expression in NSCLC. The median surviv

showed low HEJU-neu and EGFR - - prt .^ , compared to 45.47 months in the

highEGER expression group, 31.10 months (95% CL: 14.77-47.43) in the high HEJU-neu expression group, and 22.03 months (95% C.I: 2.30-41.16; RO.003) in

the high HEJU-neu and EGFR expression group.

Figure 4. Table showing high and low EGFR and HER2-neu expression in patients and tumors.

Figure5. Table showing the survival of patients based on clinical and molecular parameters .

Figure 6. Table showing Cox-proportional h « dgression models. Double

marker refers to both EGFR and HEJU-neu e a.

Figure 7 is a chart illustrating h/ calculate EGFR expression relative to

an internal control gene. The chart ^r is data obtained with two test samples,

(unknowns 1 and 2), and illustrotcs how to determine the uncorrected gene

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

by the TaqMan® instrument with known relative EGFR values determined by pre-

TaqMan® technology. This is accomplished by multiplying UGE to a correction factor Lsam- The internal control gene in the figure is β-actin and the calibrator

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

Figure 8 is a chart illustrating how to calculate HEJU-neu expression relative

to an internal control gene. The chart contains data obtained with two test samples,

(unknowns 1 and 2), and illustrates how to determine the uncorrected gene

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

by the TaqMan® instrument with previously published HEJU-neu values. This is

accomplished by multiplying UGE to a correction factor ^~ _HER2._πeu. The internal

control gene in the figure is β-actin and the calibrator RNA is Human Liver Total

RNA (Stratagene, Cat #735017).

Figure 9 is a graph showing the corrected EGFR expression values of 5 different colon cancer patients' tumors. The patients were on a CPT-11/C225 receptor tyrosine kinase targeted treatment regimen. Patient 1 was determined to have a corrected EGFR expression level of 2.08 x 10'³ and had a completed response (CR). Patient 2 had a corrected EGFR expression level of 8.04 x 10'³ and had a

partial response (PR). Patient 3 had a corrected EGFR expression level of 1.47 x

10"³ and also showed a partial response (PR). Patient 4 had a corrected EGFR

expression level of 0J6 x x 10^"3 and had stable disease (SD) showing no response.

Patient 5 had a no EGFR expression ( 0.0 x 10^"3) and had progressive disease (PR). DETAILED DESCRIPTION OF THE INVENTION

Tumors expressing high levels of HER2-neu and/or EGFR mRNA are

considered likely to be sensitive to receptor tyrosine kinase targeted chemotherapy.

Conversely, those tumors expressing low amounts of HER2-neu and EGFR mRNA

are not likely to be sensitive to receptor tyrosine kinase targeted chemotherapy. A

patient's differential HEJU-neu and EGFR mRNA expression status is judged by

comparing it to a predetermined threshold expression level.

The invention provides a method of quantifying the amount of HEJU-neu and/or EGFR mRNA expression in fresh, frozen, fixed or fixed and paraffϊn- embedded (FPE) tissue relative to gene expression of an internal control The present inventors have developed oligonucleotide primers that allow accurate assessment of HER2-neu and EGFR gene expression in fresh, frozen, fixed or fixed and embedded tissues. The oligonucleotide primers, EGFR-1753F (SEQ ID NO: 1),

EGFR-1823R (SEQ ID NO: 2), or oligonucleotide primers substantially identical thereto, preferably are used together with RNA extracted from fresh, frozen, fixed or fixed and paraffin embedded (FPE) tumor samples. The invention also provides

oligonucleotide primers, HER2-neu 267 IF (SEQ ID NO: 4), HER2-neu 2699R

(SEQ ID NO: 5), or oligonucleotide primers substantially identical thereto,

preferably are used together with RNA extracted from fresh, frozen, fixed or fixed

and paraffin embedded (FPE) tumor samples. This measurement of HEJU-neu

and or EGFR gene expression may then be used for prognosis of receptor tyrosine

kinase targeted chemotherapy

This embodiment ofthe invention involves, a method for reliable extraction

of RNA from fresh, frozen, fixed or FPE samples, determination ofthe content of EGFR mRNA in the sample by using a pair of oligonucleotide primers, preferably

oligionucleotide primer pair EGFR-1753F (SEQ ID NO: 1) and EGFR-1823R (SEQ

ID NO: 2), or oligonucleotides substantially identical thereto, for carrying out

reverse transcriptase polymerase chain reaction.

Another embodiment ofthe invention involves a method for reliable

extraction of RNA from fresh, frozen, fixed or FPE samples, and determination of

the content of HEJU-neu mRNA in the sample by using a pair of oligonucleotide primers oligonucleotide primers, HER2-neu 267 IF (SEQ ID NO: 4), HER2-neu

2699R (SEQ ID NO: 5), or oligonucleotide primers substantially identical thereto. " 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% ofthe 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).

The methods ofthe 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 ^'HEJU-neu and/or EGFR are determined in

individual patient samples and response to various chemotherapeutics is predicted.

Preferably, the methods ofthe invention are applied to solid tumors, most preferably

NSCLC tumors. A "differential expression level" as defined herein refers to the difference in

the level of expression of either EGFR or HEJU-neu in a tumor with respect to the

level of expression of either EGFR or HEJU-neu in a matching non-malignant tissue

sample, respectively. The differential expression level is determined by dividing the

UGE of a particular gene from the tumor sample with the UGE of the same gene

from a matching non-malignant tissue sample.

A "predetermined threshold level", as defined herein relating to EGFR

expression, is a level of differential EGFR expression above which (i.e., high), tumors are likely to be sensitive to a receptor tyrosine kinase targeted chemotherapeutic regimen. A high differential EGFR expression level is prognostic of lower patient survivability. Tumors with expression levels below this threshold level are not likely to be affected by a receptor tyrosine kinase targeted chemotherapeutic regimen. A low differential EGFR expression level is prognostic of higher patient survivability. Whether or not differential expression is above or below a "predetermined threshold level" is determined by the method used by

Mafune et al, who calculated individual differential tumor/normal (T N) expression

ratios in matching non-malignant tissues obtained from patients with squamous cell

carcinoma ofthe esophagus. Mafune et al, Clin Cancer Res 5:4073-4078, 1999.

This method of analysis leads to a precise expression value for each patient, being

based on the individual background expression obtained from matching non-

malignant tissue. The differential expression of EGFR is considered "high" and

indicative of low survivability if the UGE of EGFR : β-actin in a tumor sample

divided by the UGE of EGFR : β-actin in a matching non-malignant tissue sample, is above the predetermined threshold value of about 1.8. The differential expression

of EGFR is considered "low" and indicative of high survivability if the UGE of

EGFR : β-actin in a tumor sample divided by the UGE of EGFR : β-actin in a

matching n+on-malignant tissue sample, is below the predetermined threshold value

of about 1.8.

A "predetermined threshold level", as defined herein relating to differential

HER2-neu expression, is a level of HEJU-neu expression above which (i.e., high),

tumors are likely to be sensitive to a receptor tyrosine kinase targeted chemotherapeutic regimen. A high differential HEJU-neu expression level is prognostic of lower patient survivability. Tumors with expression levels below this threshold level are not likely to be affected by a receptor tyrosine kinase targeted chemotherapeutic regimen. A low differential HEJU-neu expression level is prognostic of higher patient survivability. The differential expression of HEJU-neu is considered "high" and indicative of low survivability if the UGE ofHER2-neu :

. β-actin in a tumor sample divided by the UGE of HER2-neu : β-actin in a matching

non-malignant tissue sample, is above the predetermined threshold value of about

1.8. The differential expression of HEJU-neu is considered "low" and indicative of

high survivability if the UGE of HEJU-neu : β-actin in a tumor sample divided by

the UGE of HEJU-neu : β-actin in a matching non-malignant tissue sample, is

below the predetermined threshold value of about 1.8.

A "threshold level" for HEJU-neu was determined using the following

results and method. The corrected HEJU-neu mRNA expression, expressed as the

-ratio between HEJU-neu and β-Actin PCR product, was 4J7 x 10^"3 (range 0.28- 23.86 x 10^"3) in normal lung and 4.35 x 10^"3 (range: 0.21-68..1 x 10'³) in tumor

tissue (P=O.Ol 9 Wilcoxon test). The maximal chi-square method by Miller and

Siegmund (Miller et al. Biometrics 38:1011-1016, 1982) and Halpern (Biometrics

35:1017-1023, 1982) determined a threshold value of 1.8 to segregate patients into

low and high differential HEJU-neu expressors. By this criterion, 29 (34.9%)

patients had a high differential HER2-neu expression and 54 (65.1 %) had a low

differential HEJU-neu expression.

A "threshold level" for EGFR was determined using the following results and method. The median corrected EGFR mRNA expression was 8J7 x 10"³(range: 0.31 -46.26 x 10"³) in normal lung and 7.22 x 10'³ (range: 0.27-97.49 x 10"³) in tumor tissue (P=n.s.). The maximal chi-square method (Miller (1982); Halpern (1982)) determined a threshold value of 1.8 to segregate patients into low and high differential EGFR expressors. By this criterion, 28 (33.7%) patients had a high differential EGFR expression and 55 (66.3%) had a low differential EGFR

expression status .

In performing the method ofthe present invention either differential EGFR

^"expression levels or differential HEJU-neu expression levels are assayed in a patient

to prognosticate the efficacy of a receptor tyrosine kinase targeted chemotherapeutic

regimen. Moreover, in the method ofthe present invention differential HER2-neu

expression levels are assayed in a patient prognosticate the efficacy of a receptor

tyrosine kinase targeted chemotherapeutic regimen. Additionally, in the method of

the present invention differential EGFR expression levels are assayed in a patient to

prognosticate the efficacy of a receptor tyrosine kinase targeted chemotherapeutic regimen. Alternatively, both differential EGFR expression levels and differential HER2-neu expression levels are assayed in a patient to prognosticate the efficacy of

a receptor tyrosine kinase targeted chemotherapeutic regimen.

"Matching non-malignant sample" as defined herein refers to a sample of

non-cancerous tissue derived from the same individual as the tumor sample to be

analyzed for differential EGFR and/or differential HEJU-neu expression. Preferably

a matching non-malignant sample is derived from the same organ as the organ from

which the tumor sample is derived. Most preferably, the matching non-malignant

tumor sample is derived from the same organ tissue layer from which the tumor sample is derived. Also, it is preferable to take a matching non-malignant tissue sample at the same time a tumor sample is biopsied. In a preferred embodiment tissues from the following two locations are analyzed: lung tumor and non- malignant lung tissue taken from the greatest distance form the tumor or colon tumor and non-malignant colon tissue taken from the greatest distance form the tumor as possible under the circumstances. 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 tumor samples is extracted from the cells

by any ofthe 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

ofthe 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. See Plenat et al,

Ann Pathol 2001 Jan;21(l):29-47. Non-embedded, fixed tissue as well as fixed and

embedded tissue may also be used in the present methods. Solid supports for

embedding fixed tissue are envisioned to be removable with organic solvents for

example, allowing for subsequent rehydration of preserved tissue.

RNA is extracted from paraffin-embedded (FPE) tissue cells by any ofthe methods as described in US Patent Application No. 09/469,338, filed December 20, 1999, which is hereby incorporated by reference in its entirety. As used herein, FPE

tissue means tissue that has been fixed and embedded in ansolid removable support, such as storable or archival tissue samples. RNA may be isolated from an archival pathological sample or biopsy sample which is first deparaffinized. An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent, such as xylene, 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 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,

for example: guanidinium compounds, urea, formamide, potassium iodiode,

potassium thiocyantate and similar compounds. The preferred chaotropic agent for the methods ofthe invention is a guanidinium compound, such as guanidinium isothiocyanate (also sold as guanidinium thiocyanate) and guanidinium hydrochloride. Many anionic counterions are useful, and one of skill in the art can prepare many guanidinium salts with such appropriate anions. The effective concentration of guanidinium solution used in the invention generally has a concentration in the range of about 1 to about 5M with a preferred value of about 4M.- If RNA is already in solution, the guanidinium solution may be of higher

concentration such that the final concentration achieved in the sample is in the range

of about 1 to about 5M. The guanidinium solution also is preferably buffered to a

pH of about 3 to about 6, more preferably about 4, with a suitable biochemical

buffer such as Tris-Cl. The chaotropic solution may also contain reducing agents,

such as dithiothreitol (DTT) and β-mercaptoethanol (BME). The chaotropic

solution may also contain RNAse inhibitors.

RNA is then recovered from the chaotropic solution by, for example, phenol

chloroform extraction, ion exchange chromatography or size-exclusion

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

techniques.

The quantification of HER2-neu or EGFR 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 HER2 -neu or EGFR 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 HER2-neu and/or EGFR mRNA via use of a PCR-free systems employing, for example fluorescent labeled probes similar to those ofthe Invader® Assay (Third Wave Technologies, Inc.). Most preferably, quantification of HER2-neu and/or EGFR 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 ofthe 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 any

constitutively or globally expressed gene whose presence enables an assessment of

HEJU-neu and/or EGFR mRNA levels. Such an assessment comprises a determination ofthe 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 HER2-neu and/or EGFR expression relative to an internal control gene

generated by the TaqMan® instrument. The equation used to determine UGE is

shown in Examples 3 and 4, and illustrated with sample calculations in Figures 7

and 8.

These numerical values allow the determination of whether or not the

differential gene expression (i.e., "UGE" or of a particular tumor sample divided by

the "UGE" of a matching non-tumor sample) falls above or below the

"predetermined threshold" level. The predetermined threshold level for EGFR and

HEJU-neu is about 1.8.

A further aspect of this invention provides a method to normalize

uncorrected gene expression (UGE) values acquired from the TaqMan® instrument

with "known relative gene expression" values derived from non-TaqMan® technology. Preferably, TaqMan® derived HEJU-neu and/or EGFR UGE values

from a tissue sample are normalized to samples with known non-TaqMan® derived

relative HER2-neu and/or EGFR : β-actin expression values.

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

EGFR expression whereby UGE is multiplied with a EGFR specific correction

factor (K_EGFR , resulting in a value that can be compared to a known range of EGFR

expression levels relative to an internal control gene. Example 3 and Figure 7

illustrate these calculations in detail. K_EGFR specific for EGFR, the internal control

β-actin and calibrator Human Liver Total RNA (Stratagene, Cat #735017), is 26.95

x 10^"3. These numerical values also allow the determination of whether or not the "Corrected Relative Expression" of a particular tumor sample divided by the "Corrected Relative Expression" of a matching non-tumor sample (i.e., differential expression) falls above or below the "predetermined threshold" level. The predetermined threshold level for HEJU-neu or EGFR is about 1.8. In determining whether the differential expression of either EGFR or HER2-neu in a tumor sample is 1.8 times greater than in a matching non-tumor sample, one will readily recognize

that either UGE values or Corrected Relative Expression values can be used. For

example, if one divides the Corrected Relative Expression level ofthe tumor with

that ofthe matching non-tumor sample, the K-factor cancels out and one is left with

same ratio as if one had used UGE values.

"Known relative gene expression" values are derived from previously

analyzed tissue samples and are based on the ratio ofthe RT-PCR signal of a target

gene to a constitutively expressed internal control gene (e.g. β-Actin, GAPDH, etc.). Preferably such tissue samples are formalin fixed and paraffin-embedded (FPE)

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

Example 1. To quantify gene expression relative to an internal control standard

quantitative RT-PCR technology known in the art is used. Pre-TaqMan®

technology PCR reactions are run for a fixed number of cycles (i.e., 30) and

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

ratio of EGFR expression to β-actin expression.

K_EGFR 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 EGFR expression levels relative to that particular internal control gene have already been determined (i.e., "known relative gene expression"). Preferably such tissue samples are formalin fixed and paraffin- embedded (FPE) samples and RNA is extracted from them according to the protocol described in Example 1. Such a determination can be made using standard pre- TaqMan®, quantitative RT-PCR techniques well known in the art. Upon such a

determination, such samples have "known relative gene expression" levels of EGFR

useful in the determining a new K_EGFR specific for the new internal control and/or

calibrator RNA as described in Example 3.

"Corrected Relative HER2-neu Expression" as used herein refers to

normalized HER2-neu expression whereby UGE is multiplied with a HEJU-neu

specific correction factor (Kmx₂-_n< )_> resulting in a value that can be compared to a known range of HEJU-neu expression levels relative to an internal control gene. Example 4 and Figure 8 illustrate these calculations in detail. K_HER2._πea specific for

HEJU-neu, the internal control β-actin and calibrator Human Liver Total RNA

(Stratagene, Cat #735017), is 13.3 x 10^"3.

K._HER2-„_eu 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 HEJU-neu 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 herein. Such a determination can be made using standard pre- TaqMan®, quantitative RT-PCR techniques well known in the art, for example. Upon such a determination, such samples have "known relative gene expression" levels o ^'HEJU-neu useful in the determining a new K._mR2._neu specific for the new internal control and/or calibrator RNA as described in Example 4.

The methods ofthe 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 NSCLC tumors.

Pre-chemotherapy treatment tumor biopsies are usually available only as

fixed paraffin embedded (FPE) tissues, generally containing only a very small

amount of heterogeneous tissue. Such FPE samples are readily amenable to microdissection, so that HER2-neu and/or EGFR gene expression may be

determined in tumor tissue uncontaminated with non-malignant stromal tissue.

Additionally, comparisons can be made between non-malignant stromal and tumor

tissue within a biopsy tissue sample, since such samples often contain both types of

tissues.

Generally, any oligonucleotide pairs that flank a region of EGFR gene, as

shown in SEQ ID NO: 10, may be used to carry out the methods ofthe invention. Primers hybridizing under stringent conditions to a region ofthe EGFR 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 primer pairs and oligonucleotide primers substantially identical thereto, that allow particularly accurate assessment of EGFR expression using fresh, frozen, fixed or FPE tissues. Preferable are oligonucleotide primers, EGFR-1753F (SEQ ID NO: 1) and EGFR-

1823R (SEQ JD NO: 2), (also referred to herein as the oligonucleotide primer pair EGFR) and oligonucleotide primers substantially identical thereto. The

oliogonucleotide primers EGFR-1753F (SEQ JD NO: 1) and EGFR-1823R, (SEQ

ID NO: 2) have been shown to be particularly effective for measuring EGFR mRNA

levels using RNA extracted from fresh, frozen, fixed or FPE cells by any ofthe

methods for mRNA isolation, for example as described Example 1.

Furthermore, any oligonucleotide pairs that flank a region of HEJU-neu

gene, as shown in SEQ ID NO: 11, may be used to carry out the methods ofthe

invention. Primers hybridizing under stringent conditions to a region ofthe HEJU- neu gene for use in the present invention will amplify a product between about 20- 1000 base pairs, preferably about 50-100 base pairs, most preferably less than about

100 base pairs.

The invention provides specific oligonucleotide primers pairs and

oligonucleotide primers substantially identical thereto, that allow particularly

accurate assessment of HEJU-neu expression in fresh, frozen, fixed or FPE tissues.

Preferable are oligonucleotide primers, HER2-neu 267 IF (SEQ ID NO: 4) and

HER2-neu 2699R (SEQ ID NO: 5), (also referred to herein as the oligonucleotide

primer pair HER2-neu) and oligonucleotide primers substantially identical thereto. The oliogonucleotide primers HER2-neu 2671F (SEQ JD NO: 4) and HER2-neu 2699R (SEQ JD NO: 5) have been shown to be particularly effective for measuring HEJU-neu mRNA levels using RNA extracted from fresh, frozen, fixed or FPE cells by any ofthe methods for mRNA isolation, for example as described herein. This invention includes substantially identical oligonucleotides that hybridize under stringent conditions (as defined herein) to all or a portion ofthe

oligonucleotide primer sequence of EGFR-1753F (SEQ ID NO: 1), its complement or EGFR-1823R (SEQ JD NO: 2), or its complement or oligonucleotide primer

sequence of HER2-neu 2671F (SEQ ID NO: 4), its complement or HER2-neu

2699R (SEQ ID NO: 5), or its complement.

Under stringent hybridization conditions, only highly complementary, i.e.,

substantially similar nucleic acid sequences as defined herein 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 ofthe nucleic acids is typically at least about 10

(e.g., 15) nucleotides in length. The hybridizing portion ofthe 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

EGFR-1753F (SEQ JD NO: 1), its complement or EGFR-1823R (SEQ JD NO: 2),

or its complement or oligonucleotide primer HER2-neu 267 IF (SEQ JD NO: 4), its

complement or HER2-neu 2699R (SEQ JD NO: 5), or its complement.

Hybridization ofthe 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 temperture 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 holmologous 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 temperatre ofthe 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 about 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 washh g in 3x SSC at about

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 (1 94).

Oligonucleotide primers disclosed herein are capable of allowing accurate

assessment of HEJU-neu and/or EGFR 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 ofthe invention are suitable for use in assaying HER2-neu and/or EGFR gene expression levels in all tissues where previously there existed no accurate and consistent way to assay HEJU-neu and/or EGFR gene in fresh and frozen tissues and no way at all to assay HEJU-neu and/or EGFR gene expression using fixed tissues. Over-activity of HER2-neu refers to either an amplification ofthe gene

encoding HER2-neu or the production of a level of HER2-neu activity which can be

correlated with a cell proliferative disorder (i.e., as the level of HER2-neu increases

the severity of one or more ofthe symptoms ofthe cell proliferative disorder

increases).

A "receptor tyrosine kinase targeted" chemotherapy or chemotherapeutic

regimen in the context ofthe present invention refers a chemotherapy comprising

agents that specifically interfere with Class I receptor tyrosine kinase function.

Preferably, such agents will inhibit EGFR and/or HER2-neu receptor tyrosine kinase signaling activity. Such agents include 4-anilinoquinazolines such as 6-acrylamido-4-anilinoquinazoline Bonvini et al. Cancer Res. 2001 Feb

15;61(4):1671-7 and derivatives, erbstatin (Toi et al, Eur. J. Cancer Clin. Oncol,

1990, 26, 722.), Geldanamycin, bis monocyclic, bicyclic or heterocyclic aryl

compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO

94/14808) and l-cycloρroρρyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992)

which have been described generally as tyrosine kinase inhibitors. Also, Styryl

compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), certain quinazoline derivatives (EP Application No. 0 566266

Al), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO

91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer.

Other agents targeting EGFR and/or HER2-neu receptor tyrosine kinase signaling activity include antibodies that inhibit growth factor receptor biological function indirectly by mediating cytotoxicity via a targeting function

Antibodies complexing with the receptor activates serum complement and/or

mediate antibody-dependent cellular cytotoxicity. The antibodies which bind the

receptor can also be conjugated to a toxin (immunotoxins). Advantageously

antibodies are selected which greatly inhibit the receptor function by binding the

steric vicinity of the ligand binding site of the receptor (blocking the receptor),

and/or which bind the growth factor in such a way as to prevent (block) the ligand

from binding to the receptor. These antibodies are selected using conventional in

vitro assays for selecting antibodies which neutralize receptor function. Antibodies that act as ligand agonists by mimicking the ligand are discarded by conducting suitable assays as will be apparent to those skilled in the art. For certain tumor cells,

the antibodies inhibit an autocrine growth cycle (i.e. where a cell secretes a growth

factor which then binds to a receptor ofthe same cell). Since some ligands, e.g.

TGF-α, are found lodged in cell membranes, the antibodies serving a targeting

function are directed against the ligand and/or the receptor

The cytotoxic moiety ofthe immunotoxin may be a cytotoxic drug or an

enzymatically active toxin of bacterial or plant origin, or an enzymatically active

fragment of such a toxin. Enzymatically active toxins and fragments thereof used are diphtheria, nonbinding active fragments of diphtheria toxin, exotoxin (from Pseudomonas aeruginosa), ricin, abrin, modeccin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPJI, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin. In another embodiment, the antibodies are conjugated to small molecule anticancer drugs. Conjugates ofthe monoclonal antibody and such cytotoxic moieties are made using a variety of bifunctional protein coupling agents. Examples of such reagents are

SPDP, IT , bifunctional derivatives of imidoesters such a dimethyl adipimidate HCI,

active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde,

bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine, bis-diazonium

derivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such

as tolylene 2,6-diisocyanate, and bis-active fluorine compounds such as

l,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin may be joined to the

Fab fragment ofthe antibodies. Cytotoxic radiopharmaceuticals for treating cancer may be made by

conjugating radioactive isotopes to the antibodies. The term "cytotoxic moiety" as

used herein is intended to include such isotopes.

In another embodiment, liposomes are filled with a cytotoxic drug and the

liposomes are coated with antibodies specifically binding a growth factor receptor.

Since there are many receptor sites, this method permits delivery of large amounts

of drug to the appropriate cell type. The exact formulation, route of administration

and dosage can be chosen by the individual physician in view ofthe patient's

condition. (See e.g. Fingl et al, in The Pharmacological Basis of Therapeutics,

1975, Ch. 1 p. 1). It should be noted that the attending physician would know how

and when to terminate, interrupt, or adjust administration due to toxicity, or organ

dysfunctions. Conversely, the attending physician would also know to adjust

treatment to higher levels if the clinical response were not adequate (precluding

toxicity). The magnitude of an administrated dose in the management ofthe

oncogenic disorder of interest will vary with the severity of the condition to be

treated and to the route of administration. The severity ofthe condition may, for

example, be evaluated, in part, by standard prognostic evaluation methods. Further,

the dose and perhaps dose frequency, will also vary according to the age, body

weight, and response ofthe individual patient.

Depending on the specific conditions being treated, such agents may be

formulated and administered systemically or locally. Techniques for formulation

and

admimstration may be found in Remington's Pharmaceutical Sciences, 18th ed.

Mack Publishing Co, Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery,

including intramuscular, subcutaneous, intramedullary injections, as well as

intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or

intraocular injections, just to name a few. For injection, the agents ofthe invention

may be formulated in aqueous solutions, preferably in physiologically compatible

buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.

For such transmucosal administration, penetrants appropriate to the barrier to be

permeated are used in the formulation. Such penetrants are generally known in the

art.

The invention 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 ofthe present invention.

EXAMPLES

EXAMPLE 1

RNA Isolation from FPE Tissue

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

procedure.

A. Deparafiinization 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 ofthe bench top centrifuge (about 10-20,000 x g).

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

dissolved. Two or more times are normally required depending on the amount of paraffin included in the original sample portion. (5) The xylene solution is removed by vigorously shaking with a lower alcohol, preferably with 100% ethanol (about 600 μL) for about 3 minutes.

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

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

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

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

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

temperature for about 5 minutes.

B. RNA Isolation with Phenol-Chloroform

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

μL dithiothreitol is added. (2) The sample is then homogenized with a tissue homogenizer (Ultra-

Turrax, TKA-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 ofthe 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 alcoholchloroform solution. The solution is shaken vigorously for about 10 seconds then cooled on ice for about 15 minutes.

(5) The solution is centrifuged for about 7 minutes at maximum speed. The upper (aqueous) phase is transferred to a new tube. (6) The RNA is precipitated with about 10 μL glycogen and with 400 μL isopropanol for 30 minutes at -20 °C.

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

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

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

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

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

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

EXAMPLE 2 mRNA Reverse Transcription and PCR

Reverse Transcription: RNA was isolated from microdissected or non-

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

Example 1, or from fresh or frozen tissue by a single step guanidinium isocyanate

method using the QuickPrep™ crø mRNA purification kit (Amersham Pharmacia

Biotech Inc, Piscataway, NJ.) according to the manufacturer's instructions. After

precipitation with ethanol and centrifugation, the RNA pellet was dissolved in 50 ul of 5 mM Tris/Cl at pH 8.0. M-MLV Reverse Transcriptase will extend an

oligonucleotide primer hybridized to a single-stranded RNA or DNA template in the presence of deoxynucleotides, producing a complementary strand. The resulting RNA was reverse transcribed with random hexamers and M-MLV Reverse Transcriptase from Life Technologies. The reverse transcription was accomplished

by mixing 25 μl ofthe RNA 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 RNA), 45 min at 42° C (for the M-MLV reverse

transcription enzymatic reaction) and 5 min at 95° C (for heat inactivation of

DNAse).

"Reverse transcription mix" consists of 10 ul 5X buffer (250 mM Tris-HCI,

pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 ul random hexamers (50 O.D. dissolved

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

dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3mg/ml in 10 mM Tris-HCL, pH 7.5), 1.25

ul RNA Guard 24,800U/ml (RNAse inhibitor) (Porcine #27-0816, Amersham Pharmacia) and_2,5.uj MML 200U/ul_.(Li e_Tech Cat #28025-02). Final concentrations of reaction components are: 50 mM Tris-HCI, pH 8.3,

75 mM KCl, 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 EGFR cDNA

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

a fluorescence based real-time detection method (ABI PRISM 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, (EGFR-1773 (SEQ JD NO: 3), T_m = 70° C;

HER2-neu 2657 (SEQ JD NO: 6), β-actin-611 (SEQ JD NO: 7) 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 ofthe 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 witiiin the purely exponential phase

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

Comparison ofthe 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 levels that are expressed as ratios between two absolute

measnrementsJ-gfine jf interest internal control gene). The PCR reaction mixture consisted 0.5μl ofthe reverse transcription

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

oligonucleoride primers EGFR-1753F (SEQ ID NOT, T_m = 59° C) and EGFR-

1823R (SEQ JD NO: 2, T_m = 58° C ) or oligonucleotide primers HER2-neu 2671F

(SEQ JD NO:4) and HER2-neu 2699R (SEQ ID NO: 5) 200 nM TaqMan® probe

(SEQ ID NO:3 or SEQ ID NO: 6), 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-Actin were β-Actin-592F

(SEQ ID NO: 8) and β-Actin-651R (SEQ JD NO: 9).

EXAMPLE 3

Determining the Uncorrected Gene Expression (UGE) for EGFR

Two pairs of parallel reactions are carried out. The "test" reactions and the

"calibration" reactions. Figure 7. The EGFR amplification reaction and the β-actin

internal control amplification reaction are the test reactions. Separate EGFR and β-

actin amplification reactions are performed on the calibrator RNA template and are

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

different cycle threshold (Ct) values: Ct_&m and Ct_p-_ac(in from the test reactions and

Ctserø and Ct_p._aotin from the calibration reactions. The differences in Ct values for

the two reactions are determined according to the following equation: ΔCt,_est = Ct_EGFR - Ctp._8Clin (From the "test" reaction)

ACt_BβBωIαr= QECMI - Ct_β._actin (From the "calibration" reaction)

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

2-^ΔC, _test (From the "test" reaction)

2^'ACt _oaiib_rator (From the "calibration" reaction)

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

TaqMan® instrument the following calculation is carried out:

Uncorrected gene expression (UGE) for EGFR = 2^"Δct _tesl / 2^"ΔCt _caHbrator

Normalizing UGE with known relative EGFR expression levels .

The normalization calculation entails a multiplication ofthe UGE with a

correction factor specific to EGFR and a particular calibrator RNA. A

correction factor K._ECFR 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_EGFR

equals 1.54.

Normalization is accomplished using a modification ofthe 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 FPE test

tissues were analyzed for EGFR expression using the TaqMan® methodology described above. The internal control gene β-actin and the calibrator RNA,Human

Liver Total RNA (Stratagene, Cat #735017) was used.

The already known relative EGFR expression level of each sample AG221,

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

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

_unaverag-_d = Known Values / UGE

Next, all ofthe K values are averaged to determine a single K._EGFR correction factor specific for EGFR, Stratgene Human Liver Total RNA (Stratagene, Cat

#735017) from calibrator RNA and β-actin.

Therefore, to determine the Corrected Relative EGFR Expression in an unknown tissue sample on a scale that is consistent with pre-TaqMan® EGFR expression studies, one merely multiplies the uncorrected gene expression data (UGE) derived from the TaqMan® apparatus with the K_EGFR specific correction factor, given the use ofthe same internal control gene and calibrator RNA.

Corrected Relative EGFR Expression = UGE x K_EGFR

A K_EGFR may be determined using any accurately pre-quantified calibrator

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

be calibrated to samples with known relative EGFR expression levels as described

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

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

described above. For example, if a subsequent K_EGFR is determined for a different internal

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

control gene and the calibrator RNA to tissue samples for which EGFR expression

levels relative to that particular internal control gene have 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_EGFR specific to the different internal control gene and/or

calibrator RNA.

EXAMPLE 4

Determining the Uncorrected Gene Expression (UGE) for HEJU-neu

Two pairs of parallel reactions are carried out. The "test" reactions and the

"calibration" reactions. Figure 8. The HEJU-neu amplification reaction and the β-

actin internal control amplification reaction are the test reactions. Separate HER2-

neu 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: Ctjjws-_neu and Ct_β._a-_lin from the

test reactions and Ct_H._r2._neu and Ct_p._actinfrom the calibration reactions. The differences

in Ct values for the two reactions are determined according to the following

equation:

ΔCt,-,, = Ct_HER2._neu - Ct_β._actin (From the "test" reaction) C _ffcβ-_neu - Ct_β._acώl (From the "calibration" reaction) Next the step involves raising the number 2 to the negative ΔCt, according to the following equations.

-ΔCt test (From the "test" reaction)

2 ca_li_brator (From the "calibration" reaction)

In order to then obtain an uncorrected gene expression for HEJU-neu from

the TaqMan® instrument the following calculation is carried out:

Uncorrected gene expression (UGE) for HEJU-neu = 2^"Act _test / 2"^Aα _caIibrator

Normalizing UGE with known relative HEJU-neu expression levels

The normalization calculation entails a multiplication ofthe UGE with a

correction factor (K _R2-_mι specific to HER2-ne and a particular calibrator RNA.

A correction factor jjgia-_mm ^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. Using β-actin and the accurately pre-quantified

calibrator RNA,Human Liver Total RNA (Stratagene, Cat #735017) the correction

factor K._{HEB WU} equals 12.6 x 10'³.

Normalization is accomplished using a modification ofthe 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 FPE test

tissues were analyzed for HEJU-neu expression using the TaqMan® methodology

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

Liver Total RNA (Stratagene, Cat #735017) was used. The already known relative HEJU-neu expression level of each sample

AG221, AG222, AG252, Adult Lung, PC3, AdCol is divided by its corresponding

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

Kun_av_erag_ed = Known Nalu^βS / UGE

Next, all ofthe K values are averaged to determine a single Y±_EGFR correction

factor specific for HER2-neu,Bxxman Liver Total RNA (Stratagene, Cat #735017)

calibrator, and β-actin.

Therefore, to determine the Corrected Relative HER2-neu Expression in an

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

expression studies, one merely multiplies the uncorrected gene expression data

(UGE) derived from the TaqMan® apparatus with the K_ffER2.„_eu specific correction

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

Corrected Relative HER2-neu Expression = UGE x

A K._HEB2-_MU ^may be determined using any accurately pre-quantified calibrator

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

be calibrated to samples with known relative EGFR expression levels as described

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

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

described above.

For example, if a subsequent K _ER2.„_eu is determined for a different internal

control gene and/or a different calibrator RNA, one should calibrate both the internal control gene and the calibrator RNA to tissue samples for which HEJU-neu

expression levels relative to that particular internal control gene have already been

determined or published. Such a determination can be made using standard pre-

TaqMan®, quantitative RT-PCR techniques well known in the art. The known

expression levels for these samples will be divided by their corresponding UGE

levels to determine a K for that sample. K values are then averaged depending on

the number of known samples to determine a new K _ER2._mu specific to the different

internal control gene and/or calibrator RNA.

EXAMPLE 5 Patient Population and Tissue Acquisition

Patients. Eighty-three patients suffering from NSCLC consisting of sixty- five (78.3%) men and 18 (21.7%) women, with a median age of 63.5 years (range, 34-82) were studied. Thirty-nine (47%) patients had squamous cell carcinomas, 32 (38.6%) had adenocarcinoma, and 12 (14.5%) had large cell carcinomas. The

primary tumors were graded histopathplogically as well-differentiated (Gl, one

patient), moderately-differentiated (G2, 18 patients), and poorly-differentiated (G3,

64 patients). Tumor staging was performed according to the International Union

Against Cancer (UICC) TNM classification: Forty-one (49.4%) had stage I tumors,

16 (19.3%) had stage II tumors, and 26 (31.3%) had stage JJIa tumors. All tumors

were completely resected (RO category), by at least a lobectomy as quality control.

Patients with histopathological stage JJIa tumors received postoperative

radiotherapy. The median follow-up was 85.9 months (min. 63.3; mar. 105.2

months) and no patient was lost to follow-up. Tissue Acquisition. Tissue for gene expression analysis was obtained

immediately after lung resection before starting mediastinal lymphadenectomy and

was immediately frozen in liquid nitrogen. Tissues were analyzed from the

following 2 locations: tumor and uninvolved lung tissue taken from the greatest

distance to the tumor. 6 μm frozen sections were taken from blocks of tumor tissue

and starting with the first section every fifth was routinely stained with HE and

histopathologically evaluated. Sections were pooled for analysis from areas of

estimated 75% malignant cells. RNA was isolated from tissue samples according to

the methods in Example 2.

EXAMPLE 6

Statistical Analysis

TaqMan® analyses yield values that are expressed as UGE. The ratio

between UGE in tumor tissue and UGE in matching non-malignant lung tissue was

used to determine differential gene expression. Associations between the two UGE

variables were tested by using Wilcox on signed rank test. The Chi-Square test was

used to analyze the associations between categorial clinicopathological variables.

Hazards ratios were used to calculate the relative risks of death. These calculations

were based on the Pike estimate, with the use ofthe observed and expected number

of events as calculated in the log-rank test statistic. Pike, J R Stat Soc Series A

735:201-203; 1972. The maximal chi-square method of Miller and Sigmund (Miller

et al, Biometrics 35:1011-1016, 1982) and Halpern (Biometrics 35:1017-1023,

1982) 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 statistics used to measure the strength ofthe grouping. To determine a P value that would be interpreted as a measure ofthe strength ofthe

association based on the maximal chi-square analysis, 1000 boot-strap-like

simulations were used to estimate the distribution ofthe maximal chi-square

statistics under the hypothesis of no association. Halpern, Biometrics 35:1017-

1023, 1982. Cox's proportional hazards modeling of factors that were significant in

anivariate analysis was performed to identify which factors might have a significant

influence on survival. The level of significance was set to p < 0.05.

HER2-neu mRNA expression was detectable by quantitative real-time RT- PCR in 83 of 83 (100%) normal lung and 83 of 83 (100%) tumor samples. The

corrected HEJU-neu mRNA expression, expressed as the ratio between HEJU-neu and β-Actin PCR product, was 4.17 x 10^"3 (range 0.28-23.86 x 10^"3) in normal lung and 4.35 x 10^'3 (range: 0.21-68J 1 x 10"³) in tumor tissue (E=0.019 Wilcoxon test). The maximal chi-square method by Miller and Siegmund (Miller et al. Biometrics 35:1011-1016, 1982) and Halpern (Biometrics 35:1017-1023, 1982) determined a

threshold value of 1.8 to segregate patients into low and high differential HEJU-neu expressors. By this criterion, 29 (34.9%) patients had a high differential HEJU-neu

expression and 54 (65 J %) had a low differential HER2-neu expression. Figure 4

shows associations between clinicopathological data and differential HEJU-neu

gene expression status. There were no statistically significant differences detectable.

Figure 1 displays a Kaplan Meier plot of the estimated probability of survival versus

the differential HEJU-neu mRNA expression status. The median survival was not

reached in the low differential HER2-neu expression group compared to 31 J

months (95% CI: 21.96- 40.24) in the high differential HEJU-neu expression

group. To determine a P value, bootstrap-like simulations were used to estimate the distribution of a maximal chi-square statistic, since the threshold value of 1.8 had

been chosen after examining the data. The resulting adjusted P value was .004 (Log-

rank test).

The accuracy of HEJU-neu as a prognostic factor was next determined by

the Cox's proportional hazards model analysis. In univariate analysis of potential

prognostic factors, high differential HEJU-neu expression as well as advanced pT

(tumor stage) classification, pN (lymph node stage) classification, and tumor stage

were significant unfavorable prognostic factors (Figure 5). In a multivariate analysis of prognostic factors (Figure 6), high differential HEJU-neu expression was a

significant and independent unfavorable prognostic factor, as well as advanced pN classification and tumor stage.

EGFR mRNA expression was detectable by quantitative real-time RT-PCR in 83 of 83 (100%) normal lung and 83 of 83 (100%) tumor samples. The median corrected EGFR mRNA expression was 8J7 x 10^'3(range: 0.31-46.26 x 10'³) in

normal lung and 7.22 x 10"³ (range: 0.27-97.49 x 10^'3) in tumor tissue (E=n.s.). The maximal chi-square method (Miller (1982); Halpern (1982)) determined a threshold

value of 1.8 to segregate patients into low and high differential EGFR expressors.

By this criterion, 28 (33.7%) patients had a high differential EGFR expression and

55 (66.3%) had a low differential EGFR expression status. There were no statistical

significant differences between clinicopathological variables and differential EGFR

mRNA expression status detectable (Figure 4). A trend towards inferior overall

survival was observable for the high differential EGFR expression group, but did

ot reach statistical significance (Figure 2). The median survival was not reached in the low differential EGFR expression group compared to 32.37 months (95% CL:

8.43-56.31) in the high differential EGFR expressor group (E=0J76).

High expression levels (above 1.8) of differential HER2-neu and EGFR were

found in 14 of 83 (16.9%) patients. Forty of 83 (48.2%) patients showed a low

differential expression status (below 1.8) for HEJU-neu and EGFR, whereas 14 of

83 (16.9%) showed a high differential expression for EGFR only, and 15 of 83

(18.1%) patients displayed a high differential expression for HEJU-neu. The median survival was not reached in the group that showed low differential HEJU-neu and

EGFR expression, compared to 45.47 months in the high differential EGFR expression group, 31J0 months (95% CL: 14.77-47.43) in the high differential HEJU-neu expression group, and 22.03 months (95% CI: 2.30; 41.76; P =0.003; log-rank test; Figures 3 and 5) in the high differential HEJU-neu and EGFR expression group. Univariate analysis displayed high differential HEJU-neu and EGFR coexpression as a significant unfavorable prognostic factor (Figure 5). In a multivariate analysis of prognostic factors (Figure 6), high differential HEJU-neu and high differential EGFR coexpression was a significant and independent

unfavorable prognostic factor, as was advanced pN classification and tumor stage.

EXAMPLE 7

Tumor response to a receptor tyrosine kinase targeted chemotherapy

Five colon cancer patients' tumors were initially identified as expressing

EGFR by immunohistochemistry. Patients were treated with Imclone JMC-C225,

400 mg/m² loading dose followed by 250 mg/m² weekly, plus CPT-11 at the same dose and schedule that the patient had previously progressed on. Previous CPT-11

dose attenuations were maintained.

Using the methodology described in Examples 1-4, Patient 1 was determined

to have a corrected EGFR expression level of 2.08 x 10^"3 and had a completed

response (CR) to a a receptor tyrosine kinase targeted chemotherapy comprising

CPT-11 (7-ethyl-10-[4-(l-piperidino)-l-piperidino] carboxycamptothecin) / C225

(an anti- EGFR monoclonal antibody effective in anti-cancer therapy; Mendelsohn, Endocr Relat Cancer 2001 Mar;8(l):3-9). Patient 2 had a corrected EGFR expression level of 8.04 x 10^"3 and had a partial response (PR) to the receptor tyrosine kinase targeted chemotherapy. Patient 3 had a corrected EGFR expression level of 1.47 x 10"³ and also showed a partial response (PR) to the receptor tyrosine kinase targeted chemotherapy. Patient 4 had a corrected EGFR expression level of 0J6 x x 10^'3 and had stable disease (SD) showing no response to the receptor tyrosine kinase targeted chemotherapy. Patient 5 had a no EGFR expression ( 0.0 x

10^'3 ) and had progressive disease (PR) showing no response to the receptor tyrosine kinase targeted chemotherapy. See figure 9.

Claims (24)

What is claimed is:

1. A method for determining a chemotherapeutic regimen comprising receptor tyrosine kinase targeted agent, for treating a tumor in a patient comprising: (a) obtaining a tissue sample ofthe tumor; (b) obtaining a non-malignant tissue sample matching said tumor;

(c) isolating mRNA from the tumor sample and non-malignant sample;

(d) subj ecting the mRNA to amplification using a pair of oligonucleotide primers that hybridize under stringent conditions to a region ofthe EGFR gene, or a pair of oligonucleotide primers that hybridize under stringent conditions to a region of the HER2-neu gene, to obtain an

EGFR tumor amplified sample and a EGFR non-malignant amplified sample, or a HEJU-neu tumor amplified sample and a HEJU-neu non-malignant amplified sample

(e) determining the amount of HEJU-neu mRNA in the HEJU-neu tumor amplified sample and HEJU-neu non-malignant amplified sample or determining the amount of EGFR mRNA in the EGFR tumor amplified sample and EGFR non-malignant amplified sample;

(f) obtaining a differential HEJU-neu epression level or obtaining a differential EGFR expression level; and (g) determining a chemotherapeutic regimen comprising a receptor tyrosine kinase targeted agent by comparing the differential HEJU- neu expression level and the threshold level for HER2-neu gene expression, or comparing the differential EGFR expression level and the threshold level for EGFR gene expression.

2. A method for determining a chemotherapeutic regimen receptor tyrosine kinase targeted agent for treating a tumor in a patient comprising:

(a) obtaining a tissue sample ofthe tumor;

(b) obtaining a non-malignant tissue sample matching said tumor;

(c) isolating mRNA from the tumor sample and non-malignant sample; (d) subj ecting the mRNA to amplification using a pair of oligonucleotide primers that hybridize under stringent conditions to a region ofthe EGFR gene, to obtain an tumor amplified sample and a non- malignant amplified sample;

(e) determining the amount of EGFR mRNA in the tumor amplified sample and non-malignant amplified sample;

(f) obtaining a differential EGFR expression level; and

(g) determining a chemotherapeutic regimen comprising a receptor tyrosine kinase targeted by comparing the differential EGFR expression level and the hreshόldJevel fό EGFT? geήe^"expressiθn.

3. A method for determining a chemotherapeutic regimen comprising receptor tyrosine kinase targeted agent for treating a tumor in a patient comprising:

(a) obtaining a tissue sample ofthe tumor;

(b) obtaining a non-malignant tissue sample matching said tumor; (c) isolating mRNA from the tumor sample and non-malignant sample;

(d) subjecting the mRNA to amplification using a pair of oligonucleotide primers that hybridize under stringent conditions to a region ofthe HEJU-neu gene, to obtain an tumor amplified sample and a non- malignant amplified sample; (e) determining the amount of HEJU-neu mRNA in the tumor amplified sample and non-malignant amplified sample;

(f) obtaining a differential HEJU-neu expression level; and

(g) determining a chemotherapeutic regimen comprising a receptor tyrosine kinase targeted agent by comparing the differential HER2- neu expression level and the threshold level for HER2-neu gene expression.

4. A method for determining a chemotherapeutic regimen comprising receptor tyrosine kinase targeted agent for treating a tumor in a patient comprising: (a) obtaining a tissue sample ofthe tumor; (b) obtaining a non-malignant tissue sample matching said tumor;

(c) isolating mRNA from the tumor sample and non-malignant sample;

(d) subjecting the mRNA to amplification using a pair of oligonucleotide primers that hybridize under stringent conditions to a region ofthe EGFR gene, and a pair of oligonucleotide primers that hybridize under stringent conditions to a region ofthe HER2-neu gene, to obtain an EGFR tumor amplified sample and an EGFR non- malignant amplified sample, and a HER2-neu tumor amplified sample and a HEJU-neu non-malignant amplified sample

(e) determining the amount of HEJU-neu mRNA in the HEJU-neu tumor amplified sample and HEJU-neu non-malignant amplified sample and determining the amount of EGFR mRNA in the EGFR tumor amplified sample and EGFR non-malignant amplified sample;

(f) obtaining a differential HEJU-neu expression level and obtaining a differential EGFR expression level; and (g) determining a chemotherapeutic regimen comprising a receptor tyrosine kinase targeted agent by comparing the differential HEJU- neu expression level and the threshold level for HER2-neu gene expression, and comparing the differential EGFR expression level and the threshold level for EGFR gene expression.

5. The method claim 2, wherein the oligonucleotide primers consist ofthe oligonucleotide 3rimer_pair EGFR, or pair of oligonucleotide primers substantially identical thereto.

6. The method of claim 3 wherein the oligonucleotide primers consist ofthe oligonucleotide primer pair HER2-neu, or pair of oligonucleotide primers substantially identical thereto.

7. The method of any one of claims 1, 2, 3, or 4 wherein the tumor is a non- small cell lung cancer tumor.

8. The method of claim 4 wherein the primers consist of both the oligonucleotide primer pair HER2-neu and oligonucleotide primer pair EGFR.

9. The method of any one of claims 1, 2, or 4 wherein the threshold level of EGFR gene expression is about 1.8 times EGFR gene expression in matching non-malignant tissue.

10. The method of any one of claims 1 , 3 , or 4 wherein, the threshold level of HEJU-neu gene expression is about 1.8 times HEJU-neu gene expression in matching non-malignant tissue.

11. The method of any one of claims 1, 2, 3 or 4 wherein the tissue samples are are fixed or fixed and paraffin embedded.

12. A method for determining the level of EGFR expression in a fixed paraffin embedded 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 ofthe EGFR gene, to obtain an amplified sample; (d) determining the quantitiy of EGFR mRNA relative to the quantity of mRNA of an internal control gene.

13. The method of claim 12 wherein, the pair of oligonucleotide primers consists of the oligonucleotide primer pair EGFR or a pair of oligonucleotide primers substantially similar thereto.

14. A method for determining the level of HEJU-neu expression in a fixed paraffin embedded tissue sample comprising;

(a) deparaffinizing the tissue sample, to obtain a deparaffinized sample; -(h). isolating mRNA from the deparaffimzed 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 ofthe HEJU-neu gene, to obtain an amplified sample;

(d) determining the quantity of HER2-neu mRNA relative to the quantity of mRNA of an internal control gene.

15. The method of claim 14 wherein, the pair of oligonucleotide primers consists ofthe oligonucleotide primer pair HER2-neu or a pair of oligonucleotide primers substantially similar thereto.

16. The method of claim 12 or 14 wherein the internal control gene is β-actin.

17. The method of claim 12 or 14 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 the chaotropic solution.

18. An oligonucleotide primer having the sequence of SEQ ID NO: 1 or and an oligonucleotide substantially identical thereto.

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

20. An oligonucleotide primer having the sequence of SEQ JD NO: 4 or and an oligonucleotide substantially identical thereto.

21. An oligonucleotide primer having the sequence of SEQ JD NO: 5 or and an oligonucleotide substantially identical thereto.

22. A kit for detecting expression of an EGFR gene comprising oligionucleotide pair EGFR or an oligonucleotide pair substantially identical thereto.

23. A kit for detecting expression of a HEJU-neu gene comprising oligionucleotide pair HER2-neu or an oligonucleotide pair substantially identical thereto.

24. A kit for detecting expression of a HER2-neu and EGFR gene comprising oligionucleotide pair HER2-neu or an oligonucleotide pair substantially identical thereto and oligionucleotide pair EGFR or an oligonucleotide pair substantially identical thereto.