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Immunohistochemical Detection of EGFR in Paraffin-embedded Tumor Tissues : Variation in Staining Intensity Due to Choice of Fixative and Storage Time of Tissue Sections

Derek Atkins, Karl-August Reiffen, Conny Lund Tegtmeier, Henrik Winther, Marcellus S. Bonato and Stephan Störkel

University Witten/Herdecke, Institute of Pathology, Wuppertal, Germany (DA,SS); Merck KGaA, Darmstadt, Germany (K-AR); DakoCytomation A/S, Glostrup, Denmark (CLT,HW); and University of Applied Sciences, Münster, Germany (MSB)

Correspondence to: Prof. Dr. Med. S. Störkel, Inst. of Pathology, University Witten/Herdecke, Helios-Klinikum, Wuppertal, Heusnerstr.40, 42283 Wuppertal, Germany. E-mail: sstoerkel{at}wuppertal.helios-kliniken.de


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid malignant tumors and its expression has been correlated with disease progression and poor survival. With the advent of targeted therapies, especially IMC-C225 (Cetuximab), a monoclonal antibody (MAb) directed against the EGFR, there is an increasing interest in immunohistochemistry (IHC)-based EGFR screening methods using paraffin-embedded tumor specimens to select cancer patients eligible for treatment with Cetuximab. With the EGFRpharmDX kit, a complete assay for demonstration of EGFR is now available. Because no information about the preservation of the EGFR under various conditions of fixation is available, we performed a prospective study on a panel of commonly used fixatives to determine optimal tissue preservation protocols. The stability of the epitope on cut tissue sections stored for a period up to 24 month was also tested using material originating from patients with head and neck cancer, non-small-cell lung carcinomas, and colorectal adenocarcinomas. Depending on the fixative used and the time of storage of cut tissue sections, a variation in the determined level of EGFR expression was demonstrated compared with the most optimal fixation procedure. (J Histochem Cytochem 52:893–901, 2004)

Key Words: immunohistochemistry • tissue preservation • fixation protocols • EGFR • stored slides


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
IN RECENT YEARS, targeted therapies for a variety of malignant tumors have achieved clinically significant response rates and have enabled oncologists to develop individual-based therapy strategies for patients (reviewed by Ciardiello and Tortora 2001Go; Yip and Ward 2002Go; Chinn et al. 2003Go).

The rationale for such therapeutic approaches is the identification of the targeted molecule in the tumor of the patient to be treated. Especially for solid malignant tumors, a growing list of target molecules is now routinely estimated by IHC staining in biopsy specimens and surgically removed tumor material, thereby not only generating important data for therapeutic decisions but also providing prognostic relevant information (reviewed by Ross et al. 2003Go). For example, during the past decade IHC staining for estrogen and progesterone receptors has widely replaced bio-chemical assays for the therapeutic stratification of breast cancer patients for anti-hormonal therapy. In addition, with the identification of the HER2/neu-receptor as a target molecule in breast cancer and the implementation of trastuzumab therapy, assessment of the overexpression of the HER2/neu-receptor by IHC staining or amplification of the respective gene by FISH techniques is now available in most pathology laboratories (Press et al. 1994Go; Pauletti et al. 1996Go). Moreover, during the past few years another member of the erbB-family, i.e., the epidermal growth factor receptor (EGFR; HER1, erbB-1) has become an interesting molecule for such therapeutic strategies and has prompted the development of a variety of agents targeting either the extracellular ligand-binding domain, the intracellular tyrosine kinase domain, the ligands, or the synthesis of the receptor (Artega 2002Go,2003Go; Baselga 2002Go). In particular, therapies based on monoclonal antibodies (MAbs) such as IMC-C225 (Cetuximab; ImClone Systems, New York, NY and Bristol-Myers Squibb; Princeton, NJ) are now tested in clinical trials on patients having head and neck cancer, colon cancer, pancreatic cancer, or non-small-cell lung (NSCL) cancer (Baselga et al. 2000Go; Shin et al. 2001Go; Bunn and Franklin 2002Go; Herbst and Hong 2002Go; O'Dweyer and Benson 2002Go; Xiong and Abbruzzese 2002Go). With the ongoing development of antibody-based therapeutics, pathologists become more and more dependent on pharmacodiagnostics using complete assays that have well-defined reagents, protocols, interpretation, and scoring guidelines. This approach is necessary to achieve comparable results among different diagnostic institutions. To ensure concordance among the IHC results, handling of specimens and fixation protocols must also be standardized. Because data concerning optimal preservation of tumor material for subsequent IHC staining for the EGFR are not available, a prospective study using eight different fixatives for tumor material originating from patients with NSCL cancer, head and neck cancer, or colorectal cancer was performed. Repeated testing for EGFR after 6, 9, 12, 18, and 24 months was performed for all differentially fixed tumor samples to correlate IHC staining results with storage time of tissue sections. The results presented provide information for optimal fixation protocols and assessment of the EGFR status after storage of cut sections.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Materials and Tissue Sampling
Unfixed tissue specimens from 10 surgically removed NSCL carcinomas (seven squamous cell carcinomas, three large cell carcinomas and one adenocarcinoma; male to female = 1; median age 64 years, range 50–80 years), 10 squamous cell carcinomas from the head and neck region (all male; median age 58 years, range 42–77 years), as well as specimens from 20 colorectal adenocarcinomas (18 of the classical type and two of the mucinous type; male to female = 1.5; median age 68 years, range 53–81 years) were collected from June 2000 until March 2001. The histopathological characteristics, anatomic localization, and TNM data for each tumor are summarized in Table 1. From each tumor lesion, at least eight tissue samples of 0.3–0.5 cm3 were collected and transferred into different fixatives (see below).


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Table 1

Characteristics of the tumors analyzed

 
Fixation Protocols and Tissue Arrays
Tissue samples from each tumor lesion were fixed for 24 hr in 4% neutral buffered formalin, Bouin's fixative, acetic formalin alcohol (AFA), 4% unbuffered formalin, or 10% unbuffered formalin, or for 4 hr in PreFer (Anatech; Battle Creek, MI) or Pen-fix (Richard Allen Scientific; Kalamazoo, MI), or for 48 hr in 4% neutral buffered formalin. After paraffin embedding, tumor specimens were cut into 5-µm sections and stained routinley with H&E to define representative tumor regions. For the preparation of tissue microarrays (TMAs), tumor tissue cylinders (3 mm in diameter) were punched from these regions of the donor blocks and placed on 25 x 35-mm paraffin blocks as representatively shown in Figure 1 . In total, 40 TMAs, each containing eight differentially fixed tumor samples from one patient, were constructed and stained with H&E to ensure that sufficient tumor tissue was represented. Consecutive paraffin slides of TMA blocks were serially cut and stored in the dark at room temperature for follow-up IHC staining according to the time schedule mentioned below.



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Figure 1

Schematic of a head and neck cancer tissue microarray (TMA) containing eight differently fixed tumor samples from one patient stained for EGFR. Tissue samples from each tumor were alternatively fixed as indicated. After paraffin embedding, tumor specimens were cut into 5-µm sections and stained with H&E to define representative tumor regions. For preparation of TMAs, tumor cylinders were punched from the donor blocks and placed on 25 x 35-mm paraffin blocks. Consecutive paraffin slides of each TMA were serially cut and stored in the dark at RT for follow-up IHC staining.

 
Immunohistochemistry
IHC staining of the tumor tissue array samples was performed by using the EGFR pharmDx kit (DakoCytomation; Carpinteria, CA) according to the manufacturer's instructions and using the reagents supplied with the kit. In brief, sections of 5 µm were mounted on silanized charged slides and allowed to dry for 1 hr at RT, followed by 1 hr in an incubator at 60C. After deparaffinization and rehydration, slides were incubated with proteinase K solution for 5 min. After a washing procedure with distilled water, tissue sections were covered for 5 min with 3% H2O2 to block endogenous peroxidase, followed by an additional washing procedure with the supplied buffer. Slides were then placed in a humid chamber and incubated for 30 min with the primary mouse anti-EGFR MAb (clone 2-18C9), which binds to a formalin-resistant epitope near the ligand-binding site on the extracellular domain of the EGFR (Spaulding and Spaulding 2002Go). After two rinses in buffer the slides were incubated with the detection system for 30 min. Tissue staining was visualized with a DAB substrate chromogen solution. Slides were counterstained with hematoxylin, dehydrated, and mounted. Negative controls were performed by using a mouse IgG1 MAb supplied with the kit. Skin tissue served as positive control. To validate each staining, positive and negative cell line controls being part of the EGFRpharmDx kit were included in each staining run. IHC staining of all tumor tissue arrays was performed within the first 3 months and then repeated after 6-, 9-, 12-, 18-, and 24-month storage of the tissue slides.

Scoring System and Statistics
Slide evaluation was independently performed by two pathologists (D.A., S.S) with one investigator (D.A.) blinded with regard to the length of storage. Overall interobserver difference was 5%. In case of differing results, consensus was reached by joint evaluation. Although occasional cytoplasmic staining of tumor cells was observed, which may result from either internalized or nascent receptor molecules, only staining of the tumor cell membranes was considered to be specific.

The staining pattern of tumor cell membranes was further classified as incomplete staining, i.e., tumor cells were stained in only part of their membrane, and complete staining, i.e., tumor cells displayed a circumferential staining of the entire tumor cell membrane. The following scoring approach in the assessment of EGFR immunostaining was used: score 0 = no staining or unspecific staining of tumor cells; score 1 = weak (intensity) and incomplete staining (quality) of more than 10% of tumor cells (quantity); score 2 = moderate and complete staining of more than 10% of tumor cells; score 3 = strong and complete staining of more than 10% of tumor cells. Representative examples for the different scores are given in Figure 2 .



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Figure 2

Typical examples of the scoring system used for the determination of EGFR expression. The staining pattern of tumor cell membranes was classified as score 0 when negative or unspecific (A), score 1 if the staining was weak and incomplete in more than 10% of tumor cells (B), score 2 if moderate and complete staining was detectable in more than 10% of the tumor cells (C), and score 3 if more than 10% of the tumor cells displayed strong and complete membrane staining (D). Magnification x400.

 
It should be stressed that this scoring system was used only for practical reasons and does not relate to a scoring system to be established at a later stage for the selection of patients eligible for treatment with targeted therapies.

IHC staining results were statistically analyzed according to their scale level: data description was based on absolute and relative frequencies for binary end points and on medians and quartiles for continuous end points. Univariate significance analysis was based on Pearson's X2 test (p value <0.05) for categorial parameters and on two sample Wilcoxon's tests (p value < 0.001, Bonferroni adjusted) for continuous parameters. Multivariate analysis was based on Friedman's test for related samples (p value <0.05). All computations were performed using SPSS release 11.0 for Windows.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Each of the 40 TMA blocks contained eight differently fixed tissue samples, giving a total of 320 different samples for IHC analysis at 3, 6, 9, 12, 18, and 24 months. At each of the six time points (see above), consecutive sections of the TMAs were IHC stained, giving a total of 1920 tissue samples for testing and scoring.

The interpretation was possible at a total of 1824 of these specimens (95%).

Quality and Quantity of IHC Staining for EGFR Are Dependent on the Fixative Used
We first determined the influence of different fixatives with respect to overall morphology and the quality and quantity of EGFR staining of tumor cells. Therefore, IHC results obtained within the first 3 months from all tumors tested were compared with respect to the different fixatives used. As representatively depicted in Figures 3 and 4 , strongest results with regard to EGFR staining of tumor cells were achieved by fixing the tissue samples in 4% unbuffered formalin or AFA for 24 hr or in Pen-fix for 4 hr. By using one of these fixatives, more than 60% of the tumor samples displayed strong positive staining (score 3) for EGFR and less than 5% of the samples stained negative (score 0).



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Figure 3

IHC staining for EGFR in colorectal adenocarcinomas obtained from differently fixed tumor samples within the first 3 months (A–D and I–L) and in slides stored for 24 months (E–H and M–P), demonstrating that the quality and quantity of EGFR immunoreactivity is inversely correlated with storage time of unstained paraffin slides. (A,E) 4% neutral buffered formalin, 24 hr; (B,F) Bouin's fixative, 24 hr; (C,G) AFA, 24 hr; (D,H) 4% neutral buffered formalin, 48 hr; (I,M) PreFer, 4 hr; (J,N) Pen-fix, 4 hr; (K,O) 4% unbuffered formalin, 24 hr; (L,P) 10% unbuffered formalin, 24 hr. Magnification x400.

 


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Figure 4

Positive staining for EGFR (score 3) at various time points is dependent on the fixative used. Comparison of the IHC results obtained at various time points revealed that the differences among the alternative fixatives was statistically significant only after 9-month storage time for Pen-fix and after 12-month storage time for AFA, 4% unbuffered formalin, Pen-fix, and 4% neutral buffered formalin (24 hr).

 
IHC for tumor specimens fixed in 10% unbuffered formalin resulted in strong positive staining for EGFR in more than 50% and negative staining in less than 10% of tumor samples. Tumor specimens fixed alternatively in 4% neutral buffered formalin for 24 hr or 48 hr, or in Bouin's fixative for 24 hr, resulted in strong positive staining for EGFR in around 40% and negative staining (score 0) in 15–20% of the lesions tested. The differences among the fixatives in terms of staining results obtained within the first 3 months were statistically not significant.

Tissue preservation and quality of IHC staining were satisfying with all the above-mentioned fixatives. However, occasional minimal nonspecific staining was observed in some of the tumor specimens independently of the fixative used. In contrast, results achieved by using PreFer as fixative were unsatisfactory for the preservation of morphology and IHC, as representatively shown in Figure 3.

For these reasons, we disclosed the IHC results of PreFer-fixed tumor samples from comparative analysis.

Immunoreactivity to Anti-EGFR Is Inversely Correlated with the Storage Time of Unstained Slides
To determine the correlation between immunoreactivity for EGFR and storage time, unstained tissue sections were stored for 6, 9, 12, 18, and 24 months. In parallel with the IHC data obtained within the first 3 months, strongest results for EGFR staining (score 3) were achieved in tissue samples fixed in either 4% unbuffered formalin or AFA for 24 hr or in Pen-fix for 4 hr. With the use of one of these fixatives, more than 60% of the tumor samples tested displayed strong positive staining for EGFR (score 3) after 6 months and more than 50% of tumor samples stained positive after 9, 12, and 18 months. Even after 24-month storage, around 55% of the tumor samples fixed in Pen-fix tested strongly positive (score 3) for the expression of EGFR. More than 50% of the tumor samples fixed in 10% unbuffered formalin for 24 hr displayed strong positive staining for EGFR after 6 and 9 months but declined to around 40% after 12, 18, and 24 months of storage. As with tumor samples fixed in 10% unbuffered formalin, samples alternatively fixed in 4% neutral buffered formalin for 24 hr or 48 hr showed strong positive reactivity (score 3) for EGFR in more than 50% of the lesions tested after 6 months of storage. In contrast, strong positive staining for EGFR was observed in 40% of the tumor samples fixed in 4% neutral buffered formalin (24 hr and 48 hr) after 9 months and declined to around 30% and 17% in slides stored for 24 months, respectively. With Bouin's fixative, around 30% of the tumor samples tested displayed strong positive staining for EGFR (score 3) after 6, 9, 12, 18, and 24 months of storage. Comparison of the IHC results obtained at various time points revealed that the differences between the alternative fixatives was statistically significant only after 9 months of storage for Pen-fix and after 12 months of storage for AFA, 4% unbuffered formalin, Pen-fix, and 4% neutral buffered formalin (24 hr; Figures 4 and 5) .



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Figure 5

EGFR staining in colorectal adenocarcinomas fixed in 4% unbuffered formalin (24 hr). Inverse relation between IHC staining according to the applied scoring system and storage time of tissue sections.

 
Type-specific Expression of EGFR in NSCL Carcinomas, Squamous Cell Carcinomas of the Head and Neck, and Colorectal Adenocarcinomas
For the different tumor entities analyzed, more than 80% of all head and neck tumor samples and more than 40% of all NSCL carcinoma samples displayed strong immunoreactivity (score 3) for the EGFR within the first 3 months. In contrast, only around one third of the colorectal tumor samples showed strong positivity for EGFR staining at that time point. As representatively illustrated in Figures 6 and 7 , comparison of the follow-up IHC results revealed that more than 70% of head and neck tumor samples but only 30% of the NSCL and 10% of the colorectal tumor samples displayed strong positive staining for EGFR in tissue sections stored for 24 months (p<0.05). The differences in staining results for EGFR obtained from various time points were statistically significant in colorectal adenocarinomas for 4% neutral buffered formalin (24 hr), 4% unbuffered formalin, and Pen-fix, but not in head and neck squamous cell carcinomas and NSCL cancers. Analysis of different EGFR expression patterns in colorectal adenocarcinomas but not in head and neck cancer and NSCL cancer showed that cancer cells at the leading edges of tumor invasion more frequently displayed high levels of EGFR than other tumor areas. However, because we did not systematically collect special tumor areas, the numbers of tissue samples representing the front of invasion were too small to reach statistical significance. For the same reasons, we were not able to evaluate a possible correlation of EGFR immunoreactivity and grade of differentiation of cancer cells.



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Figure 6

Type-specific and storage time-dependent expression patterns of EGFR in tissue samples from head and neck carcinomas (A,B), colorectal adenocarcinomas (C,D), and NSCL cancers (E,F) fixed in 4% unbuffered formalin (24 hr). IHC staining was performed within 3 months (A,C,E) and on paraffin slides stored for 24 months (B,D,F). Magnification x400.

 


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Figure 7

Type-specific expression of EGFR (score 3) in squamous cell carcinomas of head and neck (H and N), NSCL cancers, and colorectal adenocarcinomas (CC). Comparison of the follow-up IHC results obtained from colorectal adenocarcinomas revealed a statistically significant decrease of EGFR immunoreactivity in tumor samples stored for 24 months.

 

    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The data from this study demonstrate that, with the DakoCytomation EGFR pharmDX kit, (a) the quality and quantity of EGFR immuno-reactivity in head and neck cancer, NSCL carcinomas and colorectal adenocarinomas is in part influenced by the fixative used, (b) is inversely correlated with storage time of unstained tissue sections, and (c) is dependent on the tumor entity tested. At the initiation of this study, no information about the influence of different fixatives on EGFR immunoreactivity in cancer cells was available. Therefore, a panel of eight commonly used fixatives was selected to evaluate the influence on IHC staining for EGFR. The results of this study revealed that demonstration of EGFR expression could be obtained from tumor samples of head and neck cancer, NSCL cancers, and colorectal adenocarcinomas alternatively fixed in Pen-fix (4 hr), 4% unbuffered formalin (24 hr), AFA (24 hr), 10% unbuffered formalin (24 hr), 4% neutral buffered formalin (24 hr or 48 hr), or Bouin's fixative (24 hr). The PreFer fixative was found to be unsuitable because of poor preservation of tissue morphology. However, in the present study Pen-Fix, AFA, and 4% unbuffered formalin in general demonstrated the strongest IHC staining. Tissue samples fixed either in 4% neutral buffered formalin (24 hr or 48 hr) or in Bouin's fixative (24 hr) displayed a slightly lower percentage of positively stained tumor cells at every time point of the study. Occasional minimal nonspecific staining was observable in some of the tumor specimens, independently of storage time and the fixative used. However, as shown in Figures 3 and 6, this was negligible and had no influence on the assessment of EGFR expression on tumor cell membranes. Until now, there has been only limited information concerning the immunoreactivity of stored paraffin slides. Reports by Bertheau et al. (1998)Go and Jacobs et al. (1996)Go, who investigated the loss of immunoreactivity for a panel of antibodies in breast carcinomas, lymphomas, and neuroendocrine tumors, showed that for the majority of epitopes tested there is a time-dependent substantial loss in stored tissue slides. We extended this data for cancers of the head and neck, NSCL cancers, and colorectal adenocarcinomas with respect to the expression of EGFR as determined by the application of the DakoCytomation EGFR pharmDx kit. In agreement with the results reported for other epitopes (Jacobs et al. 1996Go; Bertheau et al. 1998Go), we observed a time-dependent loss of immunoreactivity for the EGFR in all tumor entities tested. However, the difference in the amount of EGFR expression between head and neck cancers, NSCL cancers, and colorectal adenocarcinomas, with the highest levels seen in head and neck cancers and lowest levels in colorectal adenocarcinomas, was statistically significant. In view of the EGFR expression levels observed in NSCL carcinomas, one must take into account the fact that the anti-EGFR antibody used in this study also binds to the EGFRvIII truncated mutant form, which is known to be expressed in NSCL and breast cancer cells but is not recognized by the IMC-C225 antibody used for clinical trials (Spaulding and Spaulding 2002Go). EGFR expression levels reported for a number of human cancers differ substantially from study to study. For example, head and neck cancers as well as lung carcinomas have been reported to express EGFR in 13% to more than 95% of the tumors tested, and for colorectal cancers the results vary between 25% and 80% (Irish and Bernstein 1993Go; Rusch et al. 1993Go; Salomon et al. 1995Go; Ke et al. 1998Go; Hsieh et al. 2000Go). Probably this is mainly due to the fact that these studies have been conducted using different antibodies, detection systems, and sampling protocols. With the exception of malignant gliomas, in which EGFR overexpression is accompanied by the respective gene amplification in the majority of cases, most studies investigating solid malignant tumors with known EGFR overexpression failed to show a similar correlation, implying that EGFR gene amplification is not the main mechanism for protein overexpression (Berger et al. 1987Go; Wong et al. 1987Go; Kearsley et al. 1991Go; al-Kasspooles et al. 1993; Schwechheimer et al. 1995Go). By preserving the morphology and without contamination problems, as with tissue-destructive techniques such as blotting- and PCR-based methods, IHC appears to be an ideal tool for assaying solid malignant tumors for EGFR expression under optimized and standardized conditions. In conclusion, the results of this study suggest that testing of head and neck cancers, NSCL carcinomas, and colorectal adenocarcinomas for EGFR expression with the DakoCytomation EGFRpharmDX kit should be preferentially carried out on tumor samples alternatively fixed in Pen-fix (4 h4), 4% unbuffered formalin (24 hr), AFA (24 hr), 10% unbuffered formalin (24 hr), or 4% neutral buffered formalin (24 hr). The PreFer fixative was found to be unsuitable owing to poor preservation of tissue morphology. The results of this study also demonstrated that evaluation of EGFR expression is dependent on storage time of archived tissue sections and might be critical, depending on the tumor entity tested. This is especially true for colorectal adenocarcinomas, which should be tested within the first 9 months to avoid false-negative results. In contrast, evaluation of the EGFR status in archived tissue sections originating from NSCL cancers and squamous cell carcinomas of the head and neck is less dependent on the storage time. However, the predictive value of this diagnostic tool for the success of EGFR-targeted therapy has to be further evaluated.


    Footnotes
 
Received for publication October 27, 2003; accepted February 18, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

al-Kasspooles M, Moore JH, Orringer MB, Beer DG (1993) Amplification and over-expression of the EGFR and erbB-2 genes in human esophageal adenocarcinomas. Int J Cancer 54:213–219[Medline]

Artega C (2002) Overview of epidermal growth factor receptor biology and its role as a therapeutic target in human neoplasia. Semin Oncol 29(suppl 14):3–9

Artega C (2003) Targeting HER I/ EGFR: a molecular approach to cancer therapy. Semin Oncol 30(suppl 7):3–14

Baselga J (2002) Why the epidermal growth factor receptor? The rationale for cancer therapy. Oncologist 7(suppl 4):2–8[Medline]

Baselga J, Pfister D, Cooper MR, Cohen R, Burtness B, Bos M, D'Andrea G (2000) Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J Clin Oncol 18:904–914[Abstract/Free Full Text]

Berger MS, Gullick WJ, Greenfield C, Evans S, Addis BJ, Waterfield MD (1987) Epidermal growth factor receptors in lung tumours. J Pathol 152:297–307[Medline]

Bertheau P, Cazals-Hatem D, Meignin V, de Roquancourt A, Vèrola O, Lesourd A, Sènè C (1998) Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol 51:370–374[Abstract]

Bunn PA Jr, Franklin W (2002) Epidermal growth factor receptor expression, signal pathway, and inhibitors in non-small cell lung cancer. Semin Oncol 29(suppl 14):38–44

Chinn P, Braslawsky G, White C, Hanna N (2003) Antibody therapy of non-Hodgkin's B-cell lymphoma. Cancer Immunol Immunother 52:257–280[Medline]

Ciardiello F, Tortora G (2001) A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 7:2958–2970[Abstract/Free Full Text]

Herbst RS, Hong WK (2002) IMC-C225, an anti-epidermal growth factor receptor monoclonal antibody for treatment of head and neck cancer. Semin Oncol 29(suppl 14):18–30

Hsieh ET, Shepherd FA, Tsao MS (2000) Co-expression of epidermal growth factor and transforming growth factor-alpha is independent of ras mutations in lung adenocarcinoma. Lung Cancer 29:151–157[Medline]

Irish JC, Bernstein A (1993) Oncogenes in head and neck cancer. Laryngoscope 103:42–52[Medline]

Jacobs TW, Prioleau JE, Stillman IE, Schnitt SJ (1996) Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst 88:1054–1059[Abstract/Free Full Text]

Ke LD, Adler-Storthz K, Clayman GL, Yung AW, Chen Z (1998) Differential expression of epidermal growth factor receptor in human head and neck cancers. Head Neck 20:320–327[CrossRef][Medline]

Kearsley JH, Leonhard JH, Walsh MD, Wright GR (1991) A comparison of epidermal growth factor receptor (EGFR) and c–cerbB-2 oncogene expression in head and neck squamous cell carcinomas. Pathology 23:189–194[Medline]

O'Dweyer PJ, Benson AB III (2002) epidermal growth factor receptor-targeted therapy in colorectal cancer. Semin Oncol 29(suppl 14):10–17

Pauletti G, Godolphin W, Press MF, Slamon DJ (1996) Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene 13:63–72[Medline]

Press MF, Hung G, Golophin W, Slamon DJ (1994) Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression. Cancer Res 54:2771–2777[Abstract]

Ross JS, Gray K, Gray GS, Worland PJ, Rolfe M (2003) Anticancer antibodies. Am J Clin Pathol 119:472–485[CrossRef][Medline]

Rusch V, Baselga J, Gordon-Cardo C, Orazem J, Zaman M, Hoda S, McIntosh J (1993) Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 53(suppl 10):2379–2385

Salomon DS, Brandt R, Ciardiello F, Normanno N (1995) Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19:183–232[CrossRef][Medline]

Schwechheimer K, Huang S, Cavenee WK (1995) EGFR gene amplification-rearrangement in human glioblastomas. Int J Cancer 62:145–148[Medline]

Shin DM, Donato NJ, Perez-Soler R, Shin HJ, Wu JY, Zhang P, Lawhorn K (2001) Epidermal growth factor receptor-targeted therapy with C225 and cisplatin in patients with head and neck cancer. Clin Cancer Res 7:1204–1213[Abstract/Free Full Text]

Spaulding DC, Spaulding BO (2002) Epidermal growth factor receptor expression and measurement in solid tumors. Semin Oncol 29(suppl 14):45–54

Wong AJ, Bigner SH, Bigner DD, Kinzler KW, Hamilton SR, Vogelstein B (1987) Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA 84:6899–6903[Abstract]

Xiong HQ, Abbruzzese JL (2002) Epidermal growth factor receptor-targeted therapy for pancreatic cancer. Semin Oncol 29(suppl 14):31–37

Yip YL, Ward RL (2002) Anti-erbB-2 monoclonal antibodies and erbB-2-directed vaccines. Cancer Immunol Immunother 50:569–587[CrossRef][Medline]