1857
Comparison of Two Antibodies for Immunohistochemical Evaluation of Epidermal Growth Factor Receptor Expression in Colorectal Carcinomas, Adenomas, and Normal Mucosa Rohit Bhargava, M.D.1 Beiyun Chen, M.D., Ph.D.1 David S. Klimstra, M.D.1 Leonard B. Saltz, M.D.2 Cyrus Hedvat, M.D., Ph.D.1 Laura H. Tang, M.D., Ph.D.1 William Gerald, M.D., Ph.D.1 Julie Teruya-Feldstein, M.D.1 Philip B. Paty, M.D.3 Jing Qin, Ph.D.4 Jinru Shia, M.D.1 1
Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.
2 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York. 3
Department of and Surgery, Memorial SloanKettering Cancer Center, New York, New York.
4
Biostatistics Research Branch, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland.
Supported in part by Grant 2 P01 CA65930-05A2 from the National Institutes of Health. Rohit Bhargava’s current address: Magee-Women’s Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. Address for reprints: Jinru Shia, M.D., Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021; Fax: (212) 717-3203; E-mail:
[email protected] Received July 14, 2005; revision received October 6, 2005; accepted October 26, 2005.
BACKGROUND. Immunohistochemical staining for epidermal growth factor receptor (EGFR) has been used as a criterion for the selection of patients with colon cancer for anti-EGFR therapy. Two antibodies, the PharmDx kit and the 31G7 clone, are used commonly for immunohistochemistry by various laboratories. No comparative studies on the performance of these 2 antibodies are available.
METHODS. EGFR status was evaluated in 744 tissue microarray core samples from primary and metastatic colorectal carcinomas, colorectal adenomas, and normal colorectal mucosa with both the PharmDx kit and the clone 31G7 monoclonal antibodies. The stains were compared for staining intensity by using an automated image-analysis system. The intensity of positive staining (brown color) was measured on a scale from 0 to 255.The staining intensity also was scored manually as 0, 1 ⫹, 2 ⫹, and 3 ⫹. RESULTS. Statistically, the median staining intensities scored by the automated system between the 2 antibodies did not differ significantly, although, within each category of samples (normal, adenoma, carcinoma, and metastases), the PharmDx antibody staining was slightly more intense than the clone 31G7 antibody staining. There was a linear correlation between automated image-analysis and manual scoring categories. The median automated image-analysis intensity scores for the 4 manual scoring categories with the PharmDx kit were as follows: 0 staining, 67.5; 1 ⫹ staining, 75.5; 2 ⫹ staining, 89.6; and 3 ⫹ staining, 106.0. The median automated image-analysis intensity scores for the 4 manual scoring categories with the clone 31G7 antibody were as follows: 0 staining, 71.3; 1 ⫹ staining, 73.6; 2 ⫹ staining, 84.6; and 3 ⫹ staining, 99.1. The classification of tumors as EGFR-negative (0 staining) or positive (1 ⫹, 2 ⫹, or 3 ⫹ staining) was concordant in 151 of 160 carcinomas (94.4%) with 2 antibodies using manual scoring. Five samples (3%) that scored 1 ⫹ with the PharmDx kit antibody scored 0 with the clone 31G7 antibody; whereas 4 samples (2.5%) that scored 1 ⫹ with the clone 31G7 antibody scored 0 with the PharmDx kit antibody. CONCLUSIONS. The EGFR expression results obtained by immunohistochemistry using both the EGFR PharmaDx kit and the 31G7 clone were comparable. Either antibody may be used for immunohistochemical detection of EGFR in colorectal carcinomas. In addition, manual scoring had an excellent correlation with automated scoring. Cancer 2006;106:1857– 62. © 2006 American Cancer Society.
KEYWORDS: epidermal growth factor receptor, colon carcinoma, immunohistochemistry, Ventana clone 31G7 antibody, PharmDx kit Dako antibody.
T
he epidermal growth factor receptor (EGFR) is a 170-kD receptor tyrosine kinase encoded by the c-erb-B (HER-1) protooncogene.1 It is expressed in various solid tumors, including colorectal, prostate,
© 2006 American Cancer Society DOI 10.1002/cncr.21782 Published online 10 March 2006 in Wiley InterScience (www.interscience.wiley.com).
1858
CANCER April 15, 2006 / Volume 106 / Number 8
head and neck, and lung cancers, and in certain normal tissues.2 When it is bound by ligands, such as epidermal growth factor (EGF) and transforming growth factor-␣, EGFR undergoes conformational changes that activate its intracellular tyrosine kinase activity, initiating autophosphorylation and downstream signal transduction pathways.3 Activation can mediate a variety of cellular responses, including gene expression, cell proliferation, and cell survival. Dysregulation of the EGFR signaling pathway because of EGFR overexpression, genetic aberrations, or other causes leads to malignant transformation. Recent studies have shown that EGFR expression is present in approximately 60% to 80% of colorectal carcinomas,4,5 and the receptor has emerged as a rational target for anticancer therapy in these tumors.6,7 Cetuximab is the first EGFR inhibitor to receive United States Food and Drug Administration approval for the treatment of colorectal cancer. Cetuximab is a human-mouse chimeric monoclonal antibody directed against the extracellular ligand-binding domain of EGFR.8 This drug competitively inhibits binding of EGFR by EGF and transforming growth factor-␣, thereby blocking downstream signal transduction pathways and arresting cell growth.9 Cetuximab is indicated for the treatment of advanced metastatic colorectal cancer that is resistant to irinotecan-based therapy and as a single agent in patients who cannot tolerate irinotecan.10 Currently, immunohistochemical stain for EGFR is the method of patient selection for cetuximab therapy, and only patients with an EGFR positive colorectal carcinoma are eligible for the use of this drug. However, recent studies have shown that immunohistochemical expression of EGFR does not correlate with clinical response to cetuximab, and response to cetuximab may be seen in patients who have EGFR-negative colorectal carcinoma.11 Thus, the use of immunohistochemistry in patient selection for the cetuximab treatment has to be reevaluated. One important aspect for the evaluation of immunohistochemistry is technical, and the type of primary antibody used is of significant technical importance. The 2 most commonly used EGFR antibodies for immunohistochemistry are the PharmDx kit antibody (DAKOCytomation, Carpinteria, CA) and the clone 31G7 antibody (Ventana Inc., Tucson, AZ). To our knowledge, no systematic assessment of the performance of the PharmDx Kit antibody versus the clone 31G7 antibody is available in the English literature. Thus, in the current study, we evaluated the EGFR status in 744 microarray tissue core samples from normal colon, colonic adenomas, primary colon cancers, and metastases using these 2 antibodies. The
staining intensity in the different colorectal tissues was detected by 2 methods: an image-analysis system and manual scoring. Then, a comparative analysis was performed on the staining intensity results obtained for the 2 antibodies.
MATERIALS AND METHODS Tissue Samples and Construction of Tissue Microarray Seven hundred forty-four tissue cores were derived from 248 samples (3 cores per patient) of normal colon (n ⫽ 49 samples), adenomas (n ⫽ 32 samples), primary carcinomas (n ⫽ 139 samples), liver metastases (n ⫽ 6 samples), and lung metastases (n ⫽ 22 samples). The tissue microarrays were constructed using 0.6-mm tissue cores as described previously.12 A hematoxylin and eosin-stained section was evaluated for the presence of specific diagnosis, and the area to be used for creation of the tissue microarray was marked on the slide and the donor block. Three cores of different areas from a single formalin fixed, paraffin embedded tissue block were sampled.
Immunohistochemistry Immunohistochemical staining for EGFR was performed on the tissue microarray slides using the EGFR PharmDx kit (DAKO Cytomation) and the 31G7 clone (Vertana, Inc.) according to the manufacturers’ instructions.
Quantitative Image Analysis Slides were scanned with the ACIS II system (ChromaVision Medical Systems, Inc., San Juan Capistrano, CA), which combines automated slide digitization and image analysis of immunohistochemically stained sections. Immunostained tissue microarray sections were scanned using the ⫻ 10 objective, and a composite digital image was produced. This image was divided into individual tissue cores using a grid. The intensity score represents the maximal intensity for each tissue core on the tissue microarray. The intensity of positive staining (brown color) is measured on a scale from 0 to 255. The minimum intensity score is 50.
Manual Scoring All samples were evaluated by a single pathologist. The stains were scored as 0 when there was no specific membrane staining within the tumor and were scored as positive when there was any staining of tumor cell membrane above background level. The positive samples were classified further into 1 ⫹, 2 ⫹, and 3 ⫹ staining based on their staining intensity. The highest staining intensity of all tissue
EGFR Antibody Comparison in Colon Ca/Bhargava et al.
1859
FIGURE 1. Examples of epidermal growth factor expression are (A) negative staining, (B) 1 ⫹ staining intensity, (C) 2 ⫹ staining intensity, (D) 3 ⫹ staining intensity.
cores from the same tumor was used as the final immunohistochemical result for that tumor. Examples of negative and positive stains are illustrated in Figure 1.
Statistical Analyses The intensity score was taken as the average of 3 intensities of positive staining for ACIS scoring. The highest staining intensity (of 3 scores) from the manual score was used as the final immunohistochemical result. Correlation between the clone 31G7 antibody and the PharmDx kit antibody was examined by using the Spearman correlation coefficient. The correlation between the ACIS score and the manual score was assessed by using the Kruskal–Wallis statistic. The correlation between ordered contingency table was assessed by using a Jonckheere–Terpstra statistic. P values ⬍ .05 were considered significant.
RESULTS PharmDx Kit versus Clone 31G7: The ACIS II Quantitative Image-Analysis System Of the 744 tissue cores, there were 147 cores from normal colon, 96 cores from adenomas, 417 cores from primary colon carcinomas, 18 cores from liver metastases, and 66 cores from lung metastases. The mean, median, and standard deviation for the staining intensity of both antibodies among different diagnostic categories are shown in Tables 1 through 4. Staining with the DAKO antibody (PharmDx kit) was slightly more intense than staining with the Ventana antibody (clone 31G7) in each category; however, the difference in staining intensity was not statistically significant.
ACIS II Automated Scoring versus Manual Scoring for Both Antibodies The slides that were stained with both the PharmDx kit antibody and the clone 31G7 antibody also were
1860
CANCER April 15, 2006 / Volume 106 / Number 8
TABLE 1 Staining Intensity According to the ACIS II Automated ImageAnalysis System for Normal Colon Tissue
TABLE 4 Staining Intensity According to the ACIS II Automated ImageAnalysis System for Metastatic Colorectal Carcinoma
Variable
Clone 31G7
PharmDx Kit
Variable
Clone 31G7
PharmDx Kit
Mean Median Standard deviation
78.2 78.0 8.6
88.1 86.0 15.8
Mean Median Standard deviation
72.8 69.5 17.9
76.7 74.0 15.4
The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA).
The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA).
TABLE 2 Staining Intensity According to the ACIS II Automated ImageAnalysis System for Adenoma
TABLE 5 Correlation between Manual Scoring and Automated Scoring with the PharmDx Kit
Variable
Clone 31G7
PharmDx Kit
Mean Median Standard deviation
79.8 78.0 8.6
91.5 90.5 13.8
The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA).
TABLE 3 Staining Intensity According to the ACIS II Automated ImageAnalysis System for Primary Colorectal Adenocarcinoma Variable
Clone 31G7
PharmDx Kit
Mean Median Standard deviation
77.3 75.0 10.4
84.6 82.0 16.7
The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA).
scored manually. For the PharmDx kit antibody, all countable tissue cores from adenomas and carcinomas were included for automated and manual scoring correlation. For the clone 31G7 antibody, all countable tissue cores from normal colon, adenomas, and carcinomas were included for automated and manual scoring correlation. Table 5 shows that, with the PharmDx kit antibody, the mean and median intensity scores of the tissue cores obtained by the ACIS II quantitative image-analysis system increased linearly according to manual grading from 0 to 3 ⫹. When the tissue cores were graded 0 manually, the average ACIS II score was only 68.9; whereas, when the tissue cores were graded 1 ⫹, 2 ⫹, and 3 ⫹, the average ACIS scores increased to 76.4, 90.6 and 109.4, respectively. Thus, the 2 scoring systems had a positive correlation (statistically
Manual Score (No. of Patients) ACIS Score*
0 (nⴝ26)
1ⴙ (nⴝ68)
2ⴙ (nⴝ72)
3ⴙ (nⴝ148)
Mean Median Standard deviation
68.9 67.5 6.6
76.4 75.5 8.1
90.6 89.6 8.7
109.4 106 12.6
ACIS: the ACIS II automated image-analysis system. The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA). Samples analyzed include all countable tissue cores from adenomas and carcinomas. * P ⬍ .0001; mean and median intensity scores obtained from the ACIS II automated image-analysis system correlated positively with the manual scores.
significant; P ⬍ .0001). A similar significant positive correlation between the ACIS II image system and the manual grading system also was observed with the clone 31G7 antibody (P ⬍ .0001). The mean and median ACIS II scores for tissues that were graded manually for staining from 0 to 3 ⫹ are listed in Table 6.
PharmDx Kit versus Clone 31G7: Manual Scoring The numbers of carcinoma samples that were scored negative or positive manually by the 2 antibodies were compared. Of 167 carcinoma samples that were included in this study, 160 were available for final comparison between the 2 antibodies. The results were concordant in 151 tumors (94%). Five tumors (3%) scored 1 ⫹ with the PharmDx kit antibody and scored 0 with the clone 31G7 antibody. Conversely, 4 tumors (2.5%) scored 1 ⫹ with the clone 31G7 antibody and scored 0 with the PharmDx kit antibody (Table 7). Among the positively stained carcinoma samples, consistent with the results obtained with the ACIS II image system (Table 3), the PharmDx kit antibody yielded a greater proportion of 2 ⫹ and 3 ⫹ results (81 of 160 samples; 51%) compared with the clone 31G7
EGFR Antibody Comparison in Colon Ca/Bhargava et al. TABLE 6 Correlation between Manual Scoring and Automated Scoring with the Clone 31G7 Antibody Manual Score (No. of Patients) ACIS Score*
0 (nⴝ27)
1ⴙ (nⴝ148)
2ⴙ (nⴝ48)
3ⴙ (nⴝ10)
Mean Median Standard deviation
71.4 71.3 3.8
75 73.6 6.4
84.4 84.6 6.1
105.7 99.1 21.1
ACIS: ACIS II automated image-analysis system. The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA). The samples analyzed included all countable tissue cores from normal mucosa, adenomas, and carcinomas. * P ⬍ .0001; mean and median intensity scores obtained from the ACIS II automated image-analysis system correlated positively with the manual scores.
TABLE 7 Correlation of Manual Grading Scores between the PharmDx Kit and the Clone 31G7 Antibody in Colonic Adenocarcinomas PharmDx Kit Score Clone 31G7 Score
0
1ⴙ
2ⴙ
3ⴙ
Total
0 1⫹ 2⫹ 3⫹ Total
17 4 0 0 21
5 53 0 0 58
0 35 24 0 59
0 1 13 8 22
22 93 37 8 160
The ACIS II automated image-analysis system is from ChromaVision Medical Systems, Inc. (San Juan Capistrano, CA), clone 31G7 is from Ventana Inc. (Tucson, AZ), and the PharmDx kit is from DAKOCytomation (Carpinteria, CA). P ⬍ .0001; P values were calculated after combining the 2⫹ and 3⫹ staining categories.
antibody (45 of 160 samples; 28%) by manual grading. Conversely, the clone 31G7 antibody yielded a greater proportion of 1 ⫹ results (93 of 160 samples; 58%) compared with the PharmDx kit antibody (58 of 160 samples; 36%). Overall, however, there was significant concordance between 0 and 3 ⫹ categorization performed by the 2 antibodies (P ⬍ .0001; Table 7).
DISCUSSION The primary objective of the current study was to compare the performance of the 2 commonly used EGFR antibodies for the assessment of EGFR expression in colorectal tissues: the EGFR PharmDx kit and clone 31G7. Both antibodies identify an epitope in the extracellular domain of the EGFR molecule. Such a study is necessary to promote our understanding of the utility of immunohistochemistry in selecting patients for treatment with cetuximab. It has been recognized that immunohistochemistry is influenced by
1861
a variety of factors, including tissue processing and handling, the specificity and sensitivity of the antibody, and the criteria in scoring the stain. The importance of standardized tissue processing and handling in immunohistochemistry for EGFR has been pointed out by some investigators.13,14 It has been shown that altered protein expression may result from prolonged storage time of tissue samples and may allow certain catalytic degradation of cell surface receptors.13,14 The importance of the antibody and the scoring system of the stain in immunohistochemistry are obvious. However, such issues remain to be resolved for EGFR immunohistochemistry. Two different types of EGFR antibodies currently are used commonly among different laboratories. Although it has been indicated that positive EGFR staining of any intensity in ⬎1% of tumor cells by the EGFR PharmDx kit is sufficient for the eligibility of cetuximab therapy, it is not known whether the other type of EGFR antibody, the clone 31G7 antibody, would have the same indication. Thus, we systematically analyzed the performance of the 2 major EGFR antibodies in a large number of colorectal tissue samples. We also compared an automated scoring system with manual scoring. Overall, our results showed that the EGFR PharmDx kit from DAKO consistently stained colorectal tissues more intensely than the 31G7 clone antibody from Ventana. However, statistically, the difference in the intensity between the 2 antibodies was insignificant. A good correlation was achieved between the staining intensities obtained by the 2 antibodies. In addition, our data showed that the ACIS II image-analysis system was a reliable tool for scoring EGFR staining in colorectal tissue. The slightly more intense EGFR staining with the PharmDx kit was observed in all colorectal tissues and in all positive stains, whether it was 1 ⫹, 2 ⫹, or 3 ⫹. Consequently, it did not affect the linear correlation between the staining intensities of the 2 antibodies, and it did not alter the final manual grading in most instances. However, we did observe a few colorectal carcinomas that were scored negative with the PharmDx kit but were scored 1 ⫹ positive with the clone 31G7 antibody (3%), or vice versa (2.5%). Theoretically, such a discrepancy between the 2 antibodies may have accounted for the lack of a response to cetuximab in some patients; i.e., approximately 2.5% of patients who had tumors that were classified as EGFR-positive by the PharmDx kit antibody and, thus, received the anti-EGFR drug, in fact, had EGFR-negative tumors and should not have responded to the drug. However, clinical trials to date have demonstrated a response rate of only 9% to 23% to cetuximab in patients with colorectal cancer.15,16 Thus, the small
1862
CANCER April 15, 2006 / Volume 106 / Number 8
frequency of discrepant immunohistochemical staining results between the 2 antibodies does not explain the lack of a response in most cetuximab nonresponders. It also does not explain the presence of a response in 25% of patients with EGFR-negative tumors.11 In contrast, among the categories of intensely stained tumors, a score of 2 ⫹ or 3 ⫹ with 1 antibody never resulted in a score of 0 with the other antibody (Table 7). In the current study, we used the ACIS II quantitative image-analysis system for the evaluation of EGFR staining and observed a direct correlation between those results and the results from manual scoring. A linear increase was observed in the median staining intensity with the ACIS system from 0 to 3 ⫹ by manual scoring. Thus, although the ACIS software does not distinguish normal tissues from tumor tissues, with the ability to adjust the dynamic range, such a system may provide an efficient modality for analyzing large quantities of tissue samples in a research setting. The main advantages of automated image analysis are the improved reproducibility of results, which limits interobserver variability,17 and high accuracy in measuring intensity.17 For our study, we did not analyze interobserver variations specifically in the manual scoring of EGFR immunohistochemistry slides, and all samples were analyzed manually by a single pathologist. However, the finding that the scores produced by the image system correlated positively with the manual results suggests that manual interpretation and interobserver variability may not be significant technical issues in the analysis of EGFR immunohistochemistry. With regard to accuracy in measuring intensity, because there is no current “gold standard” with which to judge the accuracy of EGFR immunohistochemistry results, it will be difficult to compare the superiority of imaging system versus manual scoring. With regard to HER-2, there are observations suggesting that imaging system scores were correlated better with HER-2 amplification by fluorescent in situ hybridization than manual scores.18 In summary, our results suggest that immunohistochemistry using both the EGFR PharmDx kit and the 31G7 clone yielded comparable results. We conclude that either antibody may be used to analyze EGFR expression in colon carcinoma. The semiquantitative manual scoring correlated well with the results obtained through the automated image-analysis system.
2.
3.
4.
5. 6.
7.
8. 9.
10. 11.
12.
13.
14. 15.
16.
17.
18.
REFERENCES 1.
Cohen S, Ushiro H, Stoscheck C, Chinkers M. A native 170,000 epidermal growth factor receptor-kinase complex
from shed plasma membrane vesicles. J Biol Chem. 1982; 257:1523-1531. Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol. 1995;19:183-232. McCune BK, Earp HS. The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of liganddependent changes in cellular location. J Biol Chem. 1989; 264:15501-15507. Goldstein NS, Armin M. Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system. Cancer. 2001;92:1331-1346. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer. 2001;37(Suppl 4):S9 –S15. Ciardiello F, Bianco R, Damiano V, et al. Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225. Clin Cancer Res. 1999;5:909-916. Ciardiello F, Tortora G. Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer. 2003;39:1348-1354. Baselga J. The EGFR as a target for anticancer therapy— focus on cetuximab. Eur J Cancer. 2001;37(Suppl 4):S16 –S22. Harding J, Burtness B. Cetuximab: an epidermal growth factor receptor chemeric human-murine monoclonal antibody. Drugs Today (Barcelona). 2005;41:107-127. Starling N, Cunningham D. Second-line therapy for advanced colorectal carcinoma. Curr Oncol Rep. 2005;7:173-180. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol. 2005;23:1803-1810. Hoos A, Urist MJ, Stojadinovic A, et al. Validation of tissue microarrays for immunohistochemical profiling of cancer specimens using the example of human fibroblastic tumors. Am J Pathol. 2001;158:1245-1251. Atkins D, Reiffen KA, Tegtmeier CL, Winther H, Bonato MS, Storkel S. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem. 2004;52:893-901. Erlichman C, Sargent DJ. New treatment options for colorectal cancer. N Engl J Med. 2004;351:391-392. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 2004;351:337-345. Saltz LB, Meropol NJ, Loehrer PJ Sr., Needle MN, Kopit J, Mayer RJ. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol. 2004;22:1201-1208. Hilbe W, Gachter A, Duba HC, et al. Comparison of automated cellular imaging system and manual microscopy for immunohistochemically stained cryostat sections of lung cancer specimens applying p53, ki-67 and p120. Oncol Rep. 2003;10:15-20. Luftner D, Henschke P, Kafka A, et al. Discordant results obtained for different methods of HER-2/neu testing in breast cancer—a question of standardization, automation and timing. Int J Biol Markers. 2004;19:1-13.
