• hepatocyte growth factor;
  • c-met;
  • adhesion;
  • scatter factor;
  • prognostic;
  • ErbB2


  1. Top of page
  2. Abstract


It has been shown that receptor tyrosine kinases (RTKs) predict outcome in patients with breast carcinoma. Although RTKs are a large family, HER-2, epidermal growth factor receptor (EGFR), Met (hepatocyte growth factor receptor), and others all have shown the ability to predict outcome. However, it remains unclear whether these markers are defining the same subpopulation of patients with breast carcinoma. In this study, the authors attempted to determine the correlation between RTKs on the basis of their ability to stratify a population according to outcome.


The authors used tissue microarray technology to study 324 patients with lymph node negative breast carcinoma who had 20–40 years of follow-up. Expression was assessed using immunohistochemical stains for Met, EFGR, fibroblast growth factor receptor (FGFR), and HER-2. Expression levels were assessed by two observers, and correlations were analyzed. Standard pathology information, including tumor size, nuclear grade, Ki-67 receptor status, and estrogen and progesterone receptor expression levels, also was collected.


RTK expression in the study cohort revealed two strong correlations. Specifically, HER-2 and EGFR showed similar expression patterns (P < 0.0001), and Met cytoplasmic domain and FGFR cytoplasmic staining showed similar expression patterns (P < 0.0001), but no correlation was found between the two groups. Of these RTKs, only high levels of Met cytoplasmic domain showed significance as a prognostic marker defining a shortened survival compared with the rest of the population (P = 0.0035; relative risk, 2.04). In the same group of patients, HER-2, hormone receptor status, and other RTK family receptors were not correlated with outcome. In multivariate analysis, only Met cytoplasmic domain and tumor size showed independent predictive value.


The current results indicate that the cytoplasmic domain of Met shows a unique staining pattern and defines a set of patients unique from the set of patients defined by overexpression of HER-2, EGFR, or hormone receptors. Furthermore, this group of patients is associated tightly and independently with worse outcome. Cancer 2003;97:1841–8. © 2003 American Cancer Society.

DOI 10.1002/cncr.11335

The single best indicator of disease free survival and overall survival in patients with breast carcinoma is lymph node status.1 Patients who have breast carcinomas with axillary lymph node metastases have a 10-year recurrence rate approaching 70%.2 However, in patients with lymph node negative breast carcinoma, there are no markers used to predict outcome that consistently show statistical significance. In this group, predicting a worse outcome is of great importance, because as many as 20% of women with lymph node negative breast carcinoma eventually will die of metastatic disease.3 Numerous prognostic markers have been tested to try to predict outcome in this group. In large studies with long follow-up, the best predictors of outcome were tumor size, tumor grade, cathepsin-D expression, Ki-67 expression, S-phase fraction, mitotic index, and vascular invasion. However, despite their marginal statistical status, tumor size and tumor grade enjoy broad acceptance as prognostic factors this group of patients.4

Tyrosine kinase receptors (RTKs) are gaining attention as prognostic markers and as possible future predictive markers as the number of trials grows for biospecific inhibitors. Among these, HER-2 (erb B2 and neu), epidermal growth factor receptor (EGFR), and Met (c-met) have been documented as prognostically significant markers for invasive breast carcinomas predicting a worse prognosis. HER-2 has been associated with outcome in patients with lymph node positive tumors but has not proven valuable in patients with lymph node negative tumors.5 Although some studies have suggested that EGFR is valuable,6 EGFR has been examined in over 25 studies, of which only approximately 50% suggest that overexpression is associated with poor outcome.7 Met has also shown mixed results. Although we and others have found Met useful in predicting worse outcome,8–10 others either have not seen the correlation or have found an opposite correlation.11 Finally, FGFR has been studied less and has shown no definitive prognostic value for patients with breast carcinoma.12

Slide-to-slide standardization is a long-standing problem in immunohistochemistry studies and may be one explanation for the variability seen in the studies described earlier. Tissue microarray technology can eliminate this problem.13, 14 Tissue microarrays are a method of placing very small samples of tissue from hundreds or thousands of patients on a single slide.15, 16 The technology has been used extensively and is the subject of multiple reviews.14, 17, 18 Tissue microarrays are suited especially well to comparisons of expression between multiple prognostic markers. In this report, we revisit the issue of the prognostic value of four RTKs in patients with lymph node negative breast carcinoma with an emphasis on the correlation between the markers. We studied expression patterns of Met, EGFR, fibroblast growth factor receptor (FGFR), and HER-2/neu on a cohort of patients with long-term follow-up.


  1. Top of page
  2. Abstract

Tissue Microarray Construction

The tissue microarrays were constructed as described previously16 and as reviewed recently.14 Briefly, formalin fixed, paraffin embedded tissue blocks containing breast carcinoma specimens were retrieved from the archives of the Yale University Department of Pathology. Areas of invasive carcinoma were identified on corresponding hematoxylin and eosin-stained slides, and the tissue blocks were cored and transferred to a recipient master block using a Tissue Microarrayer (Beecher Instruments, Sun Prairie, WI). Each core measured 0.6 mm in greatest dimension, and cores were spaced 0.8 mm apart. After cutting the recipient block and transfer with an adhesive tape to coated slides for subsequent ultraviolet cross linkage (Instrumedics, Inc., Hackensack, NJ), the slides were dipped in a layer of paraffin to prevent oxidation. The array for the cohort of patients with lymph node negative breast carcinoma was constructed from paraffin embedded, formalin fixed tissue blocks from the Yale University Department of Pathology archives. The specimens were resected between 1962 and 1980, with a follow-up that ranged between 4 months and 53.8 years, with a mean follow-up of 15.6 years and a median follow-up of 14.3 years.


Nuclear grade was evaluated by one observer (I.T.O.) according to the methods described by Fisher et al.19 on each spot. Due to the age of the specimens, they were not assigned a nuclear grade previously. Presence or absence of necrosis, mitotic count, or histologic parameters could not be included in the grading criteria because of the small size of the area evaluated. Eighty of 306 included specimens (26%) were Grade 1, 170 specimens (56%) were Grade 2, and 56 specimens (18%) were Grade 3. Similar to previous studies of nuclear grade in lymph node negative tumors, no statistical significance was achieved. However, a trend was seen between high nuclear grade and worse outcome (5-year survival).

Tumor Size

Information regarding tumor size was obtained from descriptions in the original pathology reports. For staging in the statistical analyses, tumor size > 2.0 cm was considered large, and others were considered small.


The tissue microarray slides were deparaffinized with xylene rinses and then transferred through two changes of 100% ethanol. Endogenous peroxidase activity was blocked by a 30-minute incubation in a 2.5% hydrogen peroxide/methanol buffer. Antigen retrieval was performed by boiling the slides in a pressure cooker filled with a sodium citrate buffer, pH 6.0. After antigen retrieval, the slides were incubated with 0.3% bovine serum albumin/1 × Tris-buffered saline (TBS) for 1 hour at room temperature to reduce nonspecific background staining, followed by a series of 2-minute rinses in 1 × TBS, TBS/0.01% Triton, and 1 × TBS. Primary antibody was applied for 1 hour at room temperature. Dilutions for the RTKs were as follows: Met, 1:1 (note that this antibody was provided as a culture supernate from Zymed Laboratories, Inc., South San Francisco, AC); EGFR, 1:200; FGFR, 1:300; and HER-2, 1:8000. After a series of TBS rinses, as described above, bound antibody was detected by using an antirabbit, horseradish peroxidase-labeled, polymer secondary antibody from the DAKO Envision TM + System (DAKO, Carpinteria, CA). The slides were rinsed in the TBS series and visualized with a 10-minute incubation of liquid 3,3′-diaminobenzidine in buffered substrate (DAKO) for 10 minutes. Finally, the slides were counterstained with hematoxylin and mounted with Immunomount (Shandon, Pittsburgh, PA). Immunohistochemical staining also was done for estrogen receptor (ER), progesterone receptor (PR), and HER-2, as described previously.20 Ki-67 expression was assessed using purified antihuman monoclonal antibody (1:200 dilution; overnight incubation; Pharmingen, San Diego, CA). The Met antibody 3D4 was provided as part of a collaborative arrangement with Zymed Laboratories, Inc.; the EGFR antibody was EGFR 1005 (catalog no. SC03) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and the FGFR1 antibody was FLG C15 (catalog no. SC121; Santa Cruz Biotechnology, Inc.).

Evaluation of Immunohistochemical Staining

For each spot, the regions of most intense and/or predominant staining pattern were scored by eye. Traditionally, immunohistochemistry scoring of stain intensity includes a variable for the area percentage stained with the specimen; however, due to the small size of the spot (0.6 mm in greatest dimension) and the fact that the spots often are homogenous, no area variable was included. Nuclear staining and/or cytoplasmic staining (ER/PR, EGFR, FGFR, Ki-67, and Met cytoplasmic domains) were determined separately for each specimen. The staining intensity was graded on the following scale: 0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining. The membranous staining was determined for HER-2 and EGFR. The staining intensity for membranous staining was graded on the following scale: 0, no staining; 1, incomplete staining; 2, weak but complete staining of the plasma membrane encircling the entire cell; and 3, intense complete staining. Again, we did not take the percentage of cells with staining in consideration because of the small size of the tumor sections. For specimens that were uninterpretable, a score of not available was given. Only FGFR showed distinctly separate nuclear and cytoplasmic staining characteristics; and, for this antibody, nuclear staining and cytoplasmic staining were scored individually. Scoring of the tissue microarrays was completed by two independent observers (I.T.O and M.D.F.) for EGFR, FGFR nuclear staining, FGFR cytoplasmic staining, and Met with a very high correlation between scorers (P < 0.0001). For the antibodies with established staining characteristics in the literature (ER, PR, HER-2, and Ki-67), scoring was performed by one observer (I.T.O. or M.D.F.). Ki-67 was considered positive if > 10% of nuclei were stained. Frequency distributions for these markers were in the range of other works seen in the literature, validating this cohort (see Table 1).

Table 1. Distribution of the Expression of Standard Prognostic Markers in the Cohort on Tissue Microarray
  • ER: estrogen receptor; PR: progesterone receptor.

  • a

    For a description of ordinal scoring of expression and the selection of cut-off values to define positive staining for each marker, see text.

Positive (%)57521460
Negative (%)43488640

Statistical Analysis

All analyses were completed using Statview software (version 5.0.1; SAS Institute Inc., Cary, NC). The correlation between the scores of both scorers and the relations among the different immunohistochemical and clinicopathologic parameters were measured using the chi-square test. The prognostic significance of parameters on overall survival was calculated by multivariate analysis using a Cox proportional hazards model. Survival curves were calculated using the Kaplan–Meier survival analysis method with the differences estimated using the Mantel–Cox log-rank test.


  1. Top of page
  2. Abstract

The RTKs analyzed in this study showed a variety of staining patterns that are summarized in Figure 1. Although all RTKs are present at the membrane, there are now numerous studies showing that RTKs can be found in other locations in the cell, including both the cytoplasm and, more recently, the nucleus.21 The patterns we saw included both conventional membranous patterns as well as both cytoplasmic and nuclear staining (for a detailed description, see above). The pattern of expression of each RTK was determined after examining the entire array to determine the most prevalent patterns. The question of how to divide subjective ordinal staining patterns always is controversial. In this study, we tried to use breakpoints suggested previously in the literature when they were present8, 10; however, in some instances (FGFR), there were not clear precedents in the literature or the literature was inconsistent (EGFR). A summary of the expression pattern of each RTK is shown in Table 2 with the definition of the cut-off values.

thumbnail image

Figure 1. Examples of the tissue microarray immunostains for high-level staining (3 +) for Met (A), epidermal growth factor receptor (B), fibroblast growth factor receptor (FGFR) cytoplasmic staining (C), and FGFR nuclear staining (D).

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Table 2. Distribution of Receptor Tyrosine Kinase Expression on Tissue Microarray
  • EGFR: epidermal growth factor receptor; FGFR-n: fibroblast growth factor receptor (FGFR) nuclear staining; FGFR-c: FGFR cytoplasmic staining; Met-c: Met cytoplasmic domain.

  • a

    For definitions for ordinal scoring of expression and the selection of cut-off values to define positive staining for each marker generally are consistant with the literature on these markers. Specifically, for Met, very strong staining was considered positive (3 +), and weak staining (1 + or 2 +) or the absence of staining (0) was considered negative. For HER-2, epidermal growth factor receptor, and fibroblast growth factor receptor, the literature defines 2 + and 3 + as positive and 1 + or 0 as negative.

Positive (%)1410.5486822
Negative (%)8689.5523278

In this study, we were concerned primarily with the correlation between each RTK and the correlation of each RTK with known prognostic markers. We calculated chi-square P values to determine the correlation among expression patterns of individual parameters. Table 3 shows the chi-square P values for all parameters studied in patients with lymph node negative breast carcinoma. Of particular note are the highly significant correlations in immunohistochemical expression patterns of HER-2 and EGFR and of Met and FGFR cytoplasmic staining (P < 0.0001). ER expression was correlated highly with PR expression, and they both showed a significant, inverse correlation with HER-2 expression.

Table 3. Chi-Square Analysis of the Correlation between Expression Levels of Each Markera
  • ER: estrogen receptor; PR: progesterone receptor; EGFR: epidermal growth factor receptor; FGFR-c: fibroblast growth factor receptor (FGFR) cytoplasmic staining; FGFR-n: FGFR nuclear staining.

  • a

    Statistical analyses of correlation patterns among the parameters studied in patients with lymph node negative breast carcinoma (n = 324 patients).

  • b

    Statistically significant.

  • c

    Inverse correlation.

PR< 0.0001
EGFR0.0016bc0.0010bc< 0.0001b
MET0.019bc0.190.540.77< 0.0001b0.12
Nuclear grade0.01bc0.017bc0.019b0.0475b0.0650.0016b< 0.0001b< 0.0001b

Survival Analyses

Cox univariate analyses at 10 years for all variables studied in patients with lymph node negative breast carcinoma are shown in Table 4. Ki-67 and tumor size, as expected, were correlated with poorer survival (Ki-67: P = 0.04; relative risk [RR], 1.648; tumor size: P = 0.0008; RR, 2.201). Among the other variables studied, only the cytoplasmic domain of Met showed a statistically significant correlation with a worse prognosis and shortened survival (P = 0.0035; RR, 2.041). Survival curves were produced for each variable, but only Met reached statistical significance using the Mantel–Cox log-rank test (Fig. 2). To determine the independent predictive value of Met expression, a multivariate analysis was conducted using the Cox proportional hazards model. In multivariate analysis, Met retained its significance as a predictor of worse outcome, even when the model contained all of the conventional prognostic variables as well as all other RTKs tested (RR, 1.86; P = 0.011).

Table 4. Univariate Analysis of Conventional and Receptor Tyrosine Kinase Markers
MarkerRRP value95% CI
  • 95% CI: 95% confidence interval; ER: estrogen receptor; PR: progesterone receptor; EGFR: epidermal growth factor receptor; FGFR-c: fibroblast growth factor receptor (FGFR) cytoplasmic staining; FGFR-n: FGFR nuclear staining.

  • a

    Statistically significant.

Nuclear grade1.0330.91200.578–1.846
Tumor size2.201a0.0008a1.391–3.481a
thumbnail image

Figure 2. Kaplan–Meier survival analysis demonstrates that Met expression, as assessed by antibodies to the cytoplasmic domain, has significant predictive value for the survival of patients with breast carcinoma (P = 0.0029; Mantel–Cox log-rank test). Time is indicated in months.

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  1. Top of page
  2. Abstract

In patients with lymph node negative breast carcinoma, we still are unable to discern the 15–20% of patients who eventually will succumb to their disease.3, 22 A long list of potential molecular markers of poorer prognosis have been suggested for this group patients with breast carcinoma, including Ki-67,23, 24 cathepsin-D,25 HER-2,23, 26–28 p53,27, 29 low levels of nm23,30 and hormone receptors.24, 27, 31 There also are reports that histologic findings (e.g., tumor grade, tumor size, mitotic index, and vascular invasion) and cytometric data (e.g., DNA ploidy and S-phase fraction) are helpful.23, 24, 27, 31–35 To date, there are no universally accepted markers for this group of patients.

We evaluated our tissue microarray cohort using these traditional markers for breast carcinoma. Hormone receptors showed similar staining patterns and frequencies compared with the widely reported values in breast carcinoma. ER was negative in 35% of patients, and PR was negative in 40% of patients. Neither ER expression nor PR expression was predictive of survival in our group. This finding is consistent with many other published studies showing limited value or no value for ER and PR as prognostic markers in patients with lymph node negative breast carcinoma.30, 36 HER-2 has been proven as a predictor of worse prognosis in patients with invasive, lymph node positive breast lesions and is a better predictor of survival compared with hormone receptors for this group of patients.37 However, its prognostic value in patients with lymph node negative lesions remains controversial, with conflicting data in the literature.23, 26–28 Our findings appear to support the hypothesis that HER-2 is not a reliable prognostic marker in this group of patients.

EGFR expression in breast carcinomas has been reported, both in patients with lymph node negative breast carcinoma and in patients with lymph node positive breast carcinoma, as associated with poor prognosis; however, the clinical significance and association with disease free survival (DFS) and overall survival statistics show mixed results in different studies.38–40 Tsutsui et al. reported that EGFR carries a prognostic significance only for DFS in patients with lymph node negative tumors on multivariate analysis; whereas, in the series by Torregrosa et al., EGFR failed to show statistical significance as a prognostic marker in patients with lymph node negative tumors but was associated with a worse prognosis, influencing DFS in patients with lymph node positive tumors. Conversely, Seshadri et al. concluded that the expression of EGFR was not at all a predictor of poor prognosis in patients with lymph node negative breast carcinoma. Our findings did not show any value for EGFR as a marker of prognosis in patients with lymph node negative tumors.

Our group has been particularly interested in the hepatocyte growth factor receptor, Met (or C-met). Met is a dimeric tyrosine kinase growth factor receptor in which activation is associated with increased invasion, motogenesis, and morphogenesis.41 In the breast, Met is expressed in normal ductal and lobular epithelium and functions in both the embryonic development and subsequent remodeling of the breast.42

Our previous studies showed that expression of the Met receptor in patients with invasive breast carcinoma is of significant prognostic value in determining patient survival, even in patients with negative lymph nodes.8, 9 The Vande Woude group also found this correlation between Met expression and outcome;10 although, along with others, we found no correlation in some cohorts (unpublished data). We believe this variability is due to antibody selection. Many studies have used antibodies to the c-terminal domain produced by Santa Cruz Biotechnology, Inc., using a peptide coding for the C-terminal 28 amino acids. We found significant lot-to-lot variability with this antibody. Recently, Zymed Laboratories, Inc. and others have produced monoclonal antibodies to the C-terminus. In another study from our laboratory,43 we compared the results from this antibody with another monoclonal antibody to the extracellular domain of Met. We showed results with the cytoplasmic domain similar to the results reported here, but the extracellular domain was very different. Although there is a high correlation of expression, overexpression, as assessed by the antibody to the cytoplasmic domain, selects a group of patients with worse outcome, whereas the extracellular domain antibody does not.43 We believe this may be a function of either cleavage or activation of Met. It is notable that the other study that found a correlation between Met expression and patient outcome also used a monoclonal antibody (generated by the Vande Woude laboratory)10 and that our own previous studies used a polyclonal antibody made to a cytoplasmic domain peptide.8

The finding that Met overexpression predicts poor outcome raises the question of its relation to other RTKs, especially EGFR and HER-2, which also have been implicated as prognostic variables. The current work shows that tumors that overexpress Met are unique from tumors that express HER-2 and EGFR. EGFR and HER-2 are closely related members of the erb B oncogene family; thus, it is not surprising that there is a high correlation between the expression of these two proteins. Met is not a member of this family; thus, its overexpression appears to be unrelated to erb B family RTKs.

Although there is not a close correlation between Met and the erbB family of RTKs, there is a tight, direct correlation between Met expression and the cytoplasmic staining pattern seen with the FGFR antibody (P < 0.0001). To our knowledge, the correlation between FGFR and Met has never been reported before in patients with breast carcinoma. Coordinated actions of several growth factors and their receptors, including FGFR and C-met, have been reported in patients with hepatic lesions44 and in normal morphogenesis in the uterus;45 however, the molecular basis of such an interaction, if present, remains unknown. Although FGFR is a membrane-based RTK, immunohistochemical evaluation for FGFR has not been standardized as a marker for immunohistochemistry. Thus, we scored cytoplasmic and nuclear staining for FGFR separately. Although neither cytoplasmic staining for FGRF nor nuclear staining for FGFR was a statistically significant prognostic marker in our study, there was a high correlation between Met expression and FGFR cytoplasmic expression.

RTKs recently have gained great interest both as markers of prognosis and as promising targets for novel chemotherapeutic options. The HER2/trastuzmab pair is the first U.S. Food and Drug Administration-approved example of numerous RTK-related therapeutics currently in clinical trials. This interest raises the question of the relations between different RTKs. Although data on the cross reactivity of kinase-based therapeutics is not widely available to date, there are many of types of RTK receptors that are overexpressed in malignancies of the breast.

The results of the current study indicate that the expression of Met, as assessed using a cytoplasmic domain monoclonal antibody, in patients with lymph node negative, invasive breast carcinoma is of significant predictive value in determining patient survival. Its predictive value exceeds all conventional prognostic markers assessed in this cohort as well as the other RTKs tested. The group of patients that overexpress this protein are unrelated to the group that overexpresses either EGFR or HER-2. Thus, in the future, determination of prognosis in patients with lymph node negative, invasive breast carcinoma may be improved by assessment of the level of expression of Met using a cytoplasmic domain monoclonal antibody.


  1. Top of page
  2. Abstract
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