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© 2001 American Society for Clinical Oncology Comparison of Methods of Measuring HER-2 in Metastatic Breast Cancer Patients Treated With High-Dose ChemotherapyFrom the Dana-Farber Cancer Institute, Boston, and Bayer Diagnostics, Walpole, MA; Duke University Medical Center, Durham; Labcorp Inc, Research Triangle Park, NC; and M.D. Anderson Cancer Center, Houston, TX. Address reprint requests to Lyndsay N. Harris, MD, Dana-Farber Cancer Institute, Dana 1210, 44 Binney St, Boston, MA 02115.
PURPOSE: HER-2 is overexpressed in 20% to 30% of human breast cancer and is associated with poor outcome. Studies suggest an association between HER-2 overexpression and resistance to alkylating agents. To further evaluate this relationship, we assessed the interaction of HER-2, measured by different methods, and outcome after dose intensification with alkylating agents in metastatic breast cancer. PATIENTS AND METHODS: From 1988 to 1995 at Duke University, 425 patients with metastatic breast cancer were enrolled in a study of high-dose alkylating agents (HDC) with autologous cellular support after doxorubicin-based therapy (AFM). HER-2 was measured in serum for shed extracellular domain (ECD) and in tissue by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). RESULTS: HER-2 ECD was positive in 29% (19 of 65) of patients pre-AFM and in 11.7% (34 of 290) pre-HDC. Higher pre-AFM and higher pre-HDC HER-2 ECD predicted worse overall survival (P = .045 and P = .0096, respectively). HER-2 overexpression by IHC and FISH showed no correlation with worse disease-free survival or overall survival. FISH and ECD were highly specific for IHC (97.3% and 97.7% respectively). However, ECD had a low sensitivity for IHC—only 22% of patients with HER-2 in the primary tumor shed ECD into the serum. CONCLUSION: These data suggest that the method of measuring HER-2 is important in predicting clinical outcome. HER2 ECD may identify a poor prognosis subgroup of HER-2–positive tumors. Lack of association of HER2 by IHC/FISH with worse outcome suggests that therapy with AFM and/or HDC therapy may be able to overcome the effect of this prognostic factor or it may not be a prognostic factor in this setting.
THE HER-2 (c-erbB-2) oncogene encodes a 185 kD transmembrane glycoprotein receptor that is structurally similar to the epidermal growth factor receptor (EGF-R) and has intrinsic tyrosine kinase activity.1-3 Overexpression of HER-2 has been shown in 20% to 80% of human breast cancers and is associated with a poor prognosis.4-7 Evidence suggests that overexpressing breast tumors may be less likely to respond to hormone treatment and may be resistant to conventional doses of chemotherapy with cyclophosphamide, methotrexate, and fluorouracil.8-12 Other data indicate a dose-response effect of adjuvant chemotherapy with the anthracycline containing a regimen of cyclophosphamide, doxorubicin, and fluorouracil (CAF) in the Cancer and Leukemia Group B 8541 study.13 In this study, three doses (high, moderate, and low) of adjuvant CAF were compared in patients with node-positive breast cancer. HER-2 overexpression in patients treated with high doses of CAF was associated with a better prognosis, including longer disease-free and overall survival. The significant dose-response effect was not seen in patients with minimal or no overexpression of HER-2. This observation raised the possibility that overexpression of HER-2 may identify patients who are most likely to respond to higher doses of chemotherapy. The extracellular domain (ECD) of the HER-2 protein has been detected as a circulating antigen in the serum of 20% to 40% of patients with metastatic breast cancer; it too has been associated with a poor response to hormonal treatment and chemotherapy.14-18 Studies have shown elevated levels of circulating HER-2 ECD to be associated with a lower likelihood of response and a shorter time to disease progression in patients with metastatic breast cancer treated with hormonal agents, including megestrol acetate, fadrazole, and droloxifene.19 Additional data now suggest that elevated levels of circulating HER-2 ECD may also predict worse outcome in patients treated with chemotherapy, and response to cyclophosphamide/methotrexate/5-fluorouracil (CMF) may be worse than that to doxorubicin-containing regimens.20 The role of high-dose chemotherapy with autologous cellular support for the treatment of metastatic breast cancer is unclear; however, high-dose chemotherapy can induce higher response rates in metastatic patients.21 It may be that certain subgroups of tumors that exhibit a steeper dose-response curve to alkylator-based chemotherapy are more likely to benefit. If molecular markers can identify patients with these tumors, the relative benefit may justify the toxicity of this therapy. With that in mind, we studied the role of HER-2 as a predictor for response to high-dose chemotherapy and autologous bone marrow support in a group of patients with metastatic breast cancer treated with cyclophosphamide, cisplatin, and carmustine after induction therapy with doxorubicin-based (AFM) therapy.
Patients and Treatment Between 1988 and 1995, 425 patients with measurable, metastatic breast cancer were enrolled in a study at Duke University of high-dose alkylating agents with autologous cellular support as consolidation after intensive AFM therapy. To be eligible for the study, patients were required to have histologically demonstrated measurable metastatic breast cancer not involving the brain or bone marrow and good performance status. Prior doxorubicin exposure during adjuvant chemotherapy was required to be < 300 mg/m2, and no prior chemotherapy for metastatic disease was permitted. The patient’s tumor was required to be estrogen receptor–negative and progesterone receptor–negative or demonstrated to be progressive despite at least one hormonal therapy. Patients received two to four cycles of doxorubicin (25 mg/m2 x 3 days), 5-fluorouracil (500 or 750 mg/m2 x 5 days), and methotrexate (250 mg/m2) and were evaluated for best response. Those who achieved a complete remission (CR, n = 113) were randomized to immediate high-dose consolidation versus observation and transplant at the time of relapse. Patients who attained a partial response (PR, n = 202) went on to immediate high-dose therapy. The high-dose regimen consisted of cyclophosphamide (5,625 mg/m2), cisplatin (165 mg/m2), and carmustine (600 mg/m2) (CPB).
Materials and Methods Tissue blocks were obtained retrospectively by request from the pathology department where the primary tumor was excised. If no primary tumor was available, metastatic lesions were accepted for analysis as the literature, although limited, suggests a high correlation between HER-2 tissue expression in primary and metastatic lesions from the same patient.22 A total of 230 tissue blocks could be retrieved out of 330 requested. The remaining cases could not be requested because of missing records. Reasons for lack of retrieval included the following: the center did not keep the paraffin blocks beyond 5 years (25%), the center did not release blocks for research purposes (5%), or inadequate pathology data were available to determine where the original pathology was located (20%). To assess possible retrieval bias, the following prognostic characteristics of patients on study were compared with the characteristics of patients whose blocks were retrieved: age, prior adjuvant therapy, estrogen receptor or progesterone receptor positivity, disease-free interval, number of visceral metastatic sites, and prior doxorubicin. No significant difference in any of these parameters was seen between the two groups (data not shown). Two hundred seven patients had available tissue for immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH). Two cases were not interpretable after CB11 (Biogenex) staining, leaving 205 cases assessable for IHC. Of blocks analyzed by FISH, 7 were uninterpretable and 10 contained in-situ disease, only leaving 190 assessable cases. For purposes of analysis, only patients who responded to initial induction therapy (CR or PR) were included, as these were the patients who subsequently went on to have high-dose chemotherapy (n = 151 IHC and n= 130 FISH). Fig 1 describes the number of cases available for each analysis.
Immunostaining for HER-2 was performed using monoclonal antibody CB11 using capillary-gap technology (OptiMax Plus Automated Cell Staining System). Briefly, after quenching endogeneous peroxidase activity with methanol/peroxide and blocking nonspecific binding with normal horse serum, the primary antibody was added in a 1:100 solution, and the sections were allowed to incubate overnight at 4°C in a humidified chamber. Following washes with phosphate-buffered solution, antigen localization was visualized using an avidin-biotin system (Vector Elite, Vector, Montana) and 3',3' diaminobenzidine as the chromogen. Tissue sections were then counterstained in dilute hematoxylin, dehydrated, cleared in xylene, and mounted with permount. Immunostains were scored on a scale of 0 to 3+ by a breast pathologist using the scoring system outlined in the DAKO Hercep-Test. Only unambiguous membrane staining was evaluated. Tumors with no staining or weak staining in less than 10% of the cells were scored as 0. Tumors with a faint or barely perceptible membrane staining in more than 10% of the cells or with noncircumferential staining were scored as 1+. Moderate circumferential membrane staining in more than 10% of the cells was scored as 2+. Strong circumferential membrane staining was scored as 3+. Scores of 0 or 1+ were considered negative for HER-2 overexpression; scores of 2+ or 3+ were considered positive for overexpression. Given recent controversy about this scoring system, we elected to divide patients into "high positive" = 3+ and "low positive" = 2+,3+, as some investigators suggest that 3+ positive tumors are those that are HER-2 dependent.4 HER-2 gene amplification was measured by FISH at Labcorp Inc (Research Triangle Park, NC) using the Ventana INFORM kit. Five micron sections of formalin-fixed, paraffin-embedded breast cancer tissues were mounted on microscope slides. A hematoxylin & eosin–stained section was evaluated for tumor, and the corresponding tumor area was etched on the FISH slide for reference during the scoring process. Sections were deparaffinized, and tissues were digested with proteinase. Target DNA in sections and the biotinylated HER-2 probe were co-denatured in a 90°C oven for 12 minutes. Target and probe sequences were hybridized overnight at 37°C. Sections were washed in 0.5X SSC to remove unhybridized probe. The hybridized probe was detected by incubation with fluorescein-labeled avidin. The nuclei were counterstained with of 4',6-Diamidino-2-phenylindole. Slides were examined with an epifluorescence microscope equipped with dual and single filters to detect the fluorescein and of 4',6-Diamidino-2-phenylindole fluorochromes. Two fields of 20 tumor cells were evaluated, and the number of HER-2 signals per nucleus was determined. The HER-2 gene was considered amplified if, on average, there were four or more signals per tumor nucleus.23 Slides were evaluated by two individuals (T.E., S.A.). We estimated overall and disease-free survival according to the Kaplan-Meier product limit method and applied the log-rank test to compare two distributions. We calculated disease-free survival as time from first cycle of chemotherapy to disease progression or death, whichever occurred first. Patients who were alive and disease free were censored at the date of last follow-up visit. Overall survival was calculated from time of first cycle of chemotherapy to death, and patients who were alive were censored at date of last follow-up visit. We used Cox proportional hazards models to determine which variables were significant predictors of overall and disease-free survival.24 We calculated sensitivity and specificity of HER-2 by different methods assuming that HER-2 measured by IHC is the standard method.25 Concordance between methods was calculated as percent agreement. Statistical significance was taken as P < .05. We made no formal adjustments for multiple comparisons and subset analyses but instead qualified the results in the text.
Influence of HER-2 on Disease-Free and Overall Survival in Breast Cancer Patients Who Underwent Transplant After Response to Induction AFM Given the limited number of cases available for HER-2 analysis and the fact that the overall cohort showed no difference in survival for immediate versus delayed HDC, we elected to combine the responders for our analysis. Comparison of HER-2 status and outcome of patients who underwent HDC after response to induction AFM indicated worse outcome only in those patients with elevated HER-2 ECD( Table 1). This was true for patients with circulating ECD both preinduction and postinduction chemotherapy with AFM. Statistically significant results were limited to the effect of HER-2 ECD on overall survival. These results are presented in Figs 2 to 6 with p-values listed in Table 1.
With respect to tissue measurement of HER-2, we looked at protein expression by IHC and gene amplification by FISH. No correlation with worse disease-free or overall survival was seen in either measure of expression. As seen in Table 1, we subdivided patients into "high positive" (3+) and "low positive" (2+, 3+) to see if any prognostic significance would arise, given previous reports of worse outcome in tumors with higher levels of HER-2 amplification.4 We did not see a worse prognosis in our cohort of patients, even in the patients whose tumors were 3+ overexpressers by immunohistochemistry.
Multivariable Analysis of Factors Influencing Disease-Free and Overall Survival
Comparison of Methods of Measuring HER-2 in 8801 Patients HER-2 as measured by FISH and IHC and immunoassay for serum ECD were compared. Levels of concordance of the different methods ranged from 63% to 88% ( Table 3). The highest concordance was seen between a positive result for preinduction chemotherapy circulating ECD and measurement of FISH in the tumor (88%). The concordance between FISH and ECD fell to 75% postinduction (pre-HDC), perhaps reflecting a population of patients whose ECD levels fell below the usual cutoff after chemotherapy. For IHC, concordance was highest with FISH (77% to 85%) and lowest with postinduction ECD. Looking at the sensitivity and specificity comparisons, we believe the latter reflects low sensitivity of the ECD test, as specificity appears to be high (86% to 98%). This implies that most tumors that shed ECD do express HER-2 but that a "threshold" effect occurs below which ECD cannot be detected above normal background. Concordance between IHC and FISH is 77%, which is similar to that reported by other groups.33 Not all tumors with protein overexpression will have gene amplification, reflected by a lower sensitivity of FISH for overexpression of HER-2 protein by IHC (58%). However, cases with HER-2 amplification are likely to overexpress the protein, leading to a high specificity (95%). The majority of HER-2 overexpressing tumors that are FISH positive are 3+ by IHC, reflected in the higher sensitivity of this comparison (87%).
The ECD of the HER-2 receptor has been found to circulate in the serum of 21% to 50% of invasive breast cancer patients, with 91% to 100% of these patients tumors overexpressing HER-2.17 The presence of this product is correlated with higher tumor burden, with an increased likelihood of ECD positivity in stage III and IV patients (33% to 51%). In addition, expression of HER-2 ECD in the serum may also correlate with resistance to some therapeutic agents.27 Studies regarding the role of HER-2 ECD and outcome after chemotherapy are inconsistent. This may reflect differences in patient populations, the specific assay used, or differences in assessment of positivity. Perhaps as important, and frequently overlooked, is the specific regimen used and doses involved because mechanisms of resistance vary from one chemotherapeutic agent to another. With respect to HER-2 overexpression in the primary tumor, data suggest that these cells are inherently resistant to alkylator-based chemotherapy but may have an improved outcome with doxorubicin.13,28,29 Whether increasing doses of alkylator-based therapy can overcome the drug resistance observed in HER-2–positive patients is unknown and the impetus for this study. The alkylator cyclophosphamide is thought to be the most active component of CMF, a standard regimen for breast cancer therapy. Three studies support the hypothesis that HER-2 ECD can predict resistance to CMF. Mehta et al looked at 79 stage II and III patients treated with either CMF or cyclophosphamide, methotrexate, fluorouacil, vinblastine, and prednisone from 1983 to 1989 and found that ECD positivity was associated with higher number of lymph nodes and higher likelihood of relapse.30 Jager’s group attempted to address the relationship of HER-2 ECD expression to specific regimens in their study of 211 stage II or III breast cancer patients treated either with CMF or mitoxantrone and cyclophosphamide or fluorouracil, mitoxantrone, and cyclophosphamide, where the anthracenedione, mitoxantrone, was used.31 Fifty nine patients were treated with CMF and 152 were treated with NC/FNC, and the groups were similarly matched for prognostic variables. HER-2 ECD was predictive for worse disease-free and overall survival in univariate analysis and in multivariate analysis for disease-free survival [Odds Ratio (OR) = 2.3 P < .01]. They saw no difference in outcome depending on the CMF versus NC-based regimen, although the numbers in each cohort were not large enough to see small differences. We have previously shown that metastatic breast cancer patients treated with CMF respond less well than to doxorubicin-based chemotherapy, particularly if they are ECD positive. Of HER-2 ECD–positive patients treated with CMF, only 30.8% responded versus 64.1% of those treated with doxorubicin (OR = 6.5) although time to progression and overall survival were not different between the two chemotherapy regimens.20 This suggests a fundamental resistance of HER-2 ECD–positive patients to alkylator-based chemotherapy, which may not be the case with doxorubicin. Based on the rapid regrowth of HER-2–positive tumors, this advantage seen with doxorubicin treatment may be overcome by "kinetic resistance" leading to no difference in disease-free or overall survival. The data from the present study suggest that the method of measuring HER-2 is important in our ability to predict outcome after HDC. While HER-2 ECD–positive patients with metastatic breast cancer appear to do worse than HER-2 ECD–negative patients after HDC, this may reflect higher disease burden rather than intrinsic resistance. That HER-2 IHC/FISH–positive patients did as well as HER-2 IHC/FISH–negative patients after HDC suggests that this otherwise negative prognostic factor may be overcome with HDC.32 Alternatively, induction therapy with doxorubicin at 75 mg/m2 may be adequate to negate the prognosis imparted by HER-2 as suggested by studies in the adjuvant setting.28,29 Our study does not allow us to distinguish between these two possible explanations. The "best" method to measure HER-2 has become a point of controversy as the value of measuring this marker for making treatment decisions has become apparent. HER-2 is currently used in clinical practice for deciding on the use of doxorubicin in the adjuvant setting and the use of Herceptin in the metastatic setting. A recent consensus conference was held in August 1999 in an attempt to resolve some of these issues.33 Data presented at this meeting indicate that concordance between IHC and FISH methods is in the 80% to 90% range, particularly comparing 3+ overexpressers and amplified cases. Our concordance rates between methods are similar at 77% (Table 4). No one method was clearly favored over another, and the general consensus was that different methods should be evaluated individually with reference to clinical outcome. It is possibly and even likely that one method is useful for prediction of response to chemotherapy where another might be more useful in predicting response to Herceptin, particularly if mechanisms of the interaction are different. We chose to compare different methods of measuring HER-2 in our patient cohort to better define the utility of different methods in this setting. IHC is a relatively easy, inexpensive method that measures protein expression in tissue sections and is used extensively for the assessment of molecular markers, including HER-2. The disadvantages of IHC are a greater likelihood for false-positive and false-negative results (based on comparisons with Southern and Western Blotting) than FISH.34 This method is also more subjective than FISH based on interobserver variability noted in comparative studies.32 False positives arise from binding of the antibody in question to nonspecific proteins. Jacobs et al have recently reported that if background staining of the normal epithelium is subtracted from that of the malignant epithelium, higher specificity of the antibody in question is seen.35 In addition, HER-2 overexpression is seen in cases where the gene is not amplified, ranging from 5% to 30% in the reported literature.33,36 False negatives arise when the test is performed improperly or when antigen is lost during fixation or processing of tissues. The antibody used can also give varying results depending on the epitope targeted (intracellular versus extracellular). In addition to technical issues, the reporting of an IHC result requires a subjective evaluation of "positivity" by a trained pathologist. The College of American Pathologists has recently submitted guidelines for standardization of the scoring systems for HER-2 by IHC that will help with this issue, although the subjectivity of the human eye will always be present. We chose to use CB11 antibody, as it has been employed to test HER-2 in specimens from several large, randomized trials, including the study that showed a dose-response effect to CAF.13 Several studies suggest that this antibody has a high sensitivity and specificity; therefore, it is now considered one of the two or three antibodies preferred for HER-2 measurement. Nonetheless, it is possible that our data would be different should a different antibody (eg, Tab-250, 4D5, Pab-1) be employed. FISH is a method of measuring DNA (or RNA) amplification using a fluorescently labeled oligonucleotide designed to be complementary to the gene in question. The advantages of this method are purportedly a high degree of reproducibility and low interobserver variability, although there is little data to support this notion. However, the method is costly and generally too labor intensive to be performed in most hospital pathology departments. This may change in the future given new automated platforms being developed. Currently, two commercially available FISH tests are available for HER-2 testing. The Ventana INFORM kit, employed in this study, does not have a centromeric control probe, unlike the Vysis Pathyvision kit. However, recent data comparing the two assays shows 97% to 100% concordance between the two methods.32 The data from this study are consistent with that of other groups, where concordance between IHC and FISH is reported to be in the range of 75% to 90%.32,33 In addition, we have found that the sensitivity of HER-2 ECD for HER-2 in the primary tumor is low, but specificity is high. This implies that a subset of tumors that express HER-2 are capable of shedding circulating ECD but that HER-2 negative tumors are rarely associated with circulating ECD. The latter result is in contradistinction with the data from Molina et al who found a 20% false-positive rate of ECD for HER-2 positivity at the level of the tumor. However, our study has larger numbers for comparison (142 patients with both serum and tissue).37 This study suffers from several perspectives. The numbers of patients in the trial, while adequate to address the clinical endpoints, are less adequate to address correlations between HER-2 expression and clinical outcome. In addition, the lack of prospective collection of tissue samples made it necessary to retrieve tumor blocks on patients retrospectively. This led to a lower sample size from which to do HER-2 analysis and introduced potential retrieval bias. While we have not seen differences in the distribution of usual prognostic factors in the subset of patients where tissue was retrieved, it is possible that other biases exist that are not apparent in our analysis. Finally, the difference in timepoints of sample collection in the course of the patient’s disease must be considered. Limited evidence suggests that HER-2 in the primary tumor and the metastatic site does not vary, however, it is possible that patients who respond to therapy and are able to undergo HDC represent a subset of HER-2–positive patients that behaves differently than the overall group. This may lead to the erroneous conclusion that all HER-2–positive patients with metastatic disease should be treated with HDC, which is not necessary justified by these data. In summary, these data suggest that an increase in dose of chemotherapy may overcome drug resistance in HER-2–positive patients. Alternatively, AFM chemotherapy may be able to negate the worse prognosis generally seen in HER-2–positive patients, even in the metastatic setting. These data also suggest that circulating HER-2 ECD may reflect higher disease burden rather than intrinsic resistance to therapy. Further studies are necessary in patients treated on controlled clinical trials where samples are collected in a prospective fashion and the trial is designed with adequate statistical power to answer these questions. Understanding the relevance of molecular markers in breast cancer and how different measurements reflect its biology will help us to make optimal treatment decisions for patients.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
