1 Translational Research Unit and 3 Data Centre, Jules Bordet Institute, Brussels, Belgium; 2 F. Hoffmann-La Roche Ltd, Basel, Switzerland
Received 22 January 2002; revised 20 March 2002; accepted 11 April 2002
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Abstract |
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The humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin®) is a new treatment modality for metastatic breast cancer, the efficacy of which is directly correlated with the HER-2 status of the tumour, evaluated either by immunohistochemistry (IHC) and/or by fluorescence in situ hybridisation (FISH). This analysis is generally performed on the primary tumour. There are few data regarding the HER-2 status in the corresponding distant metastases.
Methods:
HER-2 status in 107 patients with a primary breast tumour and at least one distant metastatic lesion was analysed by IHC and FISH.
Results:
We found similar levels of amplification (25% and 24%) and overexpression (13% and 19%) of HER-2 in primary and metastatic samples, respectively. Among paired primary/metastatic tumours, six (6%) showed discordance by HercepTestTM (n = 100): all six cases showed greater Her-2 overexpression in the metastatic tissue. By FISH (n = 68), five (7%) cases were discordant: two cases were amplified in the primary tumour but not in the metastasis, and three samples showed amplification in the metastasis but not in the primary. Finally, we analysed HER-2 status in different metastatic lesions from 17 patients that had at least two distant metastatic sites. Discordance between different sites from the same patient was 18% by IHC and 19% by FISH.
Conclusions:
Between the paired primary tumour and distant metastatic lesions, 94% and 93% of samples had concordant HER-2 status when analysed by IHC or FISH, respectively. These results do not support routine determination of HER-2 on metastatic sites, particularly when FISH results from the primary tumour are available.
Key words: breast cancer, HER-2, metastatic, primary
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Introduction |
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The main targets of any therapy in MBC are the metastases. However, in the great majority of cases, HER-2 status is determined on the primary tumour, and there are few published data regarding the comparison of HER-2 status between the primary and the metastatic sites. Studies have reported a high level of consistency, although not complete, in HER-2 status between primary tumours and locoregional metastases using immunohistochemistry (IHC) [1620], fluorescence in situ hybridisation (FISH) or both [2124]. Data referring to distant metastases are extremely scarce [18, 20, 22, 25, 26].
In this study, we compared HER-2 overexpression and amplification in primary tumours and their distant metastases from a series of 107 breast cancer patients with at least one distant metastatic site, using both IHC (HercepTestTM) and FISH (triple probe LSI HER-2/topoII/CEP17) methods. Additionally, the evaluation of HER-2 status in different metastatic sites from the same patient was performed in 17 cases. This is, to our knowledge, the first study to compare HER-2 status, evaluated by both these techniques, between primary tumours and their distant metastases (locoregional metastases not included), in a relatively large number of patients.
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Materials and methods |
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Immunohistochemistry
IHC was performed using the HercepTestTM (Dako, Glostrup, Denmark) according to the manufacturers instructions. A score of 3+ was considered to be a true positive. Each run included positive and negative controls. The slides were reviewed and scored by one pathologist (D.L.), and only once. The observer was blinded with regard to the FISH data and to the HER-2 status in the paired samples.
Fluorescence in situ hybridisation
Slides were deparaffinised in xylene for 5 min, three times, and dehydrated in two changes of 100% ethanol for 5 min. After incubation in a solution of 0.2 M HCl for 20 min at room temperature, slides were washed in deionised water for 1 min and incubated in the pre-treatment reagent (1 M NaSCN) for 30 min at 80°C, and rinsed in deionised water for 3 min. Slides were then incubated in a solution of 0.2 M HCl/4 mg/ml protease (Paraffin Pre-treatment kit; Vysis, Inc., Downers Grove, IL, USA) at 37°C for 1020 min, and rinsed in deionised water for 3 min. After controlling the digestion, slides were incubated in neutral-buffered formalin for 10 min, rinsed in deionised water and dehydrated through graded ethanol series (70, 85 and 100%), 1 min each. Slides were placed on a hot plate (73°C) for 5 min with a 10 µl probe (Vysis multicolor-probe Topo II Spectrum Green, HER-2 Spectrum Orange and CEP17 Spectrum Aqua), coverslipped and sealed. Denaturation was followed by incubation in humid atmosphere at 37°C for 1618 h. The next day, the rubber cement was removed and coverslips floated off by soaking the slides at room temperature in 2x SSC/0.3% Nonidet P-40 for 2 min. Slides were then incubated in a solution of 2x SSC/0.3% Nonidet P-40 at 73°C for 2 min, dried in a dark place and counterstained with 10 µl of 0.2 µM 4,6-diamidino-2-phenylindole (DAPI) in antifade solution (Vectashield; Vector Laboratories, Inc., Burlingame, CA, USA). Signals were counted in 60 nuclei using a Leica DMRB or an Olympus BX51 epifluorescence microscope equipped with the following filter sets: DAPI, aqua, fluorescein isothiocyanate (FITC), Texas Red, DAPI/FITC/TRITC, and 40-, 63- and 100-fold magnification oil immersion objectives. Gene amplification was defined as a HER-2 to CEP17 ratio >2. The slides were analysed and classified by two observers (D.G. and G.R.) who were blinded with regard to the IHC data and to the HER-2 status in the paired sample. In only two cases was there discordance between both observers, and in both cases the FISH analysis was repeated and concordance obtained. Owing to problems with tissue preparation that prevent FISH analysis, such as Bouin fixation or decalcification treatment, FISH was successfully performed on 68 primary-metastasis pairs out of the 107 originally available.
Statistical analysis
Theoretical proportions of HER-2 positivity were estimated on the basis of the observed proportions in the samples. These estimations were carried out using the samples obtained from the primary tumour and using the samples obtained for the first (chronologically) developed metastatic site. The assessments done on other metastatic sites from the same patient were not considered for estimation of the positivity rates in order to avoid dependency between the observed data (positivity on one metastatic site being most probably linked to positivity on the other sites). Inclusion of multiple sites from the same patient could consequently lead to a possible over- or underestimation of positivity rates. The 95% confidence intervals (CIs) were calculated for the theoretical proportion on the basis of asymptotic normal distributions. Rates of positivity for the primary tumours and the first metastatic site were compared using two-sided paired McNemars test at a usual 5% level. Discordance rates between the primary tumour/first metastatic site pairs were similarly analysed. Finally, a descriptive analysis was performed for the 17 patients for whom several metastatic sites were available, in order to assess the possible discordance between the sites. No inferential analysis was performed due to the small number of patients and the fact that the sites were not synchronous.
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Results |
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HER-2 status in primary and metastatic breast tumours (Table 1)
Using IHC (Table 1A), we found that 13% (95% CI 6% to 20%) of the primary tumours (n = 100) revealed Her-2 overexpression (score 3+). In metastatic lesions (n = 100), 19% (95% CI 11% to 27%) showed 3+ Her-2 overexpression. FISH analysis (Table 1B) of the primary (n = 85) and metastatic (n = 84) samples demonstrated 25% (95% CI 16% to 34%) and 24% (95% CI 15% to 33%) of HER-2/neu amplification, respectively.
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Regarding the primary tumours, there were nine (11%) discordant cases between the two techniques. All these nine cases were negative by IHC (score 0 to 2) but positive by FISH.
Among the 76 evaluable samples of the first metastatic site (71% of the 107 available), there were seven (9%) cases of discordance between IHC and FISH results: five out of seven of these cases were negative by IHC but amplified by FISH, while two out of seven were positive by IHC but not amplified by FISH.
For the samples collected from the second metastatic site, 16 of 17 were evaluable by both techniques. Only three of 16 presented discordant results, two of which were negative by IHC and amplified by FISH, while the inverse was seen in the remaining one.
Five patients had samples available from a third metastatic site. In all of these IHC and FISH could be performed and yielded concordant results.
Overall, more samples were HER-2 positive when evaluated by FISH than by IHC (P = 0.01). While this occurred in both the primary tumours (P = 0.04) and the metastatic sites (P = 0.73), the difference reached statistical significance only for the primary tumours.
Discordance in HER-2 status between primary and metastases (Table 2)
One hundred paired samples were available for IHC analysis (Table 2A). Seven cases were not evaluable due to detachment of the tissue during the pre-treatment. There were 13 and 19 cases with 3+ Her-2 overexpression in the primary tumours and in the metastatic lesions, respectively. Six of these 100 cases (6%) scored differently in the primary tumour and its respective first metastatic site. All cases showed greater Her-2 overexpression in the metastatic site as compared with the primary tumour. This increase was statistically significant (P = 0.03).
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No correlation was observed between changes in the HER-2 status and the location of the metastases.
Comparison of HER-2 status in multiple metastatic sites from the same patient (Table 3)
Seventeen cases with multiple metastatic sites were evaluable by IHC. Twelve patients had two distinct metastatic sites, and five patients had three metastases. We found discordant Her-2 overexpression in three of 17 (18%; 95% CI 0% to 26%) cases. These three cases, when evaluated by FISH, showed concordant results.
When FISH was used, 16 of 17 cases were successfully hybridised. We found differences in HER-2 amplification between different metastatic sites in three of 16 (19%; CI 0% to 38%) cases. These cases were not the same as those that had been found discordant with IHC and, when evaluated by IHC, they showed concordant results.
The discordant results among the different metastases were not associated with a particular location of the metastases.
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Discussion |
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This issue has become important in recent years due to the development and approval of trastuzumab for the treatment of HER-2-overexpressing MBC. In the metastatic setting, where the disease is virtually incurable, few drugs have shown a significant advantage, not only in terms of response rates but particularly in terms of overall survival. Trastuzumab, when given in combination with paclitaxel, achieved a survival benefit of 5 months compared with single-agent treatment of paclitaxel [14]. On the other hand, although very well tolerated, trastuzumab has a non-negligible risk of cardiotoxicity. Since trastuzumab activity is directly dependent on the HER-2 status of the tumour, a careful selection of patients is crucial for increasing its clinical benefit and avoiding unnecessary treatment of patients who most likely will not benefit from it. To date, the HER-2 status is almost always evaluated on the primary tumour sample, since routine biopsy of metastatic sites is not a standard procedure. Thus, it becomes imperative to determine whether homogenous HER-2 expression exists between the primary tumour and its distant metastases.
Most published studies have compared HER-2 status in primary tumours with their locoregional or concurrent lymph node metastases. Only five studies have presented data concerning distant metastases [18, 20, 22, 25, 26], and only one of these [25] focused exclusively on distant metastases. However, it should not be assumed that both types of metastases are biologically equivalent. Distant metastases often represent clonal outgrowths with genetic modifications not detected in the primary tumour, and are generally identified years after resection (and therapy) of the primary cancer. In addition, cells that metastasise locally via the lymphatic system may possess biological properties distinct from those that have the capacity to invade vasculature, travel to distant sites and establish clinically significant disease. Cytogenetic analyses have demonstrated extreme genetic heterogeneity (up to 70% of polyclonality) in breast cancer [2731]. Kuukasjärvi et al. [4] reported that the genetic composition of 31% of metastases differed almost completely from that of the paired primary tumours.
Our group has previously compared the Her-2 status, among other biological markers, between 370 primary breast cancer sites and their metastatic ipsilateral axillary lymph nodes [16]. Her-2, evaluated by IHC, was the marker with the best overall concordance (98%); no biological marker showed 100% concordant results.
Similarly high levels of concordance have been reported by other authors [17, 19, 21, 23, 24]. However, as has been pointed out, all these studies were restricted to locoregional metastases. Three of these studies determined HER-2 status by both IHC and a DNA hybridisation technique [21, 23, 24], and two by IHC alone [17, 19]. Another common pitfall is the number of evaluated patients: only two of the six studies had >100 evaluable patients [23, 24], and none had >200; the number of paired samples (primary/locoregional metastasis) is even smaller.
Only five studies compared the HER-2 status in breast primary tumours and distant metastases [18, 20, 22, 25, 26]. In 1993, Niehans et al. [25] retrospectively evaluated Her-2 expression in primary breast cancer and different metastatic sites in autopsy tumour samples from 30 patients who had died from the disease. Eight of these patients (27%) were Her-2 positive and, among those, there was a single case of discordant staining (3%) between the primary site and two of its metastatic lesions. The authors concluded that Her-2 expression is generally congruent at different metastatic sites. This is the only published study that evaluated exclusively distant metastases. Unfortunately, the actual number of evaluable patients was very low, and the uncontrolled fixation time of autopsy samples jeopardizes the IHC data that can be obtained from these samples. In 2000, Shimizu et al. [18] evaluated Her-2 protein levels, by IHC, in primary and metastatic breast cancer samples from 21 patients. Only seven of the metastatic samples were from distant sites, with the remaining 14 belonging to locoregional relapses. The authors found no significant differences in the Her-2 expression between the primary tumours and their metastases. Also in 2000, Masood et al. [20] evaluated Her-2 overexpression in 56 patients by IHC. Once again, only a minority (11 cases) of the metastases were from distant sites. The pattern and intensity of Her-2 overexpression in the primary tumours and their metastases were found to be almost identical, with heterogeneity present in only one case. Recently, Tanner et al. [22] analysed HER-2 amplification in 46 breast primary tumours and their metastases, using IHC and DNA in situ hybridisation techniques. The authors found complete concordance regarding HER-2 amplification between the primary tumours and their metastases. However, in only 12 cases were the analysed metastases from a distant site. In the 2000 San Antonio Breast Cancer Meeting, Edgerton et al. [26] presented some preliminary results of a study comparing HER-2 status, determined by IHC and FISH, on 193 primary breast cancers and corresponding local recurrences (68), lymph node metastases (32) and distant metastases (93). Overall, the authors found 25% discordant cases. Publication of detailed results and methodology of this study would be of interest.
In the present study, we analysed both Her-2 protein overexpression by IHC and HER-2 oncogene amplification by FISH in 107 patients with at least one distant metastatic site. Owing to technical difficulties, the number of evaluable pairs (primary/distant metastasis) was 100 for IHC and 68 for FISH. The relatively large number of samples not evaluable is an important limitation, encountered in all studies performed in paraffin-embedded tumour samples, and was mainly due to bouin fixation or decalcification treatment (needed for bone metastases), which compromise hybridisation. Seventeen cases with multiple metastatic sites were also comparatively studied. The Vysis multicolour FISH probe, which includes the topoisomerase II gene analysis, was used since the original plan was the evaluate the status of both the HER-2 and the topoisomerase II
gene in the primary tumour and its corresponding metastatic sites. Topoisomerase II
gene results are not reported, since the probe was found to be inadequate to evaluate this gene. A new topoisomerase II
probe has been developed by Vysis and is now being used to evaluate the status of this gene in the present series.
Similar levels of amplification of the HER-2 oncogene were found in the primary tumours and in the metastatic samples (25% and 24%, respectively) using FISH (Table 1B). Overexpression of Her-2, assessed by HercepTestTM, was found in 13% of the primary and 19% of the metastatic samples when only 3+ scores were considered to be positive. When 2+ scores were included, Her-2 overexpression was found in 29% of the primary tumours and 27% of the metastatic lesions (Table 1A). This means that using strong staining (3+) with the HercepTestTM as the sole indicator of Her-2 positivity was very stringent and that it underestimated the number of samples with HER-2 gene amplification (P = 0.01). Controversy related to the best IHC protocol, in particular the best antibody, to use for Her-2 determination is still ongoing. However, we have adopted an IHC protocol using the monoclonal antibody CB-11 with antigen retrieval, which allows us to decrease the rate of false negatives; all positive results are confirmed by FISH, therefore reducing the rate of false positives. This attitude seems well balanced in terms of efficiency and costs.
HER-2 status in the primary and corresponding metastatic lesions revealed a high level of concordance (94% and 93% when analysed by IHC or FISH, respectively). Discordant results were observed in only six of 100 cases (6%) by IHC (Table 2A) and five of 68 cases (7%) by FISH (Table 2B). Regarding IHC, all six discordant cases presented an increase in staining in the metastatic site in comparison to the primary tumour. This increase in IHC staining when the tumour disseminates was statistically significant (P = 0.03). Four of these six cases had a 2+ score in the primary tumour. Among the five discordant cases by FISH, in three the HER-2 gene became amplified in the metastatic site while it was non-amplified in the primary tumour, and in two the opposite occurred (HER-2 gene amplification in the primary tumour but not in the metastasis).
Including all metastatic sites in the analysis, our IHC data suggest that Her-2-positive metastatic lesions with negative primary tumours were more frequent than the converse situation (Table 2A). This is in line with the biological fact that Her-2 is correlated with enhanced tumour aggressiveness. Another possible explanation may be an underestimation of Her-2 protein overexpression in the primary tumour by HercepTestTM; this could be due to the fact that most primary samples were collected many years before the present analysis (see Table 2 for specific dates), and therefore some protein degradation might have occurred. Previous studies have shown that the sensitivity of IHC when used in paraffin-embedded fixed tumour samples varies widely, depending not only on the antibody used but also on the tissue processing, namely tissue fixation and the age of the samples, which interferes greatly with their quality [32]. This explanation is supported by the FISH data, which show a very similar percentage of HER-2-positive samples between pairs and therefore seems not to support the hypothesis of enhanced tumour aggressiveness. In fact, when FISH was used, among the 16 of 68 primary samples with HER-2 gene amplification (Table 2B), two (13%) had corresponding distant metastases with a non-amplified HER-2 gene, while among the 52 of 68 HER-2-negative primary samples, three (6%) had corresponding metastatic sites with HER-2 gene amplification.
The HER-2 status in samples from multiple distant metastatic sites belonging to the same patient were analysed in 17 cases (Table 3). Thirteen percent and 18% discordant results between samples from different metastases were found when assessed by FISH and IHC, respectively. Previously, only the study by Niehans et al. [25] had looked into this issue, but did so using solely the IHC technique: they found a single case among the 30 evaluated that presented different staining among the four metastatic sites (two were Her-2 positive and two were Her-2 negative).
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Acknowledgements |
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Footnotes |
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