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Her2 Amplification is Significantly More Frequent in Lymph Node Metastases From Urothelial Bladder Cancer Than in the Primary Tumours
EUROPEAN UROLOGY 60 (2011) 350–357
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Urothelial Cancer
Her2 Amplification is Significantly More Frequent in Lymph Node Metastases From Urothelial Bladder Cancer Than in the Primary Tumours Achim Fleischmann a,*, Diana Rotzer a, Roland Seiler a, Urs E. Studer b, George N. Thalmann b a
Institute of Pathology, University of Bern, Bern, Switzerland
b
Department of Urology, University of Bern, Bern, Switzerland
Article info
Abstract
Article history: Accepted May 15, 2011 Published online ahead of print on May 25, 2011
Background: Her2, an alias for the protein of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), might be an attractive therapeutic target in metastasising bladder cancer. Genotype and phenotype of primary tumours and their metastases may differ. Objectives: Determine Her2 status in both tumour components to better assess the potential of anti-Her2 therapies. Design, setting, and participants: Histologic examination revealed lymph node metastases in 150 patients with urothelial bladder cancer clinically staged as N0M0. A tissue microarray was constructed with four tumour samples per patient: two from the primary tumour and two from nodal metastases. Her2 status was determined at the gene level by fluorescence in situ hybridisation (FISH) and at the protein level by immunohistochemistry (IHC). Interventions: All patients underwent cystectomy and standardised extended lymphadenectomy. Measurements: Overall survival was assessed according to HER2 gene status and protein expression in primary bladder cancers and lymph node metastases. Results and limitations: Her2 amplification was significantly more frequent in lymph node metastases (15.3%) than in matched primary bladder cancers (8.7%; p = 0.003). Her2 amplification in primary tumours was highly preserved in the corresponding metastases as indicated by only one amplified primary tumour without amplification of the metastasis. There was a high concordance in HER2 FISH results between both samples from the primary tumour (k = 0.853) and from the metastases (k = 0.930). IHC results were less concordant (k = 0.539 and 0.830). FISH and IHC results were poorly correlated in primary tumours (k = 0.566) and metastases (k = 0.673). While Her2 amplification in the primary tumour significantly predicted poor outcome ( p = 0.044), IHC-based survival prediction was unsuccessful. Conclusions: Her2 amplification in metastasising bladder cancer is relatively frequent, is homogeneous in each tumour component, and predicts early death. This suggests a high potential for anti-Her2 therapies. For patient selection, FISH might be more accurate than IHC. # 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Keywords: Her2 Bladder cancer Metastases Survival Tissue microarray FISH
* Corresponding author. Institute of Pathology, University of Bern, Murtenstr.31, CH-3010 Bern, Switzerland Tel. +41 31 632 8765; Fax: +41 31 632 4995. E-mail address: achim.fl[email protected] (A. Fleischmann). 0302-2838/$ – see back matter # 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2011.05.035
EUROPEAN UROLOGY 60 (2011) 350–357
1.
Introduction
[()TD$FIG]
Her2 protein, a member of the epidermal growth factor receptor family and an alias for the protein of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), is a prognostic factor and a standard therapeutic target when overexpressed in breast cancers [1]. Amplification of the HER2 gene on chromosome 17q21 is the primary mechanism for its overexpression [2], which activates intracellular pathways that promote proliferation, survival, mobility, and invasiveness of tumour cells [3]. These oncogenic features translate into reduced survival. Inhibition of Her2 protein-mediated actions as well as antibody-dependent cytotoxicity are the mechanisms of successful anti-Her2 therapies [3]. More recently, incidence of Her2 alterations has also been evaluated in bladder cancer to assess the therapeutic potential of this target [4–8]. These investigations focused on primary tumours; however, cancer death is generally due to metastases. The genetic disparity between these two tumour
351
components is well known [9]; consequently, primary tumours cannot naturally serve as surrogates for metastases. Up to 25% of patients undergoing cystectomy and lymphadenectomy for clinically staged N0M0 bladder cancer reveal lymph node metastases on histologic examination [10] and at least two-thirds of those patients will die from cancer [10,11]. The benefit from chemotherapy in metastasising bladder cancer is limited, and new the rapeutic options are urgently needed [12,13]. Therefore, we evaluated frequency and prognostic impact of Her2 amplification and protein overexpression in 150 urothelial bladder cancers with lymph node metastases treated by radical cystectomy and extended pelvic lymphadenectomy. 2.
Materials and methods
2.1.
Patients and follow-up
The cohort comprised 150 bladder cancer patients without preoperative evidence of metastases (ie, physical examination, chest x-ray, intravenous
Fig. 1 – (A) Her2 amplification in a bladder cancer with a high number of red HER2 gene signals (red arrows) and, in general, two green signals (centromere 17; green arrows) per tumour cell. (B) Tumour cells without Her2 amplification. (C) An immunohistochemically strongly positive bladder cancer (Her2 score 3+). (D) Her2-negative tumour (score 0).
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EUROPEAN UROLOGY 60 (2011) 350–357
urography, bone scan, and pelvic computed tomography, when available)
between subgroups were analysed using the log-rank test. Multivariate
but with lymph node metastases on pathologic examination. All patients
analyses (Cox proportional hazards regression model) included Her2
underwent extended pelvic lymphadenectomy with cystectomy as a
FISH status (for better interpretability and due to its small sample size,
single procedure between January 1985 and April 2008 at the Department
the group of patients with borderline-amplification was removed for
of Urology, University of Bern. No neoadjuvant therapy was given.
analysis) and known risk factors in node-positive bladder cancer
Postoperatively, the patients were followed prospectively according to a
(primary tumour stage [15] and extracapsular extension of lymph node
standard protocol with examinations at the Department of Urology or by
metastases [10]). P values <0.05 were considered significant for all tests.
urologists in private practice at 3 mo and 6 mo, then at 6-mo intervals until
Statistical analyses were performed using R software package v.2.9.1
5 yr, and then at yearly intervals thereafter.
(GNU Project, Free Software Foundation, Boston, MA, USA).
2.2.
3.
Results
3.1.
Cohort
Surgical technique and pathology
All patients were treated by bilateral extended pelvic lymphadenectomy and cystectomy according to standard protocols described previously in detail [10,14]. The surgical specimens were processed as described previously [10]. The tumours were staged according to the 7th Union Internationale Contre le Cancer classification [15].
2.3.
Construction of the tissue microarray
The tissue microarray [16] was constructed with six tissue cores (0.6-mm diameter) per patient: two each from normal urothelium, primary tumour (centre and invasion front), and a nodal metastasis.
2.4.
Fluorescence in situ hybridisation and
immunohistochemistry Two consecutive sections of 3-mm thickness were taken for fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) assays in our laboratory, which has validated these tests and takes part in external proficiency testing. For FISH analysis of the HER2 gene, the PathVysion HER2 DNA Probe Kit was used (Vysis Inc, Downers Grove, IL, USA). It is a mixture of the HER2 probe labelled in SpectrumOrange and the CEP17 probe, which is labelled in SpectrumGreen and directed against the centromeric satellite repeat of chromosome 17. Detailed instructions for hybridisation procedures are provided by the manufacturer. A minimum of 25 nonoverlapping nuclei were evaluated on each tissue spot and HER2 was considered normal, borderline amplified, or amplified (Fig. 1A–1B), according to the HER2/CEP17 fluorescence ratios < 1.8, 1.8–2.2, or >2.2 as recommended [2]. Separate HER2 mean ratios were assigned for each primary tumour and the corresponding metastasis based on the counts on their respective spots. The original Hercep test (DAKO, Glostrup, Denmark) was used for IHC stains, which were performed according to the manufacture’s protocol.
Cohort data are given in Table 1. 3.2.
Her2 gene status and protein expression
Normal urothelium always showed normal HER2 FISH status, and, in IHC, no protein overexpression. In 147 primary tumours, HER2 status could be determined by FISH: 127 tumours (86.4%) showed no amplification, 7 tumours (4.8%) showed borderline amplification, and 13 tumours (8.8%) showed amplification. Corresponding numbers from the 139 patients with evaluable metastases were 105 (75.6%), 13 (9.3%), and 21 (15.1%). Her2 IHC was obtained from 149 primary tumours: 119 tumours (79.9%) with score 0, 17 tumours (11.4%) with score 1+, 7 tumours (4.7%) with score 2+, and 6 tumours (4%) with score 3+. Corresponding numbers from the 139 patients with evaluable metastases were 78 (56.1%), 26 (18.7%), 14 (10.1%), and 21 (15.1%). In 137 patients, HER2 FISH status of the primary tumour could be compared with the corresponding nodal metastases; for Her2 IHC this was possible in 139 patients (Table 2). In the metastases, amplification (15.3%) and borderline amplification (8.8%) were significantly ( p = 0.003) more frequent when compared with the corresponding primary tumours (8.7% and 5.1%, respectively). The same was true for Her2 protein overexpression defined as score 2+ or 3+ (25.2% vs 9.4%; p < 0.001). Patients with two evaluable tissue spots in the primary tumour (n = 114) or in the lymph node metastases (n = 111) showed a high concordance in HER2 FISH results between
Her2 protein expression per tissue spot was classified as recommended [2]: negative (0/1+), weekly positive (2+), and positive (3+) (Fig. 1C–1D) with a cut-off for score 3+ of >30% strongly positive cells. Separate Her2
Table 1 – Clinicopathologic data of 150 lymph node–positive patients with urothelial bladder cancer
mean scores were assigned for each primary tumour and the corresponding metastases based on the findings on their respective spots.
2.5.
Statistical analyses
The McNemar test measured the differences in FISH and IHC results between primary tumour and nodal metastases. Cohen’s kappa assessed the concordance of Her2 status of the gene and protein, respectively, between the two samples from the primary tumor, between the two samples from the lymph node metastasis and measured the concordance between FISH and IHC assay. Her2 status was correlated with categorical (Fisher exact test) and quantitative (paired Wilcoxon test) clinicopathologic parameters. Kaplan-Meier curves were used to assess overall survival (OS) from surgery to the date of death. Patients still living were censored at the date of the last follow-up. Differences in survival
Patient data (n = 150) Age at surgery, yr, median (range) Female/male, No. Follow-up, yr, median Cystectomy data Tumour stage, No. pT1 pT2 pT3 pT4 Lymphadenectomy data Evaluated nodes per patient, median (range) Positive nodes per patient, median (range) Extracapsular extension of metastases, No. No Yes
67 (35–89) 29/121 7.1
4 17 92 37 27 (10–56) 3 (1–46) 71 79
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EUROPEAN UROLOGY 60 (2011) 350–357
Table 2 – Comparison of primary tumours and corresponding lymph node metastases* FISH
Metastases Normal
Primary tumours
Normal Borderline Amplified
103 0 1
Borderline
Amplified
8 2 2
7 5 9
p = 0.003
IHC
Metastases
Primary tumours
0 1+ 2+ 3+
0
1+
2+
3+
75 3 0 0
20 5 1 0
7 5 2 0
8 3 4 6
p < 0.001 FISH = fluorescence in situ hybridization; IHC = immunohistochemistry. Her2 amplification and borderline amplification and Her2 protein overexpression are significantly more frequent in lymph node metastases than in primary tumours. *
Table 3 – Fluorescence in situ hybridization (FISH) assay* FISH in primary tumours
Tumour centre Normal
Invasion front
Normal Borderline Amplified
100 1 0
Borderline
Amplified
2 0 0
2 0 9
Cohen’s k = 0.853
FISH in metastases
Spot b Normal
Spot a
Normal Borderline Amplified
Borderline
81 2 0
Amplified
1 3 3
1 0 20
Cohen’s k = 0.930 * High concordance between Her2 gene status (normal, borderline amplified, amplified) in the two samples from the primary tumour and in the two samples from the lymph node metastases.
Table 4 – Immunohistochemistry (IHC) assay* IHC in primary tumours
Tumour centre
Invasion front
0 1+ 2+ 3+
0
1+
100 8 2 0
9 1 2 0
2+ 1 3 0 2
3+ 1 0 2 0
Cohen’s k = 0.539
IHC in metastases
Spot a
Spot b
0 1+ 2+ 3+
0
1+
2+
3+
53 8 3 1
4 18 3 0
0 1 8 2
0 2 2 14
Cohen’s k = 0.830 * Concordance between Her2 protein expression scores (0–3+) in the two samples is relatively poor in the primary tumour and is high in the two samples from the lymph node metastases.
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EUROPEAN UROLOGY 60 (2011) 350–357
Table 5 – Comparison of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) assays in primary tumours and corresponding lymph node metastases* FISH vs IHC in primary tumours
FISH
IHC
Normal Borderline Amplified
0/1+
2+
3+
124 6 5
3 0 4
0 1 4
Cohen’s k = 0.566
FISH vs IHC in metastases
FISH
IHC
Normal Borderline Amplified
0/1+
2+
3+
89 9 4
12 1 1
2 3 16
Cohen’s k = 0.673 *
Relatively poor concordance between HER2 gene status (normal, borderline amplified, amplified) and Her2 protein expression scores (0–3+) in primary tumours and better correlation in lymph node metastases.
[()TD$FIG] (A)
Primary tumours 1.0
(B) p = 0.044
0.8
p = 0.241
0.8
0.6
Her2 nonamplified
0.4
Her2 borderline amplified
Probability
Probability
Lymph node metastases
1.0
0.2
0.6
Her2 nonamplified 0.4
Her2 borderline amplified
0.2
Her2 amplified
0.0 0
5
Her2 amplified
0.0 10
15
0
Overall survival (yr)
Primary tumours
(C) 1.0
10
15
Lymph node metastases
(D) 1.0
p = 0.406
0.8
p = 0.721
0.8
Probability
Probability
5
Overall survival (yr)
0.6
Her2 IHC negative 0.4
0.2
0.6
Her2 IHC negative
0.4
0.2
Her2 IHC positive
Her2 IHC positive
0.0
0.0 0
5
10
Overall survival (yr)
15
0
5
10
15
Overall survival (yr)
Fig. 2 – Prognostic relevance of Her2 status: (A) Overall survival is significantly poorer in amplified than nonamplified primary bladder cancer. (B) The same trend, but not significant, is found in patients with Her2 amplification in lymph node metastases. (C and D) Immunohistochemically (IHC) determined Her2 status does not segregate significantly into low- and high-risk patients regarding survival.
EUROPEAN UROLOGY 60 (2011) 350–357
these spots (primary tumour: k = 0.853; for metastases: k = 0.930) (Table 3). For IHC, the results for corresponding spots were less concordant, particularly in the primary tumours (k = 0.539) (Table 4). The concordance between FISH and IHC assays (Table 5) was relatively poor in the primary tumours (k = 0.566) and somewhat better in the metastases (k = 0.673). Importantly, 38.5% (5 of 13) of the amplified primary tumours and 19% (4 of 21) of the amplified metastases were Her2 negative in IHC; these cases were evenly distributed over the entire time period, and there was no evidence of weaker membrane staining in older cases than in more recent ones. In contrast, all primary tumours with IHC 3+ were amplified or borderline amplified (5 of 5), whereas 9.5% (2 of 21) of the IHC 3+ metastases were not amplified; however, these two cases showed high chromosome 17 copy numbers. 3.3.
Association of Her2 status with tumour characteristics
Patients with Her2 amplified metastases had more metastases (median: 5 vs 3; p = 0.262), larger total diameter of metastases (median: 4.6 cm vs 2.3 cm; p = 0.180), and greater maximum diameter of metastases (median: 1.5 cm vs 1.0 cm; p = 0.263) than patients without such amplification; however, these differences were not significant. The same trend with smaller differences was observed for IHCbased data. Tumour stage and extracapsular extension of lymph node metastases were not associated with Her2 status. 3.4.
Univariate and multivariate survival analyses
In univariate analyses, OS was significantly ( p = 0.044) reduced for patients with Her2 amplification in the primary tumours compared with those patients without amplification (Fig. 2A). A similar trend for patients with amplified metastases was not significant ( p = 0.241) (Fig. 2B). Her2 IHC did not correlate with outcome (Fig. 2C–2D). In multivariate analyses, advanced primary tumour stage (pT3/4 vs pT1/2; p = 0.041) and extracapsular extension of lymph node metastases ( p < 0.001) were the only independent adverse risk factors for OS. Her2 amplification in primary tumours ( p = 0.074) and metastases ( p = 0.108) failed to add independent prognostic information. 4.
Discussion
Her2 status has important implications for prognosis and therapy in breast cancer. The beneficial effect of anti-Her2 therapies in the approximately 20% of breast cancers with HER2 gene amplification [2] suggests that other cancers with this alteration also may be treated successfully in this way. Larger cohorts of invasive bladder cancers have rarely been investigated for Her2 amplification by FISH and frequencies between 7% and 13.8% were reported [4,6–8]. However, these studies focused on primary tumours and did not evaluate Her2 status in the therapeutically most relevant metastases. The most important finding in our
355
study was that Her2 amplification is significantly more frequent in lymph node metastases from urothelial bladder cancer than in the primary tumours. While Her2 amplification, defined according to recently published strict criteria [2], was detected in 8.7% of the primary tumours, this genetic alteration was present in matched metastases of 15.3% of the patients. Remarkably, if Her2 amplification was found in primary tumours, then this was usually also the case in the corresponding metastases, suggesting a high stability of this alteration after occurrence in the primary tumour. Conversely, metastases with newly emerging Her2 amplifications may originate from a late-developed subclone in the primary tumour, which was not sampled but which has a high metastatic potential, or, alternatively, this genotype was acquired only at the metastatic site. Interestingly, a similar phenomenon of newly occurring Her2 amplifications has also been observed in distant metastases of breast cancers [17]. Previously, two larger series investigated Her2 status in lymph node–positive bladder cancer treated by cystectomy [18,19]. The cohorts comprised 42 [18] and 60 [19] patients, respectively, and results were conflicting. While Jimenez et al. [19], who evaluated only Her2 protein status, reported a higher incidence of Her2 overexpression in lymph node metastases when compared with matched primary bladder cancers, Hansel et al. [18] did not detect such differences at the genetic or protein levels. However, Her2 alterations in primary tumours were also highly conserved in the lymph node metastases in both series [18,19]. Her2 status in breast cancer predicts response to antiHer2 therapy [2]. This therapy acts on the protein level and therefore IHC might be considered as the method of choice for Her2 determination. Nevertheless, the appropriate assay to determine Her2 status in breast cancer remains controversial [2,20,21] because IHC results and HER2 gene status can be contradictory in paraffinembedded tissues. These discrepancies have been attributed to tissue processing artefacts at the protein level by some investigators [20]. They regard FISH as the gold standard for Her2 testing because DNA is better conserved in paraffin-embedded tissues than proteins and there is evidence that the gene status reflects the true protein status [20]. By contrast, proponents of IHC testing in breast cancer point out that optimised protocols for Her2 protein detection achieve a high concordance between FISH and IHC results [21] and that Her2 overexpression might not necessarily result from gene amplification only [21]. In our cohort, incidence of Her2 amplification in all tested primary tumours (8.8%) was in the range of other studies (7–13.8%) [4,6–8], whereas Her2 protein overexpression (8.7% with score 2+ or 3+) was lower (32–57%) [4,6–8]. However, a more recent study reported Her2 protein overexpression in 9.2% of 1005 patients with invasive bladder cancer [5]. In that study, strict definitions for Her2 scores were applied and the antibody had been calibrated in breast cancer samples using FISH results as reference. The large variability in reported Her2 results from bladder cancer has been suggested to reflect heterogeneity between kits, antibodies, protocols, and interpretations of
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EUROPEAN UROLOGY 60 (2011) 350–357
staining [5]. In our study, specificity of IHC score 3+ for detecting Her2 borderline amplification or amplification was 100% in the primary tumours and 90.5% in the metastases. However, sensitivity was low: Her2 amplification without protein overexpression was present in 38.5% of the primary tumours and in 19% of the metastases. A relatively high rate of false-negative IHC results (20–24%) in bladder cancer has also been noted by others [6,8]. This contrasts with data from breast cancer studies where this constellation is rare [2]. The reason for the higher inconsistence between FISH and IHC results in bladder cancer compared to breast cancer is unknown. It has been suggested that tissue processing might play a role [8] and fixation artefacts might be more intense in bladder cancer than in breast cancer. In contrast, a true lack of Her2 expression in amplified bladder cancers due to additional genetic alterations cannot be excluded [8]. Finally, the discrepancies between FISH and IHC results in bladder cancer raise the question of which test best identifies the Her2-positive subgroup with prognostic and predictive relevance. In line with Jimenez et al. [19], survival of the immunohistochemically Her2-positive and -negative subgroups was not significantly different in our series. However, survival stratification according to FISH results was discriminating: Patients without Her2 amplification in the primary tumours did significantly better than patients with Her2 amplification. HER2 gene status in the metastases showed a similar survival trend; however, this was not significant. The successful survival stratification by FISH but not by IHC suggests that FISH is superior for identifying patients at risk and patients with high potential to benefit from anti-Her2 therapies. Our study has limitations. Although our cohort is relatively large compared with other lymph node–positive bladder cancer cohorts, it might be underpowered for analyses of biomarkers with low incidence or limited impact on survival. This might be the reason why we did not detect a significant prognostic impact of HER2 FISH status in the metastases and why Her2 amplification in the primary tumours failed to add independent prognostic information on multivariate analysis. Similar findings have been noted in breast cancer, where the prognostic relevance of Her2 status was merely detected in larger cohorts but not in smaller ones [22]. To better understand the significance of Her2 status in bladder cancer, including the controversially discussed borderline group [2,20], these data should be validated by others.
Author contributions: Achim Fleischmann had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Fleischmann. Acquisition of data: Rotzer, Seiler. Analysis and interpretation of data: Fleischmann. Drafting of the manuscript: Fleischmann. Critical revision of the manuscript for important intellectual content: Studer, Thalmann. Statistical analysis: Fleischmann. Obtaining funding: Fleischmann. Administrative, technical, or material support: Studer, Thalmann. Supervision: None. Other (specify): None. Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/ affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None. Funding/Support and role of the sponsor: This project received funding from Novartis Stiftung fu¨r medizinisch-biologische Forschung.
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5.
Conclusions
Her2 amplification in bladder cancer is significantly more frequent in lymph node metastases than in the primary tumours, is homogeneous in the different tumour components, and is associated with early death. This suggests a high potential for anti-Her2 therapies in the subset of patients with Her2 amplification. Finally, FISH diagnostics might be more accurate for patient selection, because IHC fails to detect a substantial proportion of the prognostically relevant amplified tumours.
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available at www.sciencedirect.com journal homepage: www.europeanurology.com
Urothelial Cancer
Her2 Amplification is Significantly More Frequent in Lymph Node Metastases From Urothelial Bladder Cancer Than in the Primary Tumours Achim Fleischmann a,*, Diana Rotzer a, Roland Seiler a, Urs E. Studer b, George N. Thalmann b a
Institute of Pathology, University of Bern, Bern, Switzerland
b
Department of Urology, University of Bern, Bern, Switzerland
Article info
Abstract
Article history: Accepted May 15, 2011 Published online ahead of print on May 25, 2011
Background: Her2, an alias for the protein of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), might be an attractive therapeutic target in metastasising bladder cancer. Genotype and phenotype of primary tumours and their metastases may differ. Objectives: Determine Her2 status in both tumour components to better assess the potential of anti-Her2 therapies. Design, setting, and participants: Histologic examination revealed lymph node metastases in 150 patients with urothelial bladder cancer clinically staged as N0M0. A tissue microarray was constructed with four tumour samples per patient: two from the primary tumour and two from nodal metastases. Her2 status was determined at the gene level by fluorescence in situ hybridisation (FISH) and at the protein level by immunohistochemistry (IHC). Interventions: All patients underwent cystectomy and standardised extended lymphadenectomy. Measurements: Overall survival was assessed according to HER2 gene status and protein expression in primary bladder cancers and lymph node metastases. Results and limitations: Her2 amplification was significantly more frequent in lymph node metastases (15.3%) than in matched primary bladder cancers (8.7%; p = 0.003). Her2 amplification in primary tumours was highly preserved in the corresponding metastases as indicated by only one amplified primary tumour without amplification of the metastasis. There was a high concordance in HER2 FISH results between both samples from the primary tumour (k = 0.853) and from the metastases (k = 0.930). IHC results were less concordant (k = 0.539 and 0.830). FISH and IHC results were poorly correlated in primary tumours (k = 0.566) and metastases (k = 0.673). While Her2 amplification in the primary tumour significantly predicted poor outcome ( p = 0.044), IHC-based survival prediction was unsuccessful. Conclusions: Her2 amplification in metastasising bladder cancer is relatively frequent, is homogeneous in each tumour component, and predicts early death. This suggests a high potential for anti-Her2 therapies. For patient selection, FISH might be more accurate than IHC. # 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Keywords: Her2 Bladder cancer Metastases Survival Tissue microarray FISH
* Corresponding author. Institute of Pathology, University of Bern, Murtenstr.31, CH-3010 Bern, Switzerland Tel. +41 31 632 8765; Fax: +41 31 632 4995. E-mail address: achim.fl[email protected] (A. Fleischmann). 0302-2838/$ – see back matter # 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2011.05.035
EUROPEAN UROLOGY 60 (2011) 350–357
1.
Introduction
[()TD$FIG]
Her2 protein, a member of the epidermal growth factor receptor family and an alias for the protein of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), is a prognostic factor and a standard therapeutic target when overexpressed in breast cancers [1]. Amplification of the HER2 gene on chromosome 17q21 is the primary mechanism for its overexpression [2], which activates intracellular pathways that promote proliferation, survival, mobility, and invasiveness of tumour cells [3]. These oncogenic features translate into reduced survival. Inhibition of Her2 protein-mediated actions as well as antibody-dependent cytotoxicity are the mechanisms of successful anti-Her2 therapies [3]. More recently, incidence of Her2 alterations has also been evaluated in bladder cancer to assess the therapeutic potential of this target [4–8]. These investigations focused on primary tumours; however, cancer death is generally due to metastases. The genetic disparity between these two tumour
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components is well known [9]; consequently, primary tumours cannot naturally serve as surrogates for metastases. Up to 25% of patients undergoing cystectomy and lymphadenectomy for clinically staged N0M0 bladder cancer reveal lymph node metastases on histologic examination [10] and at least two-thirds of those patients will die from cancer [10,11]. The benefit from chemotherapy in metastasising bladder cancer is limited, and new the rapeutic options are urgently needed [12,13]. Therefore, we evaluated frequency and prognostic impact of Her2 amplification and protein overexpression in 150 urothelial bladder cancers with lymph node metastases treated by radical cystectomy and extended pelvic lymphadenectomy. 2.
Materials and methods
2.1.
Patients and follow-up
The cohort comprised 150 bladder cancer patients without preoperative evidence of metastases (ie, physical examination, chest x-ray, intravenous
Fig. 1 – (A) Her2 amplification in a bladder cancer with a high number of red HER2 gene signals (red arrows) and, in general, two green signals (centromere 17; green arrows) per tumour cell. (B) Tumour cells without Her2 amplification. (C) An immunohistochemically strongly positive bladder cancer (Her2 score 3+). (D) Her2-negative tumour (score 0).
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urography, bone scan, and pelvic computed tomography, when available)
between subgroups were analysed using the log-rank test. Multivariate
but with lymph node metastases on pathologic examination. All patients
analyses (Cox proportional hazards regression model) included Her2
underwent extended pelvic lymphadenectomy with cystectomy as a
FISH status (for better interpretability and due to its small sample size,
single procedure between January 1985 and April 2008 at the Department
the group of patients with borderline-amplification was removed for
of Urology, University of Bern. No neoadjuvant therapy was given.
analysis) and known risk factors in node-positive bladder cancer
Postoperatively, the patients were followed prospectively according to a
(primary tumour stage [15] and extracapsular extension of lymph node
standard protocol with examinations at the Department of Urology or by
metastases [10]). P values <0.05 were considered significant for all tests.
urologists in private practice at 3 mo and 6 mo, then at 6-mo intervals until
Statistical analyses were performed using R software package v.2.9.1
5 yr, and then at yearly intervals thereafter.
(GNU Project, Free Software Foundation, Boston, MA, USA).
2.2.
3.
Results
3.1.
Cohort
Surgical technique and pathology
All patients were treated by bilateral extended pelvic lymphadenectomy and cystectomy according to standard protocols described previously in detail [10,14]. The surgical specimens were processed as described previously [10]. The tumours were staged according to the 7th Union Internationale Contre le Cancer classification [15].
2.3.
Construction of the tissue microarray
The tissue microarray [16] was constructed with six tissue cores (0.6-mm diameter) per patient: two each from normal urothelium, primary tumour (centre and invasion front), and a nodal metastasis.
2.4.
Fluorescence in situ hybridisation and
immunohistochemistry Two consecutive sections of 3-mm thickness were taken for fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) assays in our laboratory, which has validated these tests and takes part in external proficiency testing. For FISH analysis of the HER2 gene, the PathVysion HER2 DNA Probe Kit was used (Vysis Inc, Downers Grove, IL, USA). It is a mixture of the HER2 probe labelled in SpectrumOrange and the CEP17 probe, which is labelled in SpectrumGreen and directed against the centromeric satellite repeat of chromosome 17. Detailed instructions for hybridisation procedures are provided by the manufacturer. A minimum of 25 nonoverlapping nuclei were evaluated on each tissue spot and HER2 was considered normal, borderline amplified, or amplified (Fig. 1A–1B), according to the HER2/CEP17 fluorescence ratios < 1.8, 1.8–2.2, or >2.2 as recommended [2]. Separate HER2 mean ratios were assigned for each primary tumour and the corresponding metastasis based on the counts on their respective spots. The original Hercep test (DAKO, Glostrup, Denmark) was used for IHC stains, which were performed according to the manufacture’s protocol.
Cohort data are given in Table 1. 3.2.
Her2 gene status and protein expression
Normal urothelium always showed normal HER2 FISH status, and, in IHC, no protein overexpression. In 147 primary tumours, HER2 status could be determined by FISH: 127 tumours (86.4%) showed no amplification, 7 tumours (4.8%) showed borderline amplification, and 13 tumours (8.8%) showed amplification. Corresponding numbers from the 139 patients with evaluable metastases were 105 (75.6%), 13 (9.3%), and 21 (15.1%). Her2 IHC was obtained from 149 primary tumours: 119 tumours (79.9%) with score 0, 17 tumours (11.4%) with score 1+, 7 tumours (4.7%) with score 2+, and 6 tumours (4%) with score 3+. Corresponding numbers from the 139 patients with evaluable metastases were 78 (56.1%), 26 (18.7%), 14 (10.1%), and 21 (15.1%). In 137 patients, HER2 FISH status of the primary tumour could be compared with the corresponding nodal metastases; for Her2 IHC this was possible in 139 patients (Table 2). In the metastases, amplification (15.3%) and borderline amplification (8.8%) were significantly ( p = 0.003) more frequent when compared with the corresponding primary tumours (8.7% and 5.1%, respectively). The same was true for Her2 protein overexpression defined as score 2+ or 3+ (25.2% vs 9.4%; p < 0.001). Patients with two evaluable tissue spots in the primary tumour (n = 114) or in the lymph node metastases (n = 111) showed a high concordance in HER2 FISH results between
Her2 protein expression per tissue spot was classified as recommended [2]: negative (0/1+), weekly positive (2+), and positive (3+) (Fig. 1C–1D) with a cut-off for score 3+ of >30% strongly positive cells. Separate Her2
Table 1 – Clinicopathologic data of 150 lymph node–positive patients with urothelial bladder cancer
mean scores were assigned for each primary tumour and the corresponding metastases based on the findings on their respective spots.
2.5.
Statistical analyses
The McNemar test measured the differences in FISH and IHC results between primary tumour and nodal metastases. Cohen’s kappa assessed the concordance of Her2 status of the gene and protein, respectively, between the two samples from the primary tumor, between the two samples from the lymph node metastasis and measured the concordance between FISH and IHC assay. Her2 status was correlated with categorical (Fisher exact test) and quantitative (paired Wilcoxon test) clinicopathologic parameters. Kaplan-Meier curves were used to assess overall survival (OS) from surgery to the date of death. Patients still living were censored at the date of the last follow-up. Differences in survival
Patient data (n = 150) Age at surgery, yr, median (range) Female/male, No. Follow-up, yr, median Cystectomy data Tumour stage, No. pT1 pT2 pT3 pT4 Lymphadenectomy data Evaluated nodes per patient, median (range) Positive nodes per patient, median (range) Extracapsular extension of metastases, No. No Yes
67 (35–89) 29/121 7.1
4 17 92 37 27 (10–56) 3 (1–46) 71 79
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Table 2 – Comparison of primary tumours and corresponding lymph node metastases* FISH
Metastases Normal
Primary tumours
Normal Borderline Amplified
103 0 1
Borderline
Amplified
8 2 2
7 5 9
p = 0.003
IHC
Metastases
Primary tumours
0 1+ 2+ 3+
0
1+
2+
3+
75 3 0 0
20 5 1 0
7 5 2 0
8 3 4 6
p < 0.001 FISH = fluorescence in situ hybridization; IHC = immunohistochemistry. Her2 amplification and borderline amplification and Her2 protein overexpression are significantly more frequent in lymph node metastases than in primary tumours. *
Table 3 – Fluorescence in situ hybridization (FISH) assay* FISH in primary tumours
Tumour centre Normal
Invasion front
Normal Borderline Amplified
100 1 0
Borderline
Amplified
2 0 0
2 0 9
Cohen’s k = 0.853
FISH in metastases
Spot b Normal
Spot a
Normal Borderline Amplified
Borderline
81 2 0
Amplified
1 3 3
1 0 20
Cohen’s k = 0.930 * High concordance between Her2 gene status (normal, borderline amplified, amplified) in the two samples from the primary tumour and in the two samples from the lymph node metastases.
Table 4 – Immunohistochemistry (IHC) assay* IHC in primary tumours
Tumour centre
Invasion front
0 1+ 2+ 3+
0
1+
100 8 2 0
9 1 2 0
2+ 1 3 0 2
3+ 1 0 2 0
Cohen’s k = 0.539
IHC in metastases
Spot a
Spot b
0 1+ 2+ 3+
0
1+
2+
3+
53 8 3 1
4 18 3 0
0 1 8 2
0 2 2 14
Cohen’s k = 0.830 * Concordance between Her2 protein expression scores (0–3+) in the two samples is relatively poor in the primary tumour and is high in the two samples from the lymph node metastases.
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EUROPEAN UROLOGY 60 (2011) 350–357
Table 5 – Comparison of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) assays in primary tumours and corresponding lymph node metastases* FISH vs IHC in primary tumours
FISH
IHC
Normal Borderline Amplified
0/1+
2+
3+
124 6 5
3 0 4
0 1 4
Cohen’s k = 0.566
FISH vs IHC in metastases
FISH
IHC
Normal Borderline Amplified
0/1+
2+
3+
89 9 4
12 1 1
2 3 16
Cohen’s k = 0.673 *
Relatively poor concordance between HER2 gene status (normal, borderline amplified, amplified) and Her2 protein expression scores (0–3+) in primary tumours and better correlation in lymph node metastases.
[()TD$FIG] (A)
Primary tumours 1.0
(B) p = 0.044
0.8
p = 0.241
0.8
0.6
Her2 nonamplified
0.4
Her2 borderline amplified
Probability
Probability
Lymph node metastases
1.0
0.2
0.6
Her2 nonamplified 0.4
Her2 borderline amplified
0.2
Her2 amplified
0.0 0
5
Her2 amplified
0.0 10
15
0
Overall survival (yr)
Primary tumours
(C) 1.0
10
15
Lymph node metastases
(D) 1.0
p = 0.406
0.8
p = 0.721
0.8
Probability
Probability
5
Overall survival (yr)
0.6
Her2 IHC negative 0.4
0.2
0.6
Her2 IHC negative
0.4
0.2
Her2 IHC positive
Her2 IHC positive
0.0
0.0 0
5
10
Overall survival (yr)
15
0
5
10
15
Overall survival (yr)
Fig. 2 – Prognostic relevance of Her2 status: (A) Overall survival is significantly poorer in amplified than nonamplified primary bladder cancer. (B) The same trend, but not significant, is found in patients with Her2 amplification in lymph node metastases. (C and D) Immunohistochemically (IHC) determined Her2 status does not segregate significantly into low- and high-risk patients regarding survival.
EUROPEAN UROLOGY 60 (2011) 350–357
these spots (primary tumour: k = 0.853; for metastases: k = 0.930) (Table 3). For IHC, the results for corresponding spots were less concordant, particularly in the primary tumours (k = 0.539) (Table 4). The concordance between FISH and IHC assays (Table 5) was relatively poor in the primary tumours (k = 0.566) and somewhat better in the metastases (k = 0.673). Importantly, 38.5% (5 of 13) of the amplified primary tumours and 19% (4 of 21) of the amplified metastases were Her2 negative in IHC; these cases were evenly distributed over the entire time period, and there was no evidence of weaker membrane staining in older cases than in more recent ones. In contrast, all primary tumours with IHC 3+ were amplified or borderline amplified (5 of 5), whereas 9.5% (2 of 21) of the IHC 3+ metastases were not amplified; however, these two cases showed high chromosome 17 copy numbers. 3.3.
Association of Her2 status with tumour characteristics
Patients with Her2 amplified metastases had more metastases (median: 5 vs 3; p = 0.262), larger total diameter of metastases (median: 4.6 cm vs 2.3 cm; p = 0.180), and greater maximum diameter of metastases (median: 1.5 cm vs 1.0 cm; p = 0.263) than patients without such amplification; however, these differences were not significant. The same trend with smaller differences was observed for IHCbased data. Tumour stage and extracapsular extension of lymph node metastases were not associated with Her2 status. 3.4.
Univariate and multivariate survival analyses
In univariate analyses, OS was significantly ( p = 0.044) reduced for patients with Her2 amplification in the primary tumours compared with those patients without amplification (Fig. 2A). A similar trend for patients with amplified metastases was not significant ( p = 0.241) (Fig. 2B). Her2 IHC did not correlate with outcome (Fig. 2C–2D). In multivariate analyses, advanced primary tumour stage (pT3/4 vs pT1/2; p = 0.041) and extracapsular extension of lymph node metastases ( p < 0.001) were the only independent adverse risk factors for OS. Her2 amplification in primary tumours ( p = 0.074) and metastases ( p = 0.108) failed to add independent prognostic information. 4.
Discussion
Her2 status has important implications for prognosis and therapy in breast cancer. The beneficial effect of anti-Her2 therapies in the approximately 20% of breast cancers with HER2 gene amplification [2] suggests that other cancers with this alteration also may be treated successfully in this way. Larger cohorts of invasive bladder cancers have rarely been investigated for Her2 amplification by FISH and frequencies between 7% and 13.8% were reported [4,6–8]. However, these studies focused on primary tumours and did not evaluate Her2 status in the therapeutically most relevant metastases. The most important finding in our
355
study was that Her2 amplification is significantly more frequent in lymph node metastases from urothelial bladder cancer than in the primary tumours. While Her2 amplification, defined according to recently published strict criteria [2], was detected in 8.7% of the primary tumours, this genetic alteration was present in matched metastases of 15.3% of the patients. Remarkably, if Her2 amplification was found in primary tumours, then this was usually also the case in the corresponding metastases, suggesting a high stability of this alteration after occurrence in the primary tumour. Conversely, metastases with newly emerging Her2 amplifications may originate from a late-developed subclone in the primary tumour, which was not sampled but which has a high metastatic potential, or, alternatively, this genotype was acquired only at the metastatic site. Interestingly, a similar phenomenon of newly occurring Her2 amplifications has also been observed in distant metastases of breast cancers [17]. Previously, two larger series investigated Her2 status in lymph node–positive bladder cancer treated by cystectomy [18,19]. The cohorts comprised 42 [18] and 60 [19] patients, respectively, and results were conflicting. While Jimenez et al. [19], who evaluated only Her2 protein status, reported a higher incidence of Her2 overexpression in lymph node metastases when compared with matched primary bladder cancers, Hansel et al. [18] did not detect such differences at the genetic or protein levels. However, Her2 alterations in primary tumours were also highly conserved in the lymph node metastases in both series [18,19]. Her2 status in breast cancer predicts response to antiHer2 therapy [2]. This therapy acts on the protein level and therefore IHC might be considered as the method of choice for Her2 determination. Nevertheless, the appropriate assay to determine Her2 status in breast cancer remains controversial [2,20,21] because IHC results and HER2 gene status can be contradictory in paraffinembedded tissues. These discrepancies have been attributed to tissue processing artefacts at the protein level by some investigators [20]. They regard FISH as the gold standard for Her2 testing because DNA is better conserved in paraffin-embedded tissues than proteins and there is evidence that the gene status reflects the true protein status [20]. By contrast, proponents of IHC testing in breast cancer point out that optimised protocols for Her2 protein detection achieve a high concordance between FISH and IHC results [21] and that Her2 overexpression might not necessarily result from gene amplification only [21]. In our cohort, incidence of Her2 amplification in all tested primary tumours (8.8%) was in the range of other studies (7–13.8%) [4,6–8], whereas Her2 protein overexpression (8.7% with score 2+ or 3+) was lower (32–57%) [4,6–8]. However, a more recent study reported Her2 protein overexpression in 9.2% of 1005 patients with invasive bladder cancer [5]. In that study, strict definitions for Her2 scores were applied and the antibody had been calibrated in breast cancer samples using FISH results as reference. The large variability in reported Her2 results from bladder cancer has been suggested to reflect heterogeneity between kits, antibodies, protocols, and interpretations of
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staining [5]. In our study, specificity of IHC score 3+ for detecting Her2 borderline amplification or amplification was 100% in the primary tumours and 90.5% in the metastases. However, sensitivity was low: Her2 amplification without protein overexpression was present in 38.5% of the primary tumours and in 19% of the metastases. A relatively high rate of false-negative IHC results (20–24%) in bladder cancer has also been noted by others [6,8]. This contrasts with data from breast cancer studies where this constellation is rare [2]. The reason for the higher inconsistence between FISH and IHC results in bladder cancer compared to breast cancer is unknown. It has been suggested that tissue processing might play a role [8] and fixation artefacts might be more intense in bladder cancer than in breast cancer. In contrast, a true lack of Her2 expression in amplified bladder cancers due to additional genetic alterations cannot be excluded [8]. Finally, the discrepancies between FISH and IHC results in bladder cancer raise the question of which test best identifies the Her2-positive subgroup with prognostic and predictive relevance. In line with Jimenez et al. [19], survival of the immunohistochemically Her2-positive and -negative subgroups was not significantly different in our series. However, survival stratification according to FISH results was discriminating: Patients without Her2 amplification in the primary tumours did significantly better than patients with Her2 amplification. HER2 gene status in the metastases showed a similar survival trend; however, this was not significant. The successful survival stratification by FISH but not by IHC suggests that FISH is superior for identifying patients at risk and patients with high potential to benefit from anti-Her2 therapies. Our study has limitations. Although our cohort is relatively large compared with other lymph node–positive bladder cancer cohorts, it might be underpowered for analyses of biomarkers with low incidence or limited impact on survival. This might be the reason why we did not detect a significant prognostic impact of HER2 FISH status in the metastases and why Her2 amplification in the primary tumours failed to add independent prognostic information on multivariate analysis. Similar findings have been noted in breast cancer, where the prognostic relevance of Her2 status was merely detected in larger cohorts but not in smaller ones [22]. To better understand the significance of Her2 status in bladder cancer, including the controversially discussed borderline group [2,20], these data should be validated by others.
Author contributions: Achim Fleischmann had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Fleischmann. Acquisition of data: Rotzer, Seiler. Analysis and interpretation of data: Fleischmann. Drafting of the manuscript: Fleischmann. Critical revision of the manuscript for important intellectual content: Studer, Thalmann. Statistical analysis: Fleischmann. Obtaining funding: Fleischmann. Administrative, technical, or material support: Studer, Thalmann. Supervision: None. Other (specify): None. Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/ affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None. Funding/Support and role of the sponsor: This project received funding from Novartis Stiftung fu¨r medizinisch-biologische Forschung.
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Conclusions
Her2 amplification in bladder cancer is significantly more frequent in lymph node metastases than in the primary tumours, is homogeneous in the different tumour components, and is associated with early death. This suggests a high potential for anti-Her2 therapies in the subset of patients with Her2 amplification. Finally, FISH diagnostics might be more accurate for patient selection, because IHC fails to detect a substantial proportion of the prognostically relevant amplified tumours.
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