Annals of Oncology Advance Access originally published online on September 4, 2007
Annals of Oncology 2007 18(12):1963-1968; doi:10.1093/annonc/mdm417
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© 2007 European Society for Medical Oncology
breast cancer |
Patients with advanced stage breast carcinoma immunoreactive to biotinylated Herceptin® are most likely to benefit from trastuzumab-based therapy: an hypothesis-generating study
1 Dipartimento di Scienze Biomediche e Oncologia Umana, Università di Torino, Torino, Italy
2 Divisione Universitaria di Oncologia Medica ed Ematologia, Istituto per la Ricerca e la Cura del Cancro, Candiolo Torino, Italy
3 Divisione di Anatomia Patologica e Medicina di Laboratorio, Istituto Europeo di Oncologia e Università degli Studi, Milano, Italy
4 Budapest Faculty of Medicine, Semmelweis University, Hungary
5 Centro Onco-Ematologico Subalpino, Ospedale San Giovanni Battista, Molinette, Torino, Italy
6 Breast Unit, Istituti Ospitalieri, Cremona, Italy
7 Istituto Nazionale Tumori, Fondazione Senatore Pascale, Napoli, Italy
8 Dipartimento di Patologia, Ospedale Regionale, Treviso, Italy
9 Royal Surrey County Hospital, Guildford, Surrey, UK
10 Istituto Nazionale per la Ricerca sul Cancro (IST), Genova, Italy
11 Divisione di Oncologia Medica, Istituto Europeo di Oncologia, Milano, Italy
* Correspondence to: Prof. A. Sapino, Department of Biomedical Sciences and Human Oncology, Via Santena 7, Turin, Italy. Tel: +39 011-6334127; Fax: +39 011-6635267; E-mail: anna.sapino{at}unito.it
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Abstract |
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Background: Biotin-labeled trastuzumab (BiotHER) can be used to test for HER2 by immunohistochemistry. We previously showed that BiotHER immunoreactivity is highly correlated with HER2 amplification and indicated that it could be associated with better clinical outcome in advanced breast cancer patients receiving trastuzumab.
Patients and methods: Tumor specimens and clinical information from 234 patients who received trastuzumab-based treatments were collected from 10 institutions. HER2 amplification and BiotHER immunoreactivity were assessed centrally. The effect of BiotHER positivity on response rate (RR), time to progression and survival were studied by univariate and multivariate analysis in patients presenting HER2-amplified breast cancer. The pathologic reviews of the assays were blinded to patient outcomes.
Results: BiotHER was positive in 109/194 (56%) HER2-amplified breast cancers and in one not amplified tumor. RRs were 74% [95% (confidence interval) CI 64%–81%] and 47% (95% CI 36%–58%) in BiotHER-positive and -negative tumors, respectively (P < 0.001). BiotHER immunoreactivity was independently associated with increased probability of tumor response (odds ratio 3.848; 95% CI 1.952–7.582), with reduced risk of disease progression [hazard ratio (HR) 0.438; 95% CI 0.303–0.633] and with reduced risk of death (HR 0.566; 95% CI 0.368–0.870) by multivariate analysis.
Conclusion: The results support a role for BiotHER testing in better tailoring trastuzumab-based treatments in patients with advanced HER2-amplified breast cancers.
Key words: biotin-labeled trastuzumab, breast neoplasms, HER2
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introduction |
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Despite the impressive clinical benefit of trastuzumab in patients with early and advanced HER2-positive breast carcinoma [1–5], not all treated patients are likely to benefit to a similar extent. Accordingly, besides the accurate assessment of HER2 status, additional predictive parameters are needed to identify more precisely the patients most likely to benefit from trastuzumab, sparing the less responsive patients the possible side-effects of this rather expensive treatment. Being a molecularly targeted therapy, the clinical efficacy of trastuzumab depends on the precise assessment of HER2 status in tumor specimens of candidate patients for treatment. Currently, the two most standardized approaches for HER2 status evaluation in the clinical setting are immunohistochemistry (IHC) and FISH [6–8]. Despite reliable selection of patients with HER2-positive tumors using the above-mentioned methods, the response rate (RR) with single-agent trastuzumab in patients with advanced breast cancer only ranges from 19% to 34% [9, 10]. It must be emphasized that neither FISH nor the most widely used IHC methods to detect HER2 overexpression can actually document the presence of the specific target site of trastuzumab, which is located in the juxtamembrane region of the extracellular domain (ECD) [11]. For this reason, we hypothesized that using trastuzumab as the primary antibody to reveal the presence of HER2 on the surface of cancer cell could result in better prediction of its clinical efficacy. We have therefore conjugated the NH2 groups of trastuzumab with biotin and directly added the biotinylated antibody to tissue sections. Biotin labeling was revealed with horseradish peroxidase (HRP)-conjugated streptavidin. In our previous study, BiotHER immunoreactivity was found to be restricted to about a half of the tumors with FISH-proven HER2 gene amplification [12]. An analysis of clinical outcome in a small series of patients with HER2-amplified advanced breast cancer receiving trastuzumab-based treatment indicated that BiotHER immunoreactivity could be associated with better clinical outcomes. With the current study, we sought to validate the preliminary clinical data, testing a larger series of advanced breast cancers from patients treated with trastuzumab-based regimens at different institutions with BiotHER.
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patients and methods |
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study population
The study was designed and coordinated by the Dipartimento di Scienze Biomediche e Oncologia Umana, University of Turin, Italy. Eleven institutions provided tumor blocks and complete demographic and clinicopathological data of women receiving trastuzumab-based therapy for HER2-positive advanced breast cancer. Best tumor response during the initial trastuzumab-based treatment was measured according to the World Health Organization criteria [13]. The study was conducted in compliance with the ethical regulatory issues of each of the participating institutions.
immunohistochemical and FISH assays
A formalin-fixed, paraffin-embedded tumor block representative of the lesions was sent to the Coordinating Center. BiotHER was prepared according to the procedure previously reported by Bussolati et al. [12].
Briefly, 1 mg of trastuzumab, commercially available as Herceptin® (Roche, Hertfordshire, UK), was diluted in saline solution (1 mg/ml concentration) and dialyzed overnight in 0.1 M Na2CO3 (pH 8.5). To 1 ml of solution, 0.12 ml of 
-caproylamido-biotin-n-hydroxy-succinimide ester (Biospa, Milano, Italia) was added. The preparation was mixed by gentle agitation for 4 h at room temperature and dialyzed in phosphate-buffered saline (PBS). The product, designated BiotHER stock solution, was kept at –80°C in 20 µl aliquots for 1 h and then sublimated. The lyophilized mAb was stored in vials.
For the IHC assay, whole tissue sections were incubated with BiotHER at a 1:100 dilution in PBS for 1 h at room temperature, washed twice for 5 min in PBS, treated with HRP-conjugated streptavidin (BioGenex, San Ramon, CA) at a 1: 50 dilution for 13 min at room temperature and finally developed with a solution of 3-3'-diaminobenzidine and H2O2 for 5 min. A tissue array (TA) of six breast cancers with different HER2 gene status and protein expression (three tumors amplified and intensely immunoreactive for BiotHER (score 3+) and three tumors not amplified and negative for BiotHER) was used as a control. The cut-off for BiotHER positivity was set up to 10% of cancer cells showing complete membrane immunoreactivity independently from intensity of the staining. Pathologists carrying out these analyses were blinded to the clinical outcome of patients.
All cases were retested by FISH at the coordinating center, either on full sections when only small biopsies were available or on TA [14] using the PathVysion HER2/neu probe kit (Vysis Inc., Downers Grove, IL). HER2 amplification was defined as a ratio of 
2.2, as suggested by recently published recommendations [8]. The amplified cases were divided into three categories depending on the following ratio values: 2.2 and 
4.0; >4.0 and 6.0; and > 6.
statistical analysis
Associations between categorical variables were studied by contingency tables and analyzed by the Fischer's exact test. Continuous variables were compared by the Mann–Whitney U test. Time to progression (TtP) was measured as the interval between the date of the first administration of trastuzumab-based therapy to the date of tumor progression or death of the patient in the absence of tumor progression. Survival was measured as the interval between the dates of the first administration of trastuzumab-based therapy to the date of patient's death, Kaplan–Meier curves of TtP and survival according to BiotHER status were drawn and compared by the log-rank test. Patients not experiencing a TtP or survival event were censored at the time of the most recent visit.
Differences in RR [proportion of patients achieving complete remission (CR) or partial remission (PR)], TtP and survival according to BioHER status were studied by univariate and multivariate analysis (logistic regression and Cox proportional regression models). In each multivariate analysis, all the variables were included and then removed through a backward elimination variable selection method using the likelihood ratio test until all the remaining factors were statistically significant (P < 0.05). When the proportional hazard assumption was not met, an interaction term between the variable violating the assumption and a time-defined variable was included in Cox proportional hazard models. Significance was set at P < 0.05. Statistical analysis was carried out using SPSS version 13.0 (SPSS Inc, Chicago, IL) software.
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HER2 status determination by method
Of the 234 cases collected and tested by central FISH, 194 tumors were amplified, 33 were not amplified and FISH was not assessable for technical reasons in seven cases. BiotHER immunoreactivity was detected in 111 (47%) cases and in 109 (56%) of the 194 FISH-positive tumors. Only one immunoreactive tumor was not amplified, while FISH was not assessable in another positive case. BiotHER immunoreactivity was invariably confined to the cell membranes of the neoplastic cells, without any staining of non-neoplastic epithelial cells.
Comparison of patients’ characteristics at the time of the first trastuzumab-based regimen according to BiotHER status is displayed in Table 1.
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clinical outcome of FISH-amplified tumors according to BiotHER
We analyzed the clinical outcome according to BiotHER immunoreactivity for the 194 patients with HER2-proven amplified tumors. RR, TtP and OS were significantly better for patients with BiotHER-positive tumors. RR (CR + PR) was 74% [95% confidence interval (CI) 64%–81%] and 47% (95% CI 36%–58%) in patients with BiotHER-positive and -negative tumors, respectively (two-tailed Fisher's exact test, P < 0.001) (Table 2).
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At a median overall follow-up of 20 months (range 1–80), 132 patients had disease progression during the initial trastuzumab-based regimen and 84 had died. Both TtP and survival were significantly longer for patients with BiotHER-positive tumors (Figure 1A and B). Median TtP was 10 months (95% CI 6–14) in BiotHER-negative patients and 17 (95% CI 12–23) months in BiotHER-positive patients (log-rank test, P < 0.001). Median survival was 26 (95% CI 21–32) and 45 (95% CI 24–66) months for BiotHER-negative and -positive patients, respectively (P = 0.015). The univariate analyses of response, TtP and survival are summarized in Table 3. Age at the first trastuzumab-based regimen and disease-free interval were dichotomized according to the median values. The type of trastuzumab-based regimen was considered as a three-level variable grouping patients receiving trastuzumab alone with patients receiving trastuzumab combined with nontaxane and nonvinorelbine agents. BiotHER positivity was significantly associated with a three-fold increase in the likelihood of response [odds ratio (OR): 3.162; 95% CI 1.705–5.866], and with reduced risk of tumor progression [hazard ratio (HR) 0.515; 95% CI 0.365–0.727) and death (HR 0.592; 95% CI 0.387–0.908).
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The independent predictive value of BiotHER positivity was confirmed by the multivariate analyses of response (OR 3.848; 95% CI 1.952–7.582), TtP (HR 0.438; 95% CI 0.303–0.633) and survival (HR 0.566; 95% CI 0.368–0.870) (Table 4). At no step in the multivariate analyses did BiotHER status lose its statistical significance for the three outcomes analyzed. Similar results were obtained excluding from the analyses the 54 patients of the original publication or correcting the multivariate analyses by treatment center (data not shown).
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discussion |
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This study confirms that biotinylated trastuzumab in IHC identifies a subset of HER2-amplified advanced breast cancer patients who are more likely to benefit from trastuzumab-based treatments with better RR, TtP and OS, independently of other possible covariates.
Standard IHC and FISH assays have fairly low predictive value in the selection of candidate patients for trastuzumab therapy due to the common overestimation of HER2-positive cases [15]. False-positive IHC assays for HER2 are most likely to be primarily due to methodological issues but inaccurate interpretation of the results may also play a major role. The use of IHC scoring systems that require a multiparametric assessment, such as percentage of positive cells, completeness of membrane staining and intensity of staining, leads to diagnostic discordance particularly in the 2+ category. BiotHER positivity most closely mirrors HER2 gene amplification (with a 97% overall concordance). Accordingly, a tumor with >10% BiotHER-positive cells is almost invariably amplified with a high HER2/CEP17 ratio. Our data support the hypothesis that BiotHER testing might identify a distinct subset of HER2 gene-amplified breast cancers. Reduced expression of hormone receptors in BiotHER-positive tumors and more frequent BiotHER positivity in tumors with higher HER2 amplification ratio (Table 1) may have occurred by chance but deserve further evaluation being potentially related to more subtle biological diversities.
Because this is a retrospective investigation on a cohort of patients treated at different institutions, it should be viewed as an exploratory hypothesis-generating study. Tumor response, for example, was provided by local investigators without any central review. This may explain why the proportion of responders in the current study is lower than that reported in our previous study [12] with patients treated at only two institutions and being mostly enrolled in clinical trials with trastuzumab. Also, most of the patients in this analysis received trastuzumab in combination with some other agent. It is therefore difficult to ascertain whether BiotHER positivity correlates with increased susceptibility to trastuzumab therapy, to chemotherapy (or type of chemotherapy) or their combination. This may be particularly relevant when analyzing tumor response data. A larger dataset allowed the inclusion of the type of trastuzumab-based treatment in our analyses and this factor was independently associated with both tumor response and TtP. Coexistence of both variables in the final model indicates that BiotHER positivity correlates with responsiveness regardless of the type of trastuzumab-based regimen used. The value of this observation is potentially limited by the small number of patients receiving trastuzumab alone.
The TtP advantage for patients with BiotHER-positive tumors is of particular interest. Univariate comparison showed a 7-month gain in median Progression Free Survival (PFS) and the final multivariable model confirmed that the risk of tumor progression was more than halved in patients with BiotHER-positive tumors. The predictive power of BiotHER immunoreactivity was independent of exposure to anthracyclines, multiple sites of metastatic disease or liver involvement that are known to strongly affect TtP in this setting of patients. Patients who achieve tumor response or stabilization after some months of trastuzumab plus chemotherapy are usually administered single-agent trastuzumab as maintenance therapy. Lack of tumor progression during this maintenance therapy may reflect, among other factors, a specific susceptibility of residual tumor cells to trastuzumab. Positive BiotHER status was also associated with a 19-month gain in median survival and with a reduced risk of death of 
40%. This finding is probably due, in part, to the clinically relevant prolongation of TtP in patients with BiotHER-positive tumors.
Several preclinical studies have indicated different mechanisms of trastuzumab resistance [16–23]. Interestingly, 22% of breast cancers express only the p95 NH2-truncated form of HER2 [21], following the proteolytic shedding of the ECD of the receptor. This portion of the molecule, that is detectable in the serum of 35%–40% of patients with metastatic breast cancer as a protein of 105 kDa, actually harbors the accessible target of trastuzumab [23]. Truncated HER2 receptor (p95) exhibits increased autokinase activity and predicts both resistance to trastuzumab and responsiveness to the dual HER2/EGFR tyrosine kinase inhibitor lapatinib [24–26]. This last issue is of particular relevance since lapatinib has been proven active in HER2-positive advanced breast cancer resistant to trastuzumab [24]. Recently, a new immunofluorescence method to asses the presence of p95 on fixed tissue samples has been retrospectively tested in a small series of 46 HER2-overexpressing breast cancer patients treated with trastuzumab as single agent [26]. RR was 51% and 11.1% for patients with p95 immunofluorescence negative and positive tumors, respectively. Based on these findings and on the results of our retrospective analysis, we believe that BiotHER testing, alone or in combination with p95 assessment, has the potential to allow a better matching of HER2-positive breast cancer patients with the appropriate anti-HER2 treatment and warrants evaluation in the context of prospective trials.
One critical scenario could be the adjuvant setting, where it may be particularly rewarding to prospectively include the BiotHER assay as a stratification factor in clinical trials randomizing patients to different forms of anti-HER2 treatments (trastuzumab, lapatinib or their combination).
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Compagnia di San Paolo special project ‘Oncology', Ministero Istruzione Università e Ricerca, Progetti di Ricerca di Interesse Nazionale 2005, Ministero dell’ Università della Ricerca Scientifica e Tecnologica (MURST) (ex 60%) and Fondazione Cassa di Risparmio di Torino, Regione Piemonte Comitato Interministeriale per la Programmazione Economica 2004 and Progetto Ricerca scientifica Finalizzata Regione. Piemonte (grant number 2006 RE R112).
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Acknowledgements |
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We thank Rosanna Lupo for immunohistochemical assistance and Patrizia Gugliotta and Cristina Botta for performing FISH testing.
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Footnotes |
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These authors contributed equally to this work. 
Received for publication June 14, 2007. Revision received July 16, 2007. Accepted for publication July 18, 2007.
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