This was a multicenter, prospective, observational study coordinated by GEICAM and performed by the Medical Oncology departments in 31 hospitals. The study protocol was approved by the Institutional Review Board and the Ethics Committee of Hospital Provincial de Castellón (Spain), according to the requirements of the Spanish regulations (GEICAM 2009-03; clinicaltrials.gov identifier: NCT01377363). Study procedures were carried out in accordance with the Declaration of Helsinki, as revised in 2008, and good clinical practice guidelines. Written informed consent was obtained from all patients before enrollment.
The study included patients over the age of 18 with a suspected diagnosis of locally recurrent or metastatic breast cancer, either at first relapse or after successive disease progressions, between December 2009 and March 2011. Patients were required to have a formalin-fixed paraffin-embedded (FFPE) tissue sample from the primary tumor. Additionally, they had to be scheduled to undergo a biopsy of the recurrent or metastatic lesion within the next 6 weeks according to the routine clinical practice of the hospital. Biopsy could be performed by fine-needle aspiration, drainage of fluid cavities, core biopsy or surgical process. Study participants had to be capable of providing written, informed consent.
Exclusion criteria included in-breast recurrences, as well as second neoplasms, except for appropriately treated in situ cervical cancer and non-melanoma skin cancer.
Registration visit was completed within 6 weeks prior to the previously planned biopsy of the metastatic lesion. For each patient, date of birth, menopausal status, tumor stage, histological grade, receptor status (ER, PR and HER2) of the primary tumor according to local laboratory, type of surgery performed and antitumor treatment, location of the metastatic lesion and whether it was a first relapse or metastatic disease progression were recorded. At this visit, the treating oncologist declared the intended treatment plan for the patient.
Biopsies of the recurrent tumors were analyzed at the local laboratory, and blinded determinations of ER, PR and HER2 were centrally performed at the Pathology Department of Hospital Clinico Universitario de Valencia, (Spain), in paired samples of the primary tumor and the metastatic lesion. Once biopsy results at local level were available, the attending physician assessed the discordances between primary and metastatic tissues and whether the treatment plan should be modified.
All primary and metastatic tissues were analyzed at the same time and using the same methodology. For the expression of ER and PR, Benchmark XT instrument and the corresponding CONFIRM® antibodies (Ventana Medical Systems, Inc) were used. Both Allred score and percentage of nuclear staining were determined. Tumors with moderate-to-intense nuclear staining of ≥1 % or an Allred score ≥3 were considered ER positive or PR positive [6, 7]. IHC analysis for the expression of HER2 was conducted using the PATHWAY® anti-HER2 (4B5) monoclonal antibody (Ventana Medical Systems, Inc), considering the expression negative (0, 1+), indeterminate (2+) or positive (3+). When IHC yielded an indeterminate result for HER2 (2+) or when a discordant result between primary tumor and metastasis was observed, FISH was carried out using HER2 FISH PharmDx® kit (Dako Denmark A/S) and results were interpreted according to manufacturer’s instructions .
Pathology report was retrieved from primary tumor diagnosis, and biopsies from metastasis were analyzed at the time of relapse. The methodology used for the analysis of these samples by IHC and FISH at local level was not standardized, and each site used instruments and commercial antibodies according to their own established criteria.
The primary objective of this study was to estimate the conversion rate of HER2 status between primary tumors and metastases in patients with advanced breast cancer. Secondary objectives included ER and PR conversion rate, evaluating the impact of the immunohistochemical subtype of the primary tumor on the conversion rate, assessing the concordance between the results obtained locally and centrally and estimating how the conversion rate of receptor status may influence the antitumor treatment. Three immunohistochemical subtypes were previously defined: (1) ‘HR-positive’ tumors (ER positive and/or PR positive and HER2 negative); (2) ‘HER2-amplified’ tumors (HER2 positive/any HR); and (3) ‘triple-negative’ tumors (ER negative, PR negative and HER2 negative).
According to previous studies, the estimated conversion rate of HER2 determined by IHC or FISH was around 10 % (range 4–20 %). Considering that this conversion rate was similar within each molecular subtype, with an alpha error of 0.05 for a bilateral contrast and an accuracy of ±0.09 %, 43 patients would be needed for each molecular subtype, requiring 129 patients. Assuming that 25 % of patients would be lost for analysis due to invalidate biopsies or inconclusive results, 172 patients were required to achieve the main objective of this study. However, in a preliminary analysis after including the first 84 patients, the observed HER2 conversion rate was 3.57 %, so the sample size was re-estimated with the same premises and 222 patients were found to be needed.
All statistical tests were performed against a two-sided, alternative hypothesis using a significance level of 0.05 and a 95 % confidence interval. The variability in the receptor expression results between local and central laboratories was measured using Cohen’s kappa index and interpreted according to Landis and Koch . All these analyses were performed using SPSS statistics software version 17.0 (SPSS Inc, Chicago, Illinois, USA).