Background
Approximately 25 % of patients with invasive breast cancer overexpress human epidermal growth factor receptor 2 (HER2) [
1]. HER2 overexpression, mainly because of
HER2 amplification, is significantly associated with aggressive disease and poor prognosis [
2]. Trastuzumab can bind the extracellular domain of HER2, and has a remarkable impact on the treatment of patients with HER2-positive breast by several pathways [
3‐
5]. Therefore, trastuzumab was approved by the United States (US) Food and Drug Administration (FDA) for the treatment of breast cancer. Hence, accurate determination of the HER2 status in patients with breast cancer is crucial [
6].
Immunohistochemistry (IHC), for measuring protein overexpression, and fluorescence
in situ hybridization (FISH), for measuring
HER2 amplification, are the most commonly applied methods for the assessment of HER2 status [
7]. Although both these methods can be applied to evaluate protein overexpression and gene amplification in relation to the morphological features of tumors, there is large inter-laboratory variation in the procedure, staff, and interpretation, which affects the analytical sensitivity and specificity of these assays, especially in the case of indeterminate samples [
8].
Ensuring the accuracy and reproducibility of HER2 detection by IHC and FISH testing is a fundamental prerequisite for targeted therapy. Recommendations for HER2 testing were developed by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2007 and were updated in 2013 [
9,
10] to improve the performance of these tests. HER2 detection requires precisely characterized and universally available reference controls [
11]. Adequate quality control is necessary to diminish inter-laboratory variation and external quality assurance.
Recently, a consensus was reached to create clinical specimen or cell line controls as reference materials for HER2 testing [
12]. Quality control of clinical specimens facilitates the assessment of variation in methodologies and laboratories. However, some disadvantages have limited the development of these quality controls, such as production and ethics. Therefore, we developed new xenograft tumor controls and checked their suitability for internal quality control (IQC) and external quality assurance (EQA). In this study, we compared three quality control samples with traditional clinical specimens. The aim of our study was to identify the most appropriate quality control that would ensure maximum accuracy and reproducibility of IHC and FISH.
Methods
Cell lines
The breast cancer cell lines MCF-7, MDA-MB-453, BT474, and SKBR-3 were obtained from the Cell Based Medical Center of Basic Medicine of Peking Union Medical College (Beijing, China). Cells were cultured as recommended by the suppliers. The HER2 status of the cell lines has been reported in previous studies [
1,
13]. MDA-MB-453, BT474, and SKBR-3 expressed HER2, while MCF-7 did not. All four cell lines were applied for the preparation of three quality-control samples as described below.
Samples
Two surgical specimen cases of invasive breast cancer with of known HER2 status were selected and obtained from the Pathology Department of Beijing Hospital, Beijing, China. Our protocol and specimen were approved by the Ethics Committee of the National Center for Clinical Laboratories. One of the samples tested negative for HER2 (A2; FISH result: negative; IHC score: 0), whereas the other sample tested positive (A12; FISH result: positive; IHC score: 3+). The HER2 status of these two samples was evaluated together with our prepared samples.
Approximately 1 × 107–2 × 107 cells were obtained and fixed in 10 % neutral-buffered formalin for approximately 2 h at room temperature. Subsequently, cell pellets were processed with gradient ethanol for dehydration and with xylene for transparency. Cell pellets were treated with melted liquid paraffin in small Eppendorf tubes and waxed thoroughly for 10 min; liquid paraffin was mixed with the cell pellets, using pipette tips. The solution was then chilled to achieve solidification, and the resulting blocks were embedded in paraffin, using a standard histochemical apparatus. Hematoxylin-eosin (HE) staining was performed to preserve the representative morphology of each sample and the number of cells to assess the uniformity of cellular distribution for each section. Each section was 5 μm in thickness. The sections were mounted onto slides and dried in an oven at 70 °C for approximately 3 h to ensure maximum adhesion.
Preparation of FFPE agarose gel samples
Approximately 1 × 106-2 × 106 cells were fixed overnight in 10 % neutral-buffered formalin at 4 °C, with the fixed cells being allowed to sediment at the base of a 15-mL tube with a conical base after centrifugation at 0.4 × g for 10 min, following which the formalin supernatant was removed. The cells were resuspended in phosphate buffered saline (PBS) solution, and the suspension was transferred to 1.5-mL Eppendorf tubes and allowed to sediment again after centrifugation at 0.4 × g for 10 min. The supernatant was removed, leaving the cells in 10–50 μL of PBS, in which the cells were resuspended. The cell suspension was embedded in prewarmed and equilibrated 3 % PBS-buffered agarose gel in 5-mm diameter × 5-mm deep iron molds from which small agarose-cell mixture cylinders were extruded after the agarose was sufficiently chilled. The agarose gel cylinders were then placed in a standard tissue-processing cassette and processed with paraffin wax. HE staining was performed; each section was 5 μm in thickness.
Preparation of FFPE xenograft tumor samples
Female severe combined immunodeficient (SCID) mice between 21 and 28 days of age (Vital River, Beijing, China) were used; the mice were quarantined for at least 3 days prior to the study. The mice were anesthetized with vaporized isoflurane, and suspensions of cells (all the four cell lines, approximately 1 × 107 cells) mixed with Matrigel™ basement membrane matrix (Corning, New York, USA) were injected subcutaneously under sterile conditions into the backs of mice, using a 5-gauge needle. Once the size of the xenograft tumor had progressed to approximately 500 mm3, the mice were euthanized via cervical dislocation, and all xenograft tumors belonging to different cell lines were removed surgically. All animal experiments were performed in compliance with the guidelines specified by the Institute for Experimental Animals, Beijing Hospital. Following surgical removal of xenograft tumors from the SCID mice, the tumors were cut into multiple pieces of approximately 250 mm3, placed into appropriately labeled disposable plastic tissue cassettes, and then immediately fixed in 10 % neutral-buffered formalin at 4 °C overnight. Subsequently, automated tissue embedding was performed using a Renaissance Tissue Processor (Ventana Medical Systems, US). This included washing in a graded ethanol series for dehydration followed by xylene washes for transparency, and heated paraffin washes prior to embedding in paraffin. HE staining was performed to ensure that the representative morphology of each sample was maintained. Each section was cut to a thickness of 5 μm.
Evaluation of HER2 status by FISH
FISH assays were performed following the manufacturers’ instructions (PathVysion HER-2 DNA Probe Kit, Abbott, Chicago, IL, USA). In brief, deparaffinization, heat pretreatment, and protease treatment were performed sequentially for all the FFPE samples. After pretreatment, the sample sections and probes were co-denatured and hybridized. A post-hybridization wash was performed for all sample sections in the dark using the wash buffer supplied by the manufacturer. 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) counter-stain was applied to the target area of the sections, and fluorescence microscopy was performed to view areas of positive hybridization. The results were evaluated qualitatively based on the kit recommendations.
Evaluation of HER2 expression by IHC analysis
The HER2 protein expression status of the sample slides was assessed using a commercial primary antibody (PATHWAY® anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody; Ventana, Tucson, AZ, USA), as per manufacturer’s instructions. Briefly, all the sample slides were processed according to a standard tissue-processing process, including deparaffinization, rehydration and antigen retrieval. After cooling for 15 min, IHC reactions were manually performed manually using the primary monoclonal antibody, 2-step plus® Poly-HRP Anti Mouse/Rabbit IgG Detection System (ZSGB-BIO, Beijing, China) and diaminobenzidine (DAB) staining. HER2 protein expressed on the membrane of tumor cells was scored as 0+, 1+, 2+, or 3+ as recommended [
10].
Discussion
Quality controls are required in HER2 status detection to ensure the accuracy and reproducibility of testing [
2,
10] and hence, choosing a suitable quality control is necessary for FISH and IHC assays. Currently, the best quality controls for HER2 testing are tissue-based controls from clinical specimens exhibiting both HER2 overexpression and
HER2 amplification that have been confirmed by IHC and FISH [
2]. Some studies have also reported that cell line samples can be used for EQA as additional quality controls instead of traditional quality controls derived from patient tumor tissue [
13,
14]. In this study, we have developed three quality controls and compared them with cell line samples and clinical specimens to identify a suitable quality control for FISH and IHC assays.
Different quality controls possess characteristic advantages and disadvantages. At present, tissue microarray (TMA)-based or FFPE-based quality controls prepared from clinical specimens are widely used for EQA or IQC in IHC or FISH testing [
15,
16]. The protocol utilizing clinical specimen quality controls is robust, cost-effective, and rapid. Moreover, it allows continuous monitoring of the performance of FISH and IHC testing [
16]. However, some potential limitations of clinical specimen quality controls should be considered. Since, EQA requires a larger number of quality controls, a collection of large clinical specimens from different patients with breast cancer is needed. Previous studies have reported that the properties of the samples being tested may influence the results and create bias in the comparison [
17]. Thus, heterogeneity among clinical specimens from different patients may affect the reproducibility of assays. Moreover, mass production of clinical specimens is difficult, and ethical problems should be taken into consideration. As additional quality controls, cell line quality controls are currently applied for IQC and EQA instead of traditional clinical specimens. Compared with traditional paraffin blocks of tumor tissues, large amounts of such controls can be obtained easily, and the reproducibility allos for sensitive detection and decreases analytical errors [
11]. In our study, we found that cell line samples can be applied for both IHC and FISH, as reported by previous studies [
11,
18], and that the protein and gene status of different cell lines can be determined accurately.
However, we found two main disadvantages in the use of cell line quality controls. First, the tumor cells showed diffuse distribution in the entire section, and the diffused cells were difficult to observe and count. The diffused cells were also prone to drop off for lack of tissue structure. Second, cultured cells were prone to polysomy, which is uncommon in patient tumor tissue. This polysomy might affect the reproducibility of cell line quality controls [
19].
To resolve these issues, agarose gel samples prepared using different cell lines were produced to optimize the basic cell line samples. Embedding in agarose gel helped gather the cells and increase the viscosity of the specimen. However, the morphological features of actual human tumors could not be simulated. Furthermore, we found that the agarose gel samples could only be applied for FISH. The use of an overheated agarose gel led to changes in the morphology of cells. Additionally, the use of agarose gel affected the positive results because of weak or faded staining. The probable reason for this is the effect of fixation or storage on the agarose gel, which has been previously reported to decrease the staining in IHC [
20]. Thus, agarose gel samples were not suitable as quality controls.
On the basis of the results of this study, we suggest xenograft tumor samples as suitable additional quality controls instead of cell line quality controls. Our observations indicate that xenograft tumor samples are similar to patient tumor specimens, providing evidence that they can be implemented for quality control. A previous study, which applied xenograft tumor samples as the quality control in the IHC assessment of estrogen receptor (ER) and progesterone receptor (PR), reported that the use of xenograft tumor samples as quality controls provides advantages in relation to reproducibility of biomarker, homogeneity of tissue histology, specimen processing, availability, and cost compared with archived tumor quality controls and cell line quality controls [
21]. Similarly, our study confirmed that xenograft tumor samples could be applied for quality control in the detection of HER2 status. Furthermore, our study proved that xenograft tumor quality controls could be applied for the detection of both protein status (IHC) and gene status (FISH). Regarding the economic factor, the cost of producing xenograft tumor samples is similar to that involved in producing cell line samples but lesser than that involved in preparing clinical specimen tissue TMA or FFPE-based quality controls (Table
1) [
15].
However, some potential disadvantages need to be considered. The characteristic features of cell lines for establishing xenografts should be monitored during long-term culture to ensure reliability. In theory, although the target tumor cells in xenografts are human-derived [
22], they might contain vessels or fibrocytes derived from mice. This indicates that the non-specificity of the secondary antibody might lead to some background staining in IHC. However, in our study, we found that the influence of this background staining was negligible. Nevertheless, poor tumorigenic potential and slow growth of HER2-positive xenograft tumors might impede its application [
23]. In our study, we chose SCID mice and Matrigel basement membrane matrix to improve the tumorigenic potential [
24]. Moreover, some distinctions were found between the xenograft tumor samples and clinical specimens in histological analysis. In the clinical specimens, a larger number of normal cells was observed and the tissue structure was more complicated. Breast catheter, breast gland, and inflammatory cell infiltration was evident. In the xenograft tumor samples, almost all the cells were tumor cells, and the tissue structure was simple. As a result, the evaluation of clinical specimens was more difficult than in the case of xenograft tumor samples. Some performance characteristics, especially the interpretation by observers in the routine work, might be neglected to be evaluated in EQA.
Acknowledgements
This work was edited by professional editors at Editage.