Diagnostic utility of snail in metaplastic breast carcinoma

The study was approved by the institutional review boards of our respective Institution. We conducted a retrospective review of 34 patients who received a diagnosis of MBC at a tertiary referral center from February 1997 to August 2007. Patient age, tumor size and grade, nodal status, hormone receptor status, initial treatment, recurrence, and follow up information were reviewed (Table 1).

The cases of MBC contained 20 cases of spindle cell type, 5 mixed spindle and squamous cell type, 4 squamous cell type, 3 matrix producing type, and 2 low-grade adenosquamous carcinoma. All patients with MBC were females with an average age of 62 yrs. (range: 32 - 90 yrs.). All tumors were characterized histologically according to Wartgotz's criteria [16‐19]. The control group consisted of 26 spindle cell lesions: 14 phyllodes tumors, 8 pseudoangiomatous stromal hyperplasias (PASH), and 4 myofibroblastomas. The majority of the patients in the control group were females (23) with only few males (3) who presented with myofibroblastoma. The average age in the control group is 53 yrs. (range: 17 - 86 yrs.).

The following immunohistochemical stains were performed: Snail, epidermal growth factor receptor (EGFR), OSCAR (a broad spectrum cytokeratin), wide spectrum cytokeratin (WS-KER) and p63, using these antibodies and positive controls listed in Table 2. Negative controls were run simultaneously and had primary antibody replaced with buffer. EGFR immunostain was performed with kits approved by the Food and Drug Administration (EGFR PharmDX;, Dako North America, Inc, Carpinteria, California) used in accordance with manufacturer instructions for antigen retrieval and immunostaining method. Antigen retrieval for all other antibodies was conducted in citrate buffer (pH, 6.0) under a pressure of 15 pounds per square inch for 3 minutes. The EnVision+ Dual Link Kit (Dako) was used for all immunostaining, with an automated slide stainer and with diaminobenzidine as chromogen and hematoxylin as counterstain.

Immunohistochemical staining was performed as follows. Formalin-fixed, paraffin-embedded samples, cut at 5 μm onto charged slides and baked at 60°C for 40 minutes prior before staining, were deparaffinized with 3 changes of xylene and rehydrated in a series of graded alcohols (100% ethanol, 95% ethanol, and 70% ethanol), then rinsed well in running distilled water. Slides were placed in a preheated citrate retrieval buffer (pH, 6.0) for 30 minutes, in a water steamer, cooled in the buffer for 5 minutes, and rinsed for 5 minutes in running distilled water.

Slides were placed on an automated slide stainer (AS100 Autostainer Plus; DAKO, Carpinteria, CA) for the following procedure (at room temperature). Sections were incubated with 3% hydrogen peroxide in ethanol for 5 minutes to inactivate endogenous peroxidases. They then were incubated in primary antibody for 30 minutes, rinsed with TBST wash buffer (S3006, Tris-Buffered Saline with Tween 20; DAKO) and incubated for 15 minutes with a peroxidase-labeled polymer conjugated to the secondary antibody. After a rinse with TBST wash buffer, sections were incubated in 3,3'-diaminobenzidine (K3468, DAB+ Substrate Chromogen; Dako) for 5 minutes, counterstained with modified Schmidt's hematoxylin for 5 minutes, followed by a 3- minute tap water rinse to set counterstain, dehydrated through graded alcohols (70% ethanol, 95% ethanol, and 100% ethanol), cleared in 3 changes of xylene, and mounted with permanent mounting media.

Antibody staining was membranous for EGFR, nuclear for PIN2 cocktail (p63) and Snail, and cytoplasmic for OSCAR and WS-KER. For all markers, scoring was performed using a score of less than 1% as negative. Several different microscopic fields (at least 10) per low and medium-power fields are examined for staining assessment.

Results were analyzed statistically with the sensitivity, specificity, and positive and negative predictive values for the different antibodies studied. Disease-free survival and overall survival were analyzed by the Kaplan-Meier survival curves. Associations of overall survival and surgery type, chemotherapy and radiotherapy were performed using the log rank test. Associations of tumor characteristics and snail percentage were assessed using Wilcoxon rank sum tests, while the association between snail percentage and age or tumor size was assessed using linear regression. In all cases, significance was defined as p-value < 0.05.

All patients had sentinel lymph node biopsy (100%), and only four patients had axillary lymph node (ALN) dissection (30.8%; 4/13). Five (15%) patients were lymph node positive. Those with positive ALNs have larger (pT2 or above) tumor size (p = 0.02). Twenty-one (62%) patients underwent mastectomy and 13 (38%) underwent breast conservation therapy (BCT). Fourteen patients (41%) received chemotherapy and seventeen (50%) patients received radiotherapy, 13 of whom received breast radiation as part of BCT. Neither chemotherapy nor radiotherapy increased survival of these patients (p = 0.87 and 0.13, respectively). Two patients were lost to follow up, with a median follow-up time of 21 months for the remaining 32. Three patients had local recurrences which occurred within 6 months, and 4 had distant metastases to lung and bone. Five patients died within the first year of diagnosis, 9 additional patients died by 5 years, and 2 more died by 10 years. The Kaplan-Meier estimated disease-free survival and 95% confidence interval (CI) was found to be 78.5% (64.4%, 95.5%) at 1 year and 71.3% (54.4%, 93.6%) at 3 years. The Kaplan-Meier estimated overall survival and 95% CI was found to be 82.8% (70.1%, 97.8%) at 1 year and 58.4% (42.0%, 81.4%) at 3 years as shown in Figure 1. Patients treated with BCT had statistically better overall survival than those with mastectomy (p = 0.029 by log rank test) as shown in Figure 2. This is partly attributed to lower tumor size (pT) in patients who underwent BCT (median tumor size is 1.5 cm) than those who had mastectomy (median tumor side is 4.8 cm) [p < 0.0001]. Although not statistically significant (p = 0.828), mastectomy patients more frequently had high grade tumors (81%) compared with BCT patients (69%). Finally, survival of these patients was not associated with age, tumor size, grade, ALN status, hormone receptor and HER2 status, and type of treatment (all p > 0.06).

The results of the immunohistochemical analyses are as shown in Tables 3 & 4 and Figures 3 & 4. Snail was a sensitive (100%) marker for identifying MBC. However it was not a specific (3.8%) marker, as it was seen in other spindle cell lesions, including myofibroblastoma (4/4; 100%); phyllodes tumor (14/14; 100%) and PASH (7/8; 87.5%).

EGFR was also a sensitive marker (100%) but had a low specificity (19.2%), whereas p63 was a very specific marker (100%) for MBC but had a lower sensitivity (67.6%) compared to both EGFR and Snail. OSCAR keratin and WS-KER were both comparable in their sensitivity (85.3% and 76.5% respectively) and specificity (92.3% and 100% respectively) for detecting MBC. When several of those immunomarkers (OSCAR, p63 and WS-KER) were combined, both the sensitivity (82.4 to 88.2%) and specificity (92.3 to 100%) were increased, with the combination of p63 and WS-KER being the best two antibodies for detecting MBC in terms of high specificity (100%).

Snail expression (percentage of tumor cells staining) did not correlate with age, tumor size, grade, type of surgery (mastectomy vs. BCT), ALN status, hormone receptor and HER2 status, and type of treatment (hormonal therapy vs. chemotherapy vs. radiotherapy) (all p > 0.10). Furthermore, it did not correlate with survival (Hazard Ratio = 1.02; 95% CI: 0.98, 1.06; p = 0.45).