br Statistical analysis The mean standard deviation median
Statistical analysis The mean, standard deviation, median, range, frequency, and percentage were used for data description. The chi-square (x2) test was used for analyses the differences in categorical variables in the 2-way table. Overall survival (OS) was defined as the time interval between the date of diagnosis and the date of death or last follow-up. The time to metastases was defined as the time interval between the curative surgery and the date of first distant metastases. Mann-Whitney test was used to compare time to metastases in relation to different prognostic factors. Kaplan-Meier method with log-rank test was used for estimation and comparing the survival rate. All analyses were performed using SPSS software, version 21.0 and p-value <0.05 considered statistically significant.
Discussion Many studies reported an absence of response to trastuzumab treatment in metastatic HER2-positive BC overexpressing p95-HER2 with loss of ECD , , . In the present study, we aimed to assess p95-HER2 Ponceau S Staining Solution australia and its prognostic effects in 32 HER2-positive patients who developed metastatic disease after trastuzumab-based adjuvant treatment. Immunohistochemistry analysis was utilized in this study as this method is the most available, simple and fast method. Out of our 32 patients, 11 (34.4%) patients were p95-HER2 positive. Comparably, Kocar et al.  used IHC analysis and found that 36.8% of metastatic patients were p95-HER2 positive. Many studies reported that detection of p95-HER2 in human BC can be accomplished only by western blot technique , , . However, it has several limitations including; a big amount of fresh-frozen tissue sample is needed and more time to consume with increasing the cost . The rate of positive expression of p95-HER2 reported in most of the studies that used western blot technique was nearly 30% , , . Also, immunofluorescence and immunoprecipitation techniques are impractical to be utilized for routine specimens due to their high costs . To overcome this limitation Scaltriti et al.  designed immunofluorescence method and recorded 100% and 94% positive and negative predictive values respectively. On comparing our results with reports in the literature that utilized technical methods other than IHC; Sáez et al.  using western blot analysis found that approximately 25% of the HER2-overexpressed BC patients express p185-HER-2 and p95-HER-2 receptors. Kallergi et al.  examined the expression of truncated HER2 on circulating tumor cells of BC patients using a triple-staining immunofluorescent method and recorded 11.1% and 39.1% p95-HER2-positive rate for early and metastatic patients respectively. Christianson et al.  recorded 22.4% of BC tissues expressing p95-HER2 with using western blotting and immunoprecipitations technique. Duchnowska et al.  using quantitative assessment of p95-HER2 expression found that 33% of metastatic BC patients treated with trastuzumab were p95-HER2 positive. Molina et al.  reported 26.7% and 45.7% p95-HER2 positivity rates in the primary HER2-positive BC and lymph nodes respectively with utilizing western blot analysis. Scaltriti et al.  using a paraffin-based immunofluorescence assay found that p95-HER2 was positive in 26% of the HER2-positive BC patients previously treated with lapatinib and capecitabine. Ozkavruk et al.  investigated real-time polymerase chain reaction (RT-PCR) to explore HER2 and p95-HER2 expression in addition to IHC and chromogenic in situ hybridization (CISH) to detect HER2 protein expression and HER2 gene amplification respectively. Out of 80 patients, 48.7% were HER2-positive and received trastuzumab treatment. Of those patients, 28.2% were trastuzumab-resistant. The mean ratio of p95-HER2 was 11.01 ± 19.73 versus 1.99 ± 1.37 in trastuzumab-resistant patients versus patients without trastuzumab resistance respectively. Although the high p95-HER2 ratio was associated with trastuzumab resistance (p = 0.210), p95-HER2 was not an independent factor of trastuzumab resistance.