RTA-408 br Conclusion br Acknowledgments This research was f

Conclusion
Acknowledgments This research was funded by Tianjin Health Institution Key Projects, grant number 15KG148” and “Tianjin Medical University General Hospital Youth Incubation Fund, grant number ZYYFY2017029”. I would like to express my heartfelt gratitude to Professor Hongtao Zhang for his generous gifting of the recombinant pUC57 vector encoding HER2-ScFv.
Introduction Amplification of HER2 gene and its receptors’ overexpression are recorded in 20–30% of BC and are related to unfavorable prognosis. HER2 overexpressing BC is potentially responsive to monoclonal RTA-408 (mAb) and small molecule tyrosine kinase inhibitors (TKI) that target different parts of the receptor [1], [2], [3]. Trastuzumab, which is a recombinant humanized mAb, has a high binding affinity to the extracellular domain (ECD) of HER2 [4]. Trastuzumab, when combined with chemotherapy, improves survival outcome in HER2-positive advanced [5] as well as early BC patients [6], [7]. Primary trastuzumab resistance may develop, and about 15% of patients treated with adjuvant trastuzumab-based chemotherapy regimens will develop distant failure [8], [9]. Trastuzumab resistance may be secondary to the expression of truncated forms of HER2 such as p95-HER2 that lacks the ECD [10] resulting in loss of trastuzumab binding target and consequently loss of its effects [11]. Carboxy-terminal fragments (CTF) sized between 90 and 115 kDa are expressed in about thirty percent of HER2-positive BC [12]. These truncated CTF are produced by metalloproteases proteolytic shedding of the HER2 receptor ECD [13] or HER2 mRNA alternative translation [14]. In HER2-positive BC, the p95-HER2 expression is usually associated with higher rate of lymph node metastases and decreased DFS compared with patients overexpressing full-length HER2 [10], [11], [12], [15]. Studies demonstrated that truncated p95-HER2 expressing cells retain their kinase activity and are responsive to lapatinib therapy [3], [16], which has a dual HER2 and HER1 TKI [17]. Geyer et al. [2] reported that combined lapatinib and capecitabine therapy for HER2-positive BC patients who previously progressed on trastuzumab-containing regimens had significantly improved time to progression, compared with capecitabine alone. Moreover, administration of lapatinib as first-line treatment for locally advanced or metastatic HER2-positive BC patients either combined with paclitaxel or as monotherapy resulted in 24% overall response rate [18].
Patients and methods
Patients’ samples
Treatment All patients were treated with trastuzumab-based CT protocol as an adjuvant therapy that consisted of doxorubicin (60 mg/m2 i.v.) plus cyclophosphamide (600 mg/m2 i.v.) on day 1, cycle repeated every 3 weeks for 4 cycles. This was followed by trastuzumab (8 mg/kg i.v.) on day 1 plus paclitaxel (80 mg/m2 i.v.) once weekly for 12 weeks with trastuzumab (6 mg/kg i.v.) every 3 weeks to complete one year.
HER2 analysis For IHC, the HER2 antibody (Clone CB11, Leica Biosystems, Newcastle-upon-Tyne, UK, 1: 80 dilutions) was utilized. Staining was scored as follow: No staining is observed, or membrane staining is observed in <10% of the tumor cells (0; negative); a faint/barely perceptible membrane staining is detected in >10% of tumor cells or the cells exhibit incomplete membrane staining (1+; negative); weak to moderate complete membrane staining observed in >10% of tumor cells (2+); strong complete membrane staining is observed in malignant cells (3+; positive). The cases that scored 2+ were further evaluated by fluorescence in situ hybridization (FISH) using the PathVysion HER-2 probe kit (Vysis, USA) where specimens with a signal ratio >2.2 were considered positive (Fig. 1a and b).
Immunohistochemical staining of p95-HER2 According to the intensity of membrane staining and its extension, p95-HER2 expression was classified from 0 to +3 as classic Her2 neu. p95-HER2 expression (+2 and +3) was considered if the samples showing moderate or intense complete membrane IHC staining in >10% of tumor cells [10]. Examples of p95-HER2 staining are shown in Fig. 2(a–d).