Subgroup analysis showed that in the previous trastuzumab subgroup, the median PFS of the pyrotinib group was significantly better than that of the lapatinib group (18.1 months vs 7.1 months, em p /em ?=?0.0031). ongoing clinical trials, findings of which may soon be available. Immunohistochemical, fluorescence in situ hybridization, chromogenic in situ hybridization, IKK-16 silver in situ hybridization, circulating tumor cells Immunohistochemistry IHC staining is the most commonly used slide-based techniques for initial testing of HER2 status in newly diagnosed breast malignancy patients. However unlike the conventional IHC assays, it is a quantitative evaluation as HER2 protein is expressed in breast epithelial cells.28 At present, U.S. Food and Drug Administration (U.S. FDA) has authorized two kits, Dako Hercep Test? (Dako Corporation, Glostrup, Denmark) and Ventana Pathway? (Ventana Medical Systems, Tucson, AZ) for making a strategic decision in determining whether the patients should undertake anti-HER2 therapy.37 IHC assays have been considered as the primary determining test for HER2 status and nearly 80% of initially diagnosed breast cancers patients in US had undertaken it.38,39 It was essential to establish a standardized IHC procedure and scoring system to provide BGN a meaningful interpretation of a HER2 immunostaining.40 Standardized IHC assay has the following advantages41: common pathologic routine, easy slide staining techniques, wide availability, and relatively IKK-16 IKK-16 low cost; while the limitations are variation of system-control standards for storage, duration, fixation, and the difficulties of a semiquantitative and subjective slide-scoring system-based application in clinical practice.42,43 Studies have proved that if microscopic process, embedding, tissue process, and storage procedure are carefully performed, appropriate correlation between protein expression status and gene-copy levels can be achieved.44 Thus, in clinical settings, errors in HER2 testing by IHC technique arises from both, difference in correlation of antigen restoration and selection of staining reagents, and variation in pathologic slide scoring. In the United Kingdom, it has been recommended that these assessments are restricted to laboratory that performs annual minimum of 250 IHC inspections (and/or 100 FISH assessments).37,45 National Surgical Adjuvant Breast and Bowel Project (NSABP) confirmed that centers undertaking high volume of HER2 testing resulted in a higher concordance between IHC and FISH outcomes.30 Despite the scoring system, several additional pitfalls in IHC interpretation must be expected. In order to eliminate false-positive results, pathologists must try to carefully avoid tissue injury in preparation, specimen edges scoring, cytoplasmic staining, fibrocystic metaplasia status, and intraductal (ductal carcinoma in situ) foci disease.46,47 Quantitative image analysis system can reduce the laboratory variability of slide scores among pathologists, which is important in IKK-16 routine microscopy.48 Fluorescence in situ hybridization The FISH technique done by IKK-16 using fluorescent-labeled probes is a morphology-driven slide-based DNA hybridization assay, to detect the HER2 gene amplification.49 It can utilize a chromosome-17 probe (CEP17) as an internal control.50 Presently, three versions of FDA-recommended FISH assessments are as follows: Ventana Inform? test (Ventana Medical Systems, Tucson, AZ), a single-probe technique that detects single HER2 gene, and the dual-probe (HER2 probe plus chromosome-17 centromere probe) kits, PathVysion? (Abbott Laboratories, Abbott Park, IL) and PHarmDX (Dako, Glostrup, Denmark).45 Previous studies proved that single-probe approach is highly correlated with dual-probe test for detection results of HER2 gene status in breast cancer, suggesting that this clinical diagnostic value of the two techniques is similar,51,52 and the simultaneous detection of HER2 and chromosome-17 could clarify the HER2 gene status.40,53 From a societal perspective, FISH is an affordable objective scoring method,54 with the advantages of two HER2 gene signals, expressed both in benign and malignant cells.55 However, the limitations of FISH technique include the higher quality for slide scoring, use of fluorescent microscope, higher test cost, and more time consuming than IHC.53 Although still debatable, several experts strongly recommend FISH over IHC in defining the HER2 status for breast malignancy, as it is more common and accurate.44 Generally, most of HER2 testing (80C85%) is done by IHC, and results is defined as 0 and 1+: negative, 2+: uncertain and require further FISH assay for confirmation, and 3+: positive.45,47 False negative FISH results are unusual, but may occur when the pathologist fails to identify the amplified areas of HER2 gene with heterogeneity.51,52 Thus, diligence and caution are required when scanning the case at low magnification analysis. Since the guidelines of HER2 testing from American Society of Clinical oncology (ASCO)-CAP were published,56 we generally considered value of 2. 0 ratio for a positive FISH cutoff instead of 2.2, which resulted by the prior expert recommended. CISH and silver in situ hybridization (SISH) The CISH approach and SISH method capture the advantages of both IHC and FISH.53 It detects HER2 gene-copy number by using a single HER2 probe. The CISH was approved by the FDA to evaluate feasibility for anti-HER2 agent.36 In addition, CISH has the lowest correlation.