Herceptest kit

Manufactured by Agilent Technologies

Sourced in Denmark, United States

The HercepTest kit is a qualitative immunohistochemical assay developed by Agilent Technologies for the detection of the human epidermal growth factor receptor 2 (HER2) protein in formalin-fixed, paraffin-embedded breast cancer tissue specimens. The kit provides reagents and instructions for the detection of HER2 protein expression in breast cancer tissue samples.

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26 protocols using herceptest kit

Metastatic Gastric Cancer HER2-Targeted Therapy

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Patients with metastatic GC were enrolled in Yonsei Cancer Center, Severance Hospital, Seoul, Korea between December 2005 and August 2015. Patients were followed up until December 2016. Patient eligibility criteria were as follows: 1) metastatic GC patients with HER2 positivity defined by either HER2 3+ in immunohistochemical (IHC) staining, or HER2 2+ in IHC staining and HER2 amplification on fluorescence in situ hybridization (FISH) (HER2:CEP17 ratio ≥2); 2) chemotherapy-naive patients with the exception of adjuvant chemotherapy 6 months prior to enrollment; 3) patients who have been treated with palliative trastuzumab in combination with either 5-fluorouracil and cisplatin (FP) or capecitabine and cisplatin (XP). Clinicopathologic parameters were reviewed from the information of the electronic medical record system as previously described [25 (link)].
HER2 status in surgical or biopsy specimen were analyzed and determined by experienced pathologists in Severance Hospital, Seoul, Korea, using the HercepTest Kit™ (DAKO, Denmark) for IHC staining and Vysis™ HER2/CEP FISH probe kit (Abbott, USA) for FISH analysis according to the manufacturers’ instructions. This study was approved by the Institutional Review Board in Severance Hospital, Seoul, Korea (IRB approval number: 4-2014-1076).

Kim C., Lee C.K., Chon H.J., Kim J.H., Park H.S., Heo S.J., Kim H.J., Kim T.S., Kwon W.S., Chung H.C, & Rha S.Y. (2017). PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer. Oncotarget, 8(69), 113494-113501.

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Breast Cancer Biomarker Assessment

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Samples were centrally stained for ER, PR (ER/PR pharmDx™ Kit, Dako), HER2 (HercepTest™ Kit, Dako) and Ki67 (clone MIB-1, dilution 1:100, DAKO). ER and PR were scored according to the Allred scoring system and considered positive when the Allred score was 3 or above. HER2 positivity was defined as 3+ if more than 10% of cells displayed a strong complete membrane staining, according to the ASCO-CAP guidelines 2013.
All samples were stained for AP2γ (TFAP2C) by immunohistochemistry. Briefly, antigen retrieval was performed by heating in Target Retrieval Solution Citrate pH 6.0 (DAKO) using a pressure cooker for 10 min. Slides were incubated with the AP2γ primary antibody (clone 6E4/4, SantaCruz) at a dilution of 1:300 for 30 min at room temperature. The DAKO REAL™ Detection System Peroxidase/DAB+ was used with an automated protocol. Tumors were scored according to the Allred score (0–8). Tumors with scores 0–2 were defined as AP-2γ negative, while tumors with scores 3–8 were defined as AP-2γ positive. Whenever present, myo-epithelial cells served as positive internal control.

Jeselsohn R., Barry W.T., Migliaccio I., Biagioni C., Zhao J., De Tribolet-Hardy J., Guarducci C., Bonechi M., Laing N., Winer E.P., Brown M., Di Leo A, & Malorni L. (2016). TransCONFIRM: Identification of a genetic signature of response to fulvestrant in advanced hormone receptor positive breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(23), 5755-5764.

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Breast Cancer Immunohistochemistry Profiling

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All cases were reviewed by two breast pathologists (DH and LY) to assess tumor grade (using the Nottingham histological three-tier grading system), tumor size, nodal status, ER, PgR, HER2, and Ki67 expression. The expression of ERα (clone EP1; Dako, Glostrup, Denmark, prediluted), PgR (clone PgR1294; Dako, prediluted), and Ki67 (clone MIB1; Dako, prediluted) were determined by immunohistochemistry (IHC) during routine pathologic examination. ER and PgR status was determined based on the percentage of positive nuclei in the invasive neoplastic compartment of the tissue. Tumors were classified as ER- or PgR-positive when ≥1% invasive tumor cells showed definite nuclear staining, regardless of staining intensity. Ki67 was evaluated as the percentage of positively stained nuclear cancer cells (regardless of staining intensity). HER2 expression was evaluated with the HercepTest kit (Dako) and scored as 0, 1+, 2+, or 3+, according to the ASCO-CAP guidelines. Tumors scored as 2+ were re-tested with FISH using the HER2 IQFISH PharmDx kit (Dako).

Peláez-García A., Yébenes L., Berjón A., Angulo A., Zamora P., Sánchez-Méndez J.I., Espinosa E., Redondo A., Heredia-Soto V., Mendiola M., Feliú J, & Hardisson D. (2017). Comparison of risk classification between EndoPredict and MammaPrint in ER-positive/HER2-negative primary invasive breast cancer. PLoS ONE, 12(9), e0183452.

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Immunohistochemical Analysis of FGFR1 in Breast Cancer

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IHC was used to detect the FGFR1 protein expression of 184 breast cancer tissues. IHC staining was performed using a Benchmark XT auto-stainer (Ventana Medical Systems Inc., Tucson, USA), and the FGFR1 antibody (ab10646, Abcam, UK) was diluted to 1:2000. Protein expression of FGFR1 was quantified as a percentage (range 0-100%) of positive cells present among all tumor cells presented. A cut-off value of 10% was selected for FGFR1 protein expression, where above 10% was defined as “high expression” and below as “low expression”.^{20 (link)} IHC staining for ER and PR was considered positive if ≥ 1% of tumor cell nuclei were stained.^{21 (link)} In addition, the expression of ER was scored as 1+, 2+, and 3+ based on its staining intensity.^{22 (link)} Expression of HER2 was evaluated with the HercepTest kit (Dako) and scored as 0, 1+, 2+, and 3+. Scores of 0 and 1+ were determined as negative, and 3+ were determined as positive.^{23 (link)} Each of the sections was scored by 2 observers, who were blinded to patients’ medical information, and disagreement between the observers was solved by discussion.

Lv Q., Guan S., Zhu M., Huang H., Wu J, & Dai X. (2021). FGFR1 Is Associated With Tamoxifen Resistance and Poor Prognosis of ER-Positive Breast Cancers by Suppressing ER Protein Expression. Technology in Cancer Research & Treatment, 20, 15330338211004935.

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Standardized HER2 and ER Testing

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The various procedures used by the Mayo and the IEO laboratories are described in Supplementary Appendix B. In the central laboratories, HER2 IHC was tested using the HercepTest® kit (Dako, Carpinteria, CA), and HER2 FISH was tested using the PathVysion HER2 DNA probe kit/HER2/CEP17 probe mixture (Abbott Molecular, Des Plaines, IL). HER2 positivity was defined according to the 2007 ASCO/CAP guidelines (IHC-positive: 3+ complete membrane staining in > 30% of invasive cells; FISH-positive: HER2/CEP17 ratio > 2.2) [3 (link)].
IEO and Mayo performed IHC for ER each according to their own methods, previously used in large multicenter trials and each recommended by ASCO/CAP ER/PR testing guidelines. IEO used a dual antibody (Dako cocktail of ER 1D5 and 2.123 monoclonal antibodies), while Mayo used a single antibody (Dako ER 1D5 monoclonal antibody). ER status was defined as positive if ≥ 1% cells stained positively vs < 1% for negative.

McCullough A.E., Dell'Orto P., Reinholz M.M., Gelber R.D., Dueck A.C., Russo L., Jenkins R.B., Andrighetto S., Chen B., Jackisch C., Untch M., Perez E.A, & Piccart-Gebhart M.J. (2014). Central Pathology Laboratory Review of HER2 and ER in Early Breast Cancer: An ALTTO Trial [BIG 2-06 / NCCTG N063D (Alliance)] Ring Study. Breast cancer research and treatment, 143(3), 485-492.

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Breast Cancer Receptor Status Analysis

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For the 1998–2008 cohort, tumour receptor status for ER, PR and HER2 were determined by immunohistochemistry of triplicate tumour cores in tissue microarrays, and scored by SSC. Antibodies were as follows; ER: Vector 6F11/2 (1:50), PR: Vector 1A6 (1:40), HER2: Dako HercepTest Kit (pre-diluted). For the 2009–2014 cohort, ER and HER2 status determined according to UK guidelines were obtained from NHS histopathology records.

Daniels S.L., Burghel G.J., Chambers P., Al-Baba S., Connley D.D., Brock I.W., Cramp H.E., Dotsenko O., Wilks O., Wyld L., Cross S.S, & Cox A. (2016). Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and “BRCA-Like” Status, in Both Blood and Tumour DNA. PLoS ONE, 11(7), e0160174.

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Biomarker Analysis of Tumor Tissues

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EGFR protein expression levels, EGFR gene amplification status, K-ras mutations, and HER-2 protein expression levels were measured in tissue specimens from tumors obtained from patients who had provided informed consent for exploratory biomarker analysis. The tumor tissues were centrally tested and classified. EGFR expression was analyzed using an immunohistochemistry (IHC) staining kit (EGFR PharmDX; Dako, Copenhagen, Denmark) and classified into four categories (0, 1+, 2+, and 3+), as previously described [21 (link)]. The EGFR gene copy number was measured by fluorescence in situ hybridization (FISH), as reported previously [22 (link)]. For K-ras mutation analysis, DNA was extracted from formalin-fixed paraffin-embedded tumor samples. The sequences of K-ras codons 12 and 13 and the surrounding region of the gene were analyzed by conventional polymerase chain reaction followed by direct sequencing. The expression status of HER-2 was analyzed using the HercepTest kit (Dako) and classified into four categories (0, 1+, 2+, and 3+).

Satoh T., Lee K.H., Rha S.Y., Sasaki Y., Park S.H., Komatsu Y., Yasui H., Kim T.Y., Yamaguchi K., Fuse N., Yamada Y., Ura T., Kim S.Y., Munakata M., Saitoh S., Nishio K., Morita S., Yamamoto E., Zhang Q., Kim J.M., Kim Y.H, & Sakata Y. (2014). Randomized phase II trial of nimotuzumab plus irinotecan versus irinotecan alone as second-line therapy for patients with advanced gastric cancer. Gastric Cancer, 18(4), 824-832.

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Immunohistochemical Analysis of Breast Cancer Biomarkers

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Immunohistochemical (IHC) analysis was performed
on 4 μm thick paraffin-embedded formalin-
fixed tissue sections. IHC for HER2 was
performed using the HercepTest kit according to
the manufacturer’s protocol (Dako, Denmark).
In brief, sections were deparaffinized and rehydrated
in graded alcohols. The slides were then
incubated with pre-diluted anti-HER2 antibody,
washed in phosphate buffered saline (PBS) and
incubated with horseradish peroxidase-conjugated
secondary antibody. Estrogen receptor (ER) and
progesterone receptor (PR) status was checked
with 1D5 and PGR-1A6 antibodies respectively
(Dako, Glostrup, Denmark). The HER2/neu expression
was scored based on the degree of membrane
staining according to previous guidelines
(13 (link)). Samples with HER2/neu scores of 2 or more
were regarded as positive. Samples with nuclear
staining for ER and/or PR in more than 10% of the
tumor cells were considered as ER and/or PR positive.
P53 status was evaluated with a commercial
antibody designed to detect the N-terminal of P53
(Dako, Denmark).

Kazemi-Oula G., Ghafouri-Fard S., Mobasheri M.B., Geranpayeh L, & Modarressi M.H. (2015). Upregulation of RHOXF2 and ODF4 Expression in Breast Cancer Tissues. Cell Journal (Yakhteh), 17(3), 471-477.

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HER2 IHC Protocol Using FDA and ASCO/CAP Criteria

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HER2 IHC was performed with the HercepTest kit (Dako, Glostrup, Denmark) according to the manufacturer’s protocols. We cut 4-μm-thick sections from the TMA block for HER2 IHC. HER2 expression was separately analyzed using FDA and ASCO/CAP 2013 criteria. Using the FDA criteria, the 2+positive cases exhibited weak-to-moderate complete membrane staining in >10% of tumor cells [18 (link)], whereas the ASCO/CAP 2013 criteria defined the 2+positive cases as circumferential membrane staining that is incomplete and/or weak-to-moderate and present within >10% of the invasive tumor cells, or complete and circumferential membrane staining that is intense and present within ≤10% of the tumor cells [12 (link)]. The HER2 IHC results were subcategorized into low (0, 1+) and high (2+, 3+) expression groups.

Kim G., Chung Y.R., Kim B., Song B, & Moon K.C. (2016). Comparison of the FDA and ASCO/CAP Criteria for HER2 Immunohistochemistry in Upper Urinary Tract Urothelial Carcinoma. Journal of Pathology and Translational Medicine, 50(6), 436-441.

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Immunohistochemical Analysis of Breast Tumor Markers

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Estrogen receptor (ER), progesterone receptor (PR) and HER-2 expression were previously analysed on tumors by immunohistochemistry (IHC) on tissue microarrays (TMAs) [24 (link)]. The TMAs were constructed as previously described (Stefansson et al. 2009). Immunohistochemistry (IHC) was then applied using 4 μm thick TMA sections using the following anti-bodies: anti-ER (1D5; DAKO), anti-PR (PgR 636; DAKO) and anti-HER2 (HercepTest Kit; DAKO). ER and PR were scored positive given any visible nuclear staining in more than 1% of tumor cell nuclei. HER-2 positivity was defined as score of 3+ according to criteria provided by the anti-body manufacturer.

Stefansson O.A., Hermanowicz S., van der Horst J., Hilmarsdottir H., Staszczak Z., Jonasson J.G., Tryggvadottir L., Gudjonsson T, & Sigurdsson S. (2017). CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer. BMC Cancer, 17, 469.

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