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Histone deacetylase (HDAC) inhibition is a novel entity in medical oncology, and several HDAC inhibitors are in clinical trials. One of them is the hydroxamic acid belinostat (PXD101) that has demonstrated therapeutic efficacy for several clinical indications. Acetylation of histones is a key event after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude mice carrying A2780 human ovarian cancer xenografts. Acetylated H4 was monitored in vivo by immunohistochemistry during treatment with belinostat, and compared with pharmacokinetics in plasma and tumor tissue. We found an increased level of acetylated H4 15 min after a single treatment (200 mg/kg i.v.) with maximum level reached after 1 h. H4 acetylation intensity reflected the belinostat concentration in plasma and tumor tissue. The threshold level for belinostat activity, indicated by acetylated H4, correlated with belinostat plasma concentrations above 1,000 ng/ml. In conclusion, examination of H4 acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors.

Background: The expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) in breast carcinomas is a strong predictor of the efficacy of hormonal therapy for breast cancer patients as well as providing a degree of prognostic information. Anti-ERα (clone EP1) and anti-PR (clone PgR 1294) configured as FLEX ready-to-use antibodies have been tested on the Dako Omnis automated staining platform. These products are in performance evaluation and are not commercially available. A series of concordance studies were performed to evaluate the performance characteristics of these monoclonal antibodies on breast cancer tissue specimens: anti-ERα clone EP1/Dako Omnis was compared to (a) anti-ERα clone EP1/Autostainer Link 48 (238 specimens) and to (b) anti-ERα clone SP1/Autostainer (116 specimens), and anti-PR clone PgR 1294/Dako Omnis was compared to (a) anti-PR clone PgR 636/Autostainer Link 48 (289 specimens) and to (b) anti-PR clone 16 (Leica Biosystems, Newcastle, UK) (144 specimens). In addition, the specificity of the ER and PR antibodies for Dako Omnis was evaluated on a set of normal tissue specimens. Methods: Formalin-fixed, paraffin-embedded (FFPE) human breast carcinoma specimens and normal tissues were obtained from commercial providers or local hospitals. The specimens had no associated personal information and were not traceable back to the tissue donors. Tissue pretreatment and immunohistochemical staining were performed using the recommended protocol for each antibody and staining platform. The stained slides were evaluated for nuclear ER or PR expression according to ASCO/CAP guidelines (≥1% cut-off for positive) by pathologists who were blinded from the staining method and specimen ID. The concordance studies included breast cancer specimens covering the clinical range of ER or PR expression with approximately half the specimens in the negative (<1%) category, and at least 10% of the specimens in the weakly positive (≥1 ≤10%) category in each study. Two-sided Wilson Score 95% Confidence Intervals were calculated using JMP software (SAS Institute, USA). For the analytical specificity studies the presence or absence of specific staining in the various normal tissue types was recorded. Results: High concordance rates were observed with both anti-ERα clone EP1/Dako Omnis and anti-PR clone PgR 1294/Dako Omnis compared to the other ER/PR antibodies, with overall agreement rates exceeding 95% in all of the comparative studies. On a set of normal tissues, specific positive nuclear staining was observed only in tissue types known to express ERα or PR. Conclusions: Monoclonal antibodies anti-ERα clone EP1 and anti-PR clone PgR 1294 configured as FLEX ready-to-use on Dako Omnis are sensitive and specific assays for detecting estrogen receptor and progesterone receptor in FFPE tissues. In comparison testing for assessment of hormonal receptor status on breast carcinoma specimens, anti-ERα clone EP1/Dako Omnis and anti-PR clone PgR 1294/Dako Omnis were highly concordant with commercially-available ER or PR antibodies. Citation Format: Viale G, Dell'Orto P, Falzon M, Fält A, Hicks D, Hoff K, Jakobsen K, Jensen LB, Levy YY, McMahon L, Miller K, Russo L. Performance evaluation of two ready-to-use antibodies under development for the Dako Omnis automated staining platform on breast carcinoma specimens: Anti-estrogen receptor α clone EP1 and anti-progesterone receptor clone PgR 1294. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-16.