Abstract B6: Advanced image analysis of H3K27 trimethylation in skin from subjects dosed with the EZH2 inhibitor tazemetostat
Authors: McDonald Alice A. , Kuznetsov Galina , Kumar Namita , Weaver Amy , Do Trevor , Dzubay Jeff , Keilhack Heike , waters Nigel , Rioux Nathalie , Blakemore Stephen J. 
Published: May 2016

Tazemetostat (EPZ-6438 or E7438) is a selective small molecule inhibitor of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase (HMT). EZH2 is the catalytic subunit of the multi-protein HMT complex known as polycomb repressive complex 2 (PRC2), which is responsible for mono-, di-, and trimethylation (me3) of histone H3 lysine 27 (H3K27). An exploratory objective for the phase 1 study of tazemetostat in subjects with advanced solid tumors or B-cell lymphomas was to explore the pharmacokinetic: pharmacodynamic (PK: PD) relationships such as correlation of H3K27me3 inhibition in patient tissue with systemic drug exposure.

Pre-clinical xenograft models of EZH2 inhibition have previously shown tumor regression correlating with dose-dependent H3K27me3 decreases in both tumor tissue and skin epidermis (Knutson 2014). H3K27me3 reduction in the epidermis was calculated as a change in percentage of H3K27me3 positive cell employing an immunohistochemistry (IHC) assay with subsequent image analysis. These studies and the low feasibility of obtaining matched pre- and post-treatment tumor biopsies, led to the selection of skin as a surrogate tissue for evaluation in the phase 1 study at screening and 28 days post dose and the use of IHC as a PD biomarker of target inhibition.

Examination of phase 1 skin biopsies exhibited dose and exposure-dependent decreases in the percentage of H3K27me3 positive cells, however, the magnitude of reduction (60%) was less than observed in pre-clinical models (80%). Further investigation of the IHC images revealed a difference of H3K27me3 levels in distinct layers or strata of the skin. The stratum spinosum exhibited a distinct dose and exposure-dependent loss of H3K27me3 staining while the stratum basale showed a concomitant minimal loss of H3K27me3. These observations gave rise to the possibility of a stratum-specific PD response in skin to tazemetostat exposure. To fully evaluate this notion, a more sophisticated, unbiased, image analysis algorithm was employed to measure H3K27me3 levels across the strata of the epidermis.

The image analysis algorithm was designed to identify three distinct regions: the full thickness epidermis, stratum basale to stratum corneum; the stratum basale alone; and the stratum spinosum. Iterative rounds of optimization were performed to identify the basale layer while excluding follicles and glands. Because skin has inherent challenges related to the chromogenic similarity between DAB and melanin, careful attention was paid to the negative control to ensure that endogenous melanin was not included in the analysis.

Segregation of the stratum basale layer from the remainder of the epidermis led to a refined H3K27me3 analysis and resulted in an improved correlation of H3K27me3 inhibition with drug exposure. This analysis highlights that the zonal layers of the epidermis may require separate evaluation when analyzing skin as a surrogate tissue for proof of mechanism studies. Although more accessible for repeat biopsy, the use of skin as a surrogate tissue does not preclude the need to understand the PD effects in the target tumor tissue. To that end, optional paired tumor biopsies are being collected in the Phase 2 tazemetostat trial and will be evaluated for PD endpoints including H3K27me3 in addition to other biomarkers.

Citation Format: Alice A. McDonald, Galina Kuznetsov, Namita Kumar, Amy Weaver, Trevor Do, Jeff Dzubay, Heike Keilhack, Nigel waters, Nathalie Rioux, Stephen J. Blakemore. Advanced image analysis of H3K27 trimethylation in skin from subjects dosed with the EZH2 inhibitor tazemetostat. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B6.