In the spring of the first year of the 18th century Fellows of the Royal College of Physicians of London may have been surprised to receive a card desiring then ‘to accompany the corpse of Mr John Dryden from the College of Physicians in Warwick Lane to Westminster Abbey on Monday the 13th of the Instant May in 1700’. Dryden (F. R. S. 1663) had died in somewhat straitened circumstances in Soho on 1 May and his family had arranged for a simple burial the next day at St Anne’s Church. Several persons of quality, hearing of this, decided that Dryden, the great poet and dramatist of the Restoration era and forerunner of the Augustan age, deserved a more imposing funeral, and a subscription was raised for a more appropriate public ceremony with subsequent interment at Westminster Abbey.

We have developed a method to assess target expression changes following exposure of plucked hairs to chemotherapeutic agents and other potential therapeutics. Successful ex vivo responses provide proof of concept data prior to clinical studies. Plucked hair is a valuable surrogate biomarker tissue to monitor pharmacodynamic (PD) responses in a clinical trial. The collection of hairs is minimally invasive, simple and is amenable to frequent sampling. Since hairs are epithelial appendages, many signaling pathways active in other epithelial tissues, including cancers, are also present in hairs. Highly proliferative plucked human scalp hair can be utilized as a surrogate tissue to measure proliferation, phosphorylation and DNA damage responses after treatment. Furthermore, hairs are highly vascularized, suggesting that administered Test Articles may be delivered efficiently to the hair in a similar manner to other highly vascularized tissues, including many tumor types. Human plucked hair (either from the scalp or beard) was placed in maintenance media in the presence of conventional or targeted chemotherapeutic agents, known for their different mechanisms of action (e.g. Gemcitabine, Carboplatin, Tarceva), for a range of time points. Hairs were then fixed post-exposure and longitudinal sections immunohistochemically labelled for various markers including p-ERK1/2, p-AKT, p-Chk1, g-H2AX, Ki67 and androgen receptor. Quantitative image analysis to measure the level of labelling was performed using an Aperio ScanScope. The relative number of positively labelled cell nuclei, or relative tissue area (depending on the labelling pattern of the target), of the hair outer root sheath labelled was analyzed, along with label intensity. In subsequent trial patients, formaldehyde fixed plucked hairs were returned to the lab, similarly sectioned, labelled and quantified. In addition, where antibody labelling is not possible or more detailed information on the modulation of a pathway may be useful, drug treated plucked hairs can be subjected to Next Generation Sequencing (NGS) gene expression analysis. The expected changes in labelling were observed. For example, Gemcitabine, which is linked to DNA polymerase inhibition, increased p-Chk1 labelling 4-fold after 4-8 hours and strongly induced p53 by 24 hours. Tarceva, an inhibitor of the EGF pathway, decreased levels of both p-ERK1/2 and p-AKT 3-fold after only 10 minutes. The alkylating agent carboplatin increased g-H2AX labelling up to 12-fold after 24 hours. We have demonstrated that plucked hair is a valuable PD biomarker tissue for various chemotherapy agents. Proof of concept studies performed ex vivo can inform the design of clinical validation studies by indicating optimum markers and timepoints. Further, each hair is an independent unit thereby allowing independent replicate tissues (hairs) to be sampled. Citation Format: Greg Tudor, Frida Ponthan, Adam Boanas, Aude-Marine Bonavita, Catherine Booth. Pharmacodynamic biomarkers: Evaluation of oncology drug target engagement in human plucked scalp and beard hair. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4350.

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The early identification of GI toxicity caused by novel therapeutics is crucial for drug development pipelines. GI toxicity is one of the most frequent adverse effects amongst chemotherapeutics. Impaired barrier function and epithelial ulceration can lead to symptoms including diarrhoea, dehydration and susceptibility to systemic infection. We have identified a gene signature that can be utilised in an in vitro organoid model to predict relevant GI toxicity. Methods: Both mice and small intestinal organoids derived from human and mouse were exposed to chemotherapeutic agents known to cause GI toxicity. For in vivo assays mice were treated with 50 or 100mg/kg CPT11 for 4 or 8 hours or 100, 200 or 400mg/kg Iressa for 8 or 24 hours before small intestinal (SI) tissue was removed for RNA-Seq analysis. SI tissue was also analysed by histological staining and damage measured to confirm induction of GI-toxicity. Human and mouse organoids were treated with CPT11 and Iressa for 8 hours at a range of concentrations. The organoids were lysed, RNA extracted and an mRNA-Seq library was generated. Samples were sequenced using 2 × 75bp PE reads on a Next-Seq550. Sequences were aligned to the relevant genomes using BWA and normalised using DESeq2. Normalised gene counts were analysed using Partek Genomics Suite and Ingenuity Pathway Analysis (IPA) was used for gene set enrichment (GSEA) and pathway analysis of differentially expressed genes. Results: For all treatments and time points the mechanism of action (MOA) of each agent was evident from the GSEA and pathway analysis. CPT11 induced DNA damage and cell cycle arrest pathways, whereas Iressa demonstrated downregulation of cell cycle and chromosomal replication and mitotic genes. Analysis of toxicity pathways in IPA identified a number of differentially expressed genes that were consistent with increased toxicity. A gene signature was identified and a multivariate scoring system used to demonstrate signature engagement. This signature was activated in all SI tissues treated with CPT11 or Iressa, irrespective of the MOA of both agents. Conclusions: Deployment of this signature on mouse and human organoids treated with chemotherapeutic agents demonstrated activation of the signature, which could be an alternative method for GI toxicity testing. Further analysis of this signature in gene expression studies from GI toxicity inducing agents submitted to the NCBI Gene expression omnibus (GEO) demonstrated that the signature was activated with a variety of agents with different MOA’s such as NSAIDs, gamma-secretase inhibitors, CDK8/19 inhibitors and CAR-T-cells. Citation Format: Gary S. Beale, Aude Marine-Bonavita, Valentina Ubertini, Greg Tudor, Francesca Philips, Catherine Booth. Identification of a gastrointestinal (GI) toxicity gene signature that predicts GI-toxicity induction in small intestinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 153.