Conclusions • This is the first study to demonstrate that quantitative measurement of pCDC2 at multiple time points is possible in specimens from patients receiving DNA-damaging cytotoxic therapy for solid tumors. • The primary tissue for pCDC2 analysis from skin punch biopsies should be epidermis as nearly all biopsies contain evaluable epidermis. • The preferred IHC parameter for analysis is the percentage of the total number of CDC2 positive cells that are positive for pCDC2 by manual

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... tion (Parameter 4). • pCDC2 levels were significantly increased from baseline to 24 hours post-chemotherapy and were significantly higher at 48 hours when compared to 24 hours, suggesting pCDC2 induction continues to increase between 24 and 48 hours post-chemotherapy. Similar results were observed in the gemicitabine-treated subgroup. • The data suggest pCDC2 to be a fit-for-purpose biomarker for assessing Wee1 target engagement when used in combination of DNA damaging agents. Conclusion: To our knowledge, this is the first clinical study to quantitatively evaluate pCDC2 with multiplex IHC methods at multiple time points in patients with solid tumors receiving DNA-damaging chemotherapy. The methods employed allow quantitative measurement of pCDC2 and have identified the primary analysis parameter and tissue type for future studies. The results suggest that pCDC2 in skin punch biopsies is a fitfor-purpose biomarker for assessing pCDC2 inhibition in early clinical studies of Wee1 kinase inhibitors.