`
`Clinical Research 2: Novel Therapeutics and Biomarkers of Response
`
`Evaluation and development of an
`immunohistochemical procedure to monitor
`phospho-S6 as a biomarker for the activity of
`RAD001.
`
`Volume 65, Issue 9 Supplement, pp. 109-110
`American Association for Cancer Research
`
`
`
`AUTHOR INFORMATION
`
`1. Michael Stumm,
`2. Terence M. O’Reilly,
`3. Sabine Zumstein-Mecker,
`4. Caroline Fux,
`5. Maya Walker, and
`6. Heidi A. Lane
`
`1. Novartis Institutes for Biomedical Research, Basel, Switzerland
`Proc Amer Assoc Cancer Res, Volume 46, 2005
`
`Abstract
`
`469
`RAD001 is an orally active mTOR pathway inhibitor in clinical evaluation. The mTOR kinase
`regulates both S6 kinase (S6K) (a translational activator) and 4E-BP1 (a translational inhibitor), so
`that mTOR inhibition will disrupt the regulation of protein synthesis. In experimental and early clinical
`studies, RAD001 has demonstrated the inhibition of S6K-1 inhibition occurs in peripheral blood
`lymphocytes (PBL) and in various tissues, including tumors, and is well-aligned with the
`pharmacokinetics of RAD001. This suggests that S6K activity is a valuable marker for the activity of
`RAD001. As immunohistochemical (IHC) assays are broadly applicable, we sought to prepare a
`phospho-S6 protein (pS6) IHC assay for clinical use. A549 cell pellets or cytospin preparations from
`cells, with or without RAD001 exposure, demonstrated “on-off” staining with anti-pS6 antibodies
`which recognize distinct phosphorylated-epitopes (Ser 235/236 or Ser 240/246), reflecting the
`activated S6K pathway. Specificity of staining was confirmed by immunoblotting. CA20948 tumors
`from rats receiving an effective RAD001 treatment demonstrated the absence of pS6 staining as
`compared with vehicle-treatment (both Ser 235/236 or Ser 240/246). In addition, skin samples from
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`these rats showed marked reduction in pS6 staining. Phosphospecificity of the Ser 240/246 mAb
`was demonstrated by calf intestinal phosphatase (CIP) pretreatment of tissue sections yielding no
`staining. Furthermore storage of tissue sections at room temperature for 2 months markedly reduced
`immunoreactivity. IHC performed with A549 cells and patient-derived tumor material with different
`lots of Ser 240/246 indicated lot-specific intensity of immunoreactivity. Tissue microarrays of rat and
`mouse organs demonstrated pS6 detected by Ser 240/246 in various organs, notably the hair
`follicles where a response to RAD001 was clearly evident by IHC. Lastly a series of human breast
`and colon cancer samples showed the utility of IHC in detecting pS6. Hence, the development of the
`IHC method for following S6 phosphorylation changes in tumors and surrogate tissues provides a
`useful marker of RAD001 activity during clinical studies.
`
`• American Association for Cancer Research
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