Protein molecular profiling of archival and frozen fine needle aspirate material in human breast cancer before and after therapy

Proc Amer Assoc Cancer Res, Volume 46, 2005 2175 Reverse phase protein microarrays (RPPA) have been applied to tissue sections however; herein we report the novel use of this technology for the analysis of both archival cytology aspirate smears as well as frozen fine need aspirate material obtained from patients with breast carcinoma both before and after treatment. This represents the innovative use of a readily obtainable cellular tissue sample for monitoring kinase driven cell signaling events. The ability to monitor the in vivo state of cell signaling proteins before and after treatment will potentially augment the ability to design individualized therapy regimens and map aberrant phenotypes for diagnoses. Reverse phase protein microarrays were conducted on a set of thirty samples from 10 patients: archival smears and frozen aspirate material before and after receiving treatment with docetaxel and capecitibine followed by doxorubicin and cyclophophosphamide (NCI clinical trial 00C0149). A set of specific, validated antibodies to phosphorylated or cleaved proteins in the c-erbB2 and c-erbB1 signal pathways used to immunostain the RPPA’s included: ErbB2, EGFR Tyr1148, AKT ser473, Bcl2 ser70, p42/44 MAPK, Cleaved Caspases 3 and 9, and STAT3 Tyr705 - all measured concomitantly from a few thousand cells. The immunostaining method utilizes a catalysed signal amplification methodology that has sensitivity in the femtomolar (10-15) range, and a Coefficient of Variation of 10%. The quantity and quality of protein obtained from the frozen fine needle aspirates was superior as compared to the fixed, archival smears. However, there was significant protein available in the archival smears for RPPA analysis. Most notably there were detectable differences in the EGFR mitogenesis pathway specifically EGFR Tyr1148, p42/44 MAPK and STAT3 Tyr705 before and after chemotherapy. Assessing the state of phosphorylation of proteins involved in oncogenic signaling pathways with material obtained from fine needle aspirates could facilitate the future of molecular medicine and the individualized treatment of cancer.