Hormone-driven expression of the ERG oncogene after fusion with TMPRSS2 occurs in 30% to 70% of therapy-naive prostate cancers. Its relevance in castration-resistant prostate cancer (CRPC) remains controversial as ERG is not expressed in some TMPRSS2-ERG androgen-independent xenograft models. However, unlike these models, CRPC patients have an increasing prostate-specific antigen, indicating active androgen receptor signaling. Here, we collected blood every month from 89 patients (54 chemotherapy-naive patients and 35 docetaxel-treated patients) treated in phase I/phase II clinical trials of an orally available, highly specific CYP17 inhibitor, abiraterone acetate, that ablates the synthesis of androgens and estrogens that drive TMPRSS2-ERG fusions. We isolated circulating tumor cells (CTC) by anti-epithelial cell adhesion molecule immunomagnetic selection followed by cytokeratin and CD45 immunofluorescence and 4 ¶,6-diamidino-2-phenylindole staining. We used multicolor fluorescence in situ hybridization to show that CRPC CTCs, metastases, and prostate tissue invariably had the same ERG gene status as therapy-naive tumors (n = 31). We then used quantitative reverse transcription-PCR to show that ERG expression was maintained in CRPC. We also observed homogeneity in ERG gene rearrangement status in CTCs (n = 48) in contrast to significant heterogeneity of AR copy number gain and PTEN loss, suggesting that rearrangement of ERG may be an earlier event in prostate carcinogenesis. We finally report a significant association between ERG rearrangements in therapy-naive tumors, CRPCs, and CTCs and magnitude of prostate-specific antigen decline (P = 0.007) in CRPC patients treated with abiraterone acetate. These data confirm that CTCs are malignant in origin and indicate that hormone-regulated expression of

Tumor cells in blood of patients with metastatic carcinomas have been associated with poor survival prospects. Further characterization of these cells may provide further insights into the metastatic process. Circulating Tumor Cells (CTC) were enumerated in 7.5 mL of blood with the CellSearch TM system. After enumeration of Cytokeratin1, CD452, nucleated cells, the cells are fixed in the cartridge while maintaining their original position. Cartridges were hybridized with FISH probes against the centromeric regions of chromosome 1, 7, 8, and 17. Next fluorescence images of the FISH probes of the previous identified CTC were acquired. Leukocytes surrounding the CTC were used as internal controls.

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Supplementary Figure Legends 1-5 from Characterization of <i>ERG</i>, <i>AR</i> and <i>PTEN</i> Gene Status in Circulating Tumor Cells from Patients with Castration-Resistant Prostate Cancer

<div>Abstract<p>Hormone-driven expression of the <i>ERG</i> oncogene after fusion with <i>TMPRSS2</i> occurs in 30% to 70% of therapy-naive prostate cancers. Its relevance in castration-resistant prostate cancer (CRPC) remains controversial as <i>ERG</i> is not expressed in some <i>TMPRSS2-ERG</i> androgen-independent xenograft models. However, unlike these models, CRPC patients have an increasing prostate-specific antigen, indicating active androgen receptor signaling. Here, we collected blood every month from 89 patients (54 chemotherapy-naive patients and 35 docetaxel-treated patients) treated in phase I/phase II clinical trials of an orally available, highly specific CYP17 inhibitor, abiraterone acetate, that ablates the synthesis of androgens and estrogens that drive <i>TMPRSS2-ERG</i> fusions. We isolated circulating tumor cells (CTC) by anti–epithelial cell adhesion molecule immunomagnetic selection followed by cytokeratin and CD45 immunofluorescence and 4′,6-diamidino-2-phenylindole staining. We used multicolor fluorescence <i>in situ</i> hybridization to show that CRPC CTCs, metastases, and prostate tissue invariably had the same <i>ERG</i> gene status as therapy-naive tumors (<i>n</i> = 31). We then used quantitative reverse transcription–PCR to show that <i>ERG</i> expression was maintained in CRPC. We also observed homogeneity in <i>ERG</i> gene rearrangement status in CTCs (<i>n</i> = 48) in contrast to significant heterogeneity of <i>AR</i> copy number gain and <i>PTEN</i> loss, suggesting that rearrangement of <i>ERG</i> may be an earlier event in prostate carcinogenesis. We finally report a significant association between <i>ERG</i> rearrangements in therapy-naive tumors, CRPCs, and CTCs and magnitude of prostate-specific antigen decline (<i>P</i> = 0.007) in CRPC patients treated with abiraterone acetate. These data confirm that CTCs are malignant in origin and indicate that hormone-regulated expression of <i>ERG</i> persists in CRPC. [Cancer Res 2009;69(7):2912–8]</p></div>