BACKGROUND: DNA microarray profiling performed on clinical tissue specimens can potentially provide significant information regarding human cancer biology. Biopsy cores, the typical source of human tumor tissue, however, generally provide very small amounts of RNA (0.3-15 microg). RNA amplification is a common method used to increase the amount of material available for hybridization experiments. Using human xenograft tissue, we sought to address the following three questions: 1) is amplified RNA representative of the original RNA profile? 2) what is the minimum amount of total RNA required to perform a representative amplification? 3) are the direct and indirect methods of labeling the hybridization probe equivalent? METHODS: Total RNA was extracted from human xenograft tissue and amplified using a linear amplification process. RNA was labeled and hybridized, and the resulting images yielded data that was extracted into two categories using the mAdb system: "all genes" and "outliers". Scatter plots were generated for each slide and Pearson Coefficients of correlation were obtained. RESULTS: Results show that the amplification of 5 microg of total RNA yields a Pearson Correlation Coefficient of 0.752 (N = 6,987 genes) between the amplified and total RNA samples. We subsequently determined that amplification of 0.5 microg of total RNA generated a similar Pearson Correlation Coefficient as compared to the corresponding original RNA sample. Similarly, sixty-nine percent of total RNA outliers were detected with 5 microg of amplified starting RNA, and 55% of outliers were detected with 0.5 microg of starting RNA. However, amplification of 0.05 microg of starting RNA resulted in a loss of fidelity (Pearson Coefficient 0.669 between amplified and original samples, 44% outlier concordance). In these studies the direct or indirect methods of probe labeling yielded similar results. Finally, we examined whether RNA obtained from needle core biopsies of human tumor xenografts, amplified and indirectly labeled, would generate representative array profiles compared to larger excisional biopsy material. In this analysis correlation coefficients were obtained ranging from 0.750-0.834 between U251 biopsy cores and excised tumors, and 0.812-0.846 between DU145 biopsy cores and excised tumors. CONCLUSION: These data suggest that needle core biopsies can be used as reliable tissue samples for tumor microarray analysis after linear amplification and either indirect or direct labeling of the starting RNA.

E2F transcription factors, including E2F3, directly modulate expression of EZH2. Recently, overexpression of the EZH2 gene has been implicated in the development of human prostate cancer. In tissue microrarray studies we now show that expression of high levels of nuclear E2F3 occurs in a high proportion (98/147, 67%) of human prostate cancers, but is a rare event in non-neoplastic prostatic epithelium suggesting a role for E2F3 overexpression in prostate carcinogenesis. Patients with prostate cancer exhibiting immunohistochemically detectable nuclear E2F3 expression have poorer overall survival (P=0.0022) and cause-specific survival (P=0.0047) than patients without detectable E2F3 expression. When patients are stratified according to the maximum percentage of E2F3-positive nuclei identified within their prostate cancers (up to 20, 21-40%, etc.), there is an increasingly significant association between E2F3 staining and risk of death both for overall survival (P=0.0014) and for cause-specific survival (P=0.0004). Multivariate analyses select E2F3 expression as an independent factor predicting overall survival (unstratified P=0.0103, stratified P=0.0086) and cause-specific survival (unstratified P=0.0288, stratified P=0.0072). When these results are considered together with published data on EZH2 and on the E2F3 control protein pRB, we conclude that the pRB-E2F3-EZH2 control axis may have a critical role in modulating aggressiveness of individual human prostate cancer.

We conducted a prospectively randomized clinical trial to compare the efficacy and safety of subcutaneous interferon-alpha2a, subcutaneous interleukin-2 and intravenous 5-fluorouracil as home therapy against oral tamoxifen in 78 patients with progressive metastatic renal cell carcinoma. Treatment courses consisted of interferon-alpha2a 5 x 10(6) IU m(-2), day 1 weeks 1 + 4; days 1, 3, 5 weeks 2 + 3; 10 x 10(6) IU m(-2), days 1, 3, 5 weeks 5-8; interleukin-2 10 x 10(6) IU m(-2), twice daily days 3-5 weeks 1 + 4; 5 x 10(6) IU m(-2), days 1, 3, 5 weeks 2 + 3; and 5-fluorouracil 1000 mg m(-2), day 1 weeks 5-8. The tamoxifen group received tamoxifen 80 mg twice daily over 8 weeks. Among 41 patients treated with interleukin-2, interferon-alpha2a and 5-fluorouracil there were 7 complete (17.1%) and 9 partial responders (21.9%), with an overall objective response rate of 39.1% (95% CI, 24.2-55.5). An additional 15 patients (36.6%) were stable throughout therapy. The overall survival was 24 months (range 5-76+). In 37 patients receiving tamoxifen no objective remissions occurred. 13 patients (35.1%) had stable disease and 24 patients (64.9%) showed continued disease progression. The overall survival was 13 months (range 3-73+). In summary, this home-based therapy regimen of interferon-alpha2a, interleukin-2 and 5-fluorouracil demonstrated significant therapeutic efficacy in patients with progressive renal cell carcinoma when compared to hormonal therapy.