Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients.

Previously, we had identified gene expression patterns that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis but better response to chemotherapy. We investigated whether tumor expression of these 21 genes and other candidate genes can predict response to docetaxel. Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Three 10-microm FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Of the 97 patients, 81 (84%) had sufficient invasive cancer, 80 (82%) had sufficient RNA for QRTPCR assay, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). A significant relationship (P<0.05) between gene expression and CR was observed for 14 genes, including CYBA. CR was associated with lower expression of the ER gene group and higher expression of the proliferation gene group from the 21 gene assay. Of note, CR was more likely with a high RS (P=0.008). We have established molecular profiles of sensitivity to docetaxel. RT-PCR technology provides a potential platform for a predictive test of docetaxel chemosensitivity using small amounts of routinely processed material.

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.

BACKGROUND: Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. RESULTS: RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. CONCLUSION: We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.

Filgrastim-mediated neutrophil recovery in patients with breast cancer treated with docetaxel and doxorubicin.

STUDY OBJECTIVES: To study the impact of filgrastim 5 microg/kg given once/day through absolute neutrophil count (ANC) recovery on the duration of grade 4 neutropenia (ANC < 0.5 x 10(3)/mm3) and time to ANC recovery. Additional objectives were to study the average number of filgrastim injections/cycle required to achieve ANC recovery and differences in outcome by cycle. DESIGN: Combined analysis of two double-blind, randomized, multicenter trials. PATIENTS: Two hundred twenty-two patients treated for breast cancer. MEASUREMENTS AND MAIN RESULTS: All patients but one were evaluable for efficacy end points. Mean +/- SD duration of grade 4 neutropenia was 1.7 +/- 1.3 days in cycle 1; the duration decreased in cycles 2-4 to between 1.0 and 1.2 days. Fifty percent of patients had ANC recovery to 10 x 10(3)/mm3 or greater by day 11 of the cycle, and 90% by day 13, corresponding to 10 and 12 days of filgrastim administration, respectively. Across all cycles, the mean +/- SD number of filgrastim injections/cycle was 10.51 +/- 1.70, with little variation among cycles. CONCLUSION: When filgrastim is administered as recommended, starting 24 hours after chemotherapy and continuing through an ANC of 10 x 10(3)/mm3 or greater, neutrophil recovery is rapid and predictable. Because the first cycle of chemotherapy has the highest rates of neutropenia and febrile neutropenia, it seems prudent to administer growth factor support preemptively.