The Oncotype DX Breast Cancer Assay for DCIS Patients

The Oncotype DX Breast Cancer Assay for DCIS is a multi-gene diagnostic assay designed to support personalized treatment planning for patients with DCIS following local excision. The assay provides an individualized estimate of the 10-year risk of local recurrence (DCIS or invasive carcinoma) to help guide treatment decision making in women with ductal carcinoma in situ treated by local excision, with or without tamoxifen.

The Oncotype DX Breast Cancer Assay is performed in the licensed Genomic Health laboratory where the assay was developed. All H&E's are reviewed by board certified surgical pathologists and the DCIS tumor is marked for manual microdissection. The dissected, enriched tumor sample then undergoes RNA extraction. Next, the RNA is analyzed using a technique called real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Finally, the DCIS Score™ result is calculated from the gene expression results.

Advantages of RT-PCR

The Oncotype DX Breast Cancer Assay analyzes the expression of a panel of 21 genes from a tumor specimen using a technique called RT-PCR. A high-throughput, real-time RT-PCR method was developed to analyze the expression of select genes. Unlike routine histopathologic H&E slide review or assessment of immunohistochemical stains for hormone receptors, RT-PCR is sensitive, specific, highly reproducible, and has a wide dynamic range. RT-PCR is a mature technology that is routinely utilized in several clinical applications including viral load testing for HIV.

To quantify gene expression, RNA is extracted from formalin-fixed, paraffin-embedded (FPET) tumor tissue and subjected to DNase I treatment. Total RNA content is measured and the absence of DNA contamination is verified. Reverse transcription is performed and is followed by quantitative TaqMan^® (Roche Molecular Systems, Inc.) RT-PCR reactions in 384-well plates. The expression of each of 16 cancer related genes is measured in triplicate and then normalized relative to a set of five reference genes.

The Oncotype DX Breast Cancer Assay for DCIS patients standardized testing methods have been optimized to minimize variability due to:

• Sources of pre-analytic variability: delay to fixation, choice of fixative, duration of fixation, age of sample

Oncotype DX Assay Development

The Oncotype DX Breast Cancer Assay for DCIS patients was developed in four steps. Each of these steps is described in detail below.

• Optimization of methods for quantifying gene expression in formalin-fixed, paraffin-embedded tissue (FPET)
• Selection of 250 candidate genes from the human genome
• Testing of candidate genes to identify an optimal gene panel for clinical validation
• Prospective clinical validation of the gene panel and DCIS Score result calculation

Step 1. Optimization of methods for quantifying gene expression in formalin-fixed, paraffin-embedded tissue

The ability to work with FPET samples is critical in the U.S., as this is the standard method for tumor preservation and storage. When tissue is preserved in paraffin, the RNA is fragmented. However, the relative ratio of RNA between genes is unchanged. By utilizing RT-PCR techniques, the expression of most genes—relative to a set of reference genes—can be measured. To develop the Oncotype DX Breast Cancer Assay, Genomic Health researchers optimized RT-PCR technology 1) for high-throughput, real-time quantitation of specific RNA in FPET, and 2) to be reproducible regardless of the variability inherent in tumor blocks.

Step 2. Selection of 250 candidate genes from the human genome

Genomic Health researchers relied on numerous sources to identify 250 candidate genes—those possibly associated with breast cancer tumor behavior—from among the approximately 25,000 genes in the human genome.

Step 3. Testing of candidate genes to identify an optimal gene panel for clinical validation

The 250 candidate genes were analyzed in a total of 447 patients from three independent clinical studies in order to identify a panel of genes strongly correlated with distant recurrence-free survival. The selection of the 16 cancer genes used for the Oncotype DX Breast Cancer Assay was based on the results of the three clinical trials, which demonstrated a consistent and strong statistical link between these genes and distant breast cancer recurrence. Five reference genes were identified to normalize the expression of these cancer-related genes. The 21- gene panel described above has been optimized for the DCIS Score, which is calculated from a subset of the genes using a DCIS-specific algorithm and coefficients.

Step 4. Prospective clinical validation of the DCIS Score

The Oncotype DX Breast Cancer Assay gene panel and DCIS Score result calculation were validated in a large, independent, multicenter clinical trial (ECOG E5194). The endpoints and analysis plan were prospectively defined. The results of this study were presented at the San Antonio Breast Conference 2011 and the results of the ECOG E5194 clinical validation study will be published in the near future.

21-Gene Panel Used to Calculate Recurrence Score^® Result

The Oncotype DX Breast Cancer Assay gene panel was selected from the published literature, a genomics database and experiments based on DNA arrays performed on fresh-frozen tissue. The DCIS Score is obtained by performing the Oncotype DX Breast Cancer Assay, using a distinct DCIS algorithm and coefficients that was pre-specified because of its ability to predict recurrence in patients with DCIS regardless of whether adjuvant tamoxifen therapy was given.

Development of the DCIS Score algorithm was based on published results for the Oncotype DX Breast Cancer Assay showing similarity in the expression profiles of the Recurrence Score genes between DCIS and Invasive Breast Cancer (IBC) when both are present within the same patient tumor. The DCIS Score algorithm was developed based on published data obtained from the Kaiser Permanente and NSABP B-14 studies in which the proliferation gene group, PR and GSTM1 were found to predict distant recurrence regardless of whether adjuvant tamoxifen therapy was given. This DCIS Score was subsequently validated as a predictor of local recurrence in patients from the ECOG E5194 study. These results were presented at the San Antonio Breast Conference 2011.