Breast Cancer Gene Expression Using Amplified Core Biopsies

Institution: Stanford University
Investigator(s): Stefanie Jeffrey, M.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5JB-0126 Award: $155,286
Award Type: IDEAS II
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (1999)
When normal breast epithelial cells turn into breast cancer there are profound changes in the pattern of genetic expression. These changes include both gain and loss of gene function, as well as changes in the level of expression for critical genes. This 'pattern' of gene expression in cancer opens the door to improved diagnosis and treatment based on a 'molecular diagnosis'. The molecular diagnosis of cancer interfaces with emerging technologies in genomic research and this has the potential to improve medical decision making at earlier stages of the disease.

Our research project utilizes a newly-developed, state-of-the-art gene microarray or "gene chip." This consists of a glass microscope slide containing over 9000 gene spots in a square area less than 0.75 inches per side. In order to survey the expression level of breast cancer genes, the RNA is extracted from a tumor sample and converted (reverse transcribed) into cDNA. This cDNA needs to be representative for diversity, quality, and quantity compared to the starting tissue sample. These cDNA samples are placed on the microarray and binding occurs between the cDNA and the individual gene 'spots' on the chip. These represent 'matches' and provides a readout for the relative expression level for over 9000 genes, many whose function is currently unknown. Based on the use of color dyes and special scanners, we can quickly determine specific patterns of gene expression in a given breast tumor sample.

Unfortunately, the current use of the 'gene chip' technology is limited to large tumors (>1.5 cm), because of the need to isolate substantial quantities of mRNA. This project seeks to improve the utility of this procedure for smaller breast tumor samples obtained from routine core biopsies. In this project we will test RNA 'scale-up' amplification from small breast samples using an antisense RNA amplification technique. We will be able to compare core biopsies from large tumors with the traditional RNA preparation method using the same tumor sample. In addition, we can perform multiple core biopsies from a single tumor to see if there are consistent gene expression patterns as a measure of tumor heterogeneity.

Development of this gene survey approach will put more diagnostic power in the hands of surgeons and oncologists to understand the molecular features of breast tumors. For example, the expression levels of genes characteristic of angiogenesis, metastasis, or aggressive growth (e.g., HER2/neu) would indicate the need for individual types of therapy. This is potentially a vast improvement over pathological appearance and today's limited biomarker technology. Thus, we eventually hope to use this amazing technology to help women with small tumors in order to predict whether or not they need chemotherapy, and if they do, which drugs would work most effectively. Ultimately, specific and currently unrecognized gene products may be identified for the development of novel targeted cytotoxic therapies.

Final Report (2000)
Progress. We created a frozen tumor bank of 28 breast cancers, freezing large pieces of tissue and associated core needle biopsies. Core biopsies were obtained from multiple sites within a single tumor in 16 cases. We tested and refined different signal amplification and RNA amplification techniques and optimized reference sample preparation. After successful RNA amplification from tumor and core needle biopsy specimen, we performed genomic analyses of amplified and unamplified RNA using 23,000 gene cDNA microarrays.

Studying both cell cultures and 6 human breast cancers, we showed that gene expression patterns of amplified RNA from core needle biopsies of a tumor recapitulates the gene expression pattern of the corresponding tumor and any associated lymph node metastases. RNA degradation may negatively affect this correlation.

Future Directions. We will continue tumor heterogeneity experiments using our frozen specimen and to test room temperature RNA preservation techniques. Our current amplification protocol will be applied to small breast cancers in order to extend our work using nonamplified RNA from larger tumors. Our protocol may also be applied to studies of patients treated with primary (preoperative) chemotherapy and to examine genomic changes in precancerous tissue and microscopically "normal" breast tissue adjacent to cancers.

Impact. Using larger tumors, we have recently shown that breast cancers subgrouped by gene expression patterns show differences in clinical outcome and sensitivity to Tamoxifen not predicted by usual methods. Adding the ability to analyze RNA from small breast cancers can potentially identify patients who do not require chemotherapy or hormone therapy, thereby avoiding morbidity and unnecessary expense. Molecular targets for novel tumor-specific therapies may be identified for those patients with tumors that lead to a poor clinical outcome.

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis.
Periodical:BMC Genomics
Index Medicus: BMC Genomics
Authors: Zhao H, Hastie T, Whitfield ML, Borresen-Dale AL, Jeffrey SS
Yr: 2002 Vol: 2002 Nbr: 3 Abs: 31-46 Pg:1