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Gene Expression Profiles Obtained from Fine-Needle Aspirations of Breast Cancer Reliably Identify Routine Prognostic Markers and Reveal Large-Scale Molecular Differences between Estrogen-negative and Estrogen-positive Tumors¹

Departments of Breast Medical Oncology [L. P., F. J. E., B. A., M. C., D. B., M. R., V. V., C. A., G. N. H.], Pathology [N. S., W. F. S.], Biomathematics [K. H., D. S.], and Radiation Oncology [T. A. B.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-009, and Millennium Predictive Medicine Inc., Cambridge, Massachusetts [M. A., J. S., E. C., A. D.]

Purpose: The purpose of this study was to determine whether comprehensive transcriptional profiles (TPs) can be obtained from single-passage fine-needle aspirations (FNAs) of breast cancer and to explore whether profiles capture routine clinicopathological parameters.

Experimental Design: Expression profiles were available on 38 patients with stage I-III breast cancer who underwent FNA at the time of diagnosis. [³³P]dCTP-labeled cDNA probes were generated and hybridized to cDNA membrane microarrays that contained 30,000 human sequence clones, including 10,890 expressed sequence tags.

Results: The median total RNA yield from the biopsies was 2 µg (range, 1–25 µg). The cellular composition of each biopsy was examined and, on average,, 79% of the cells were cancer cells. TP was successfully performed on all 38 of the biopsies. Unsupervised complete-linkage hierarchical clustering with all of the biopsies revealed an association between estrogen receptor (ER) status and gene expression profiles. There was a strong correlation between ER status determined by TP and measured by routine immunohistochemistry (P = 0.001). A similar strong correlation was seen with HER-2 status determined by fluorescent in situ hybridization (P = 0.0002). Using the first 18 cases as the discovery set, we established a cutoff of 2.0 and 18.0 for ER and HER-2 mRNA levels, respectively, to distinguish clinically-negative from -positive cases. We also identified 105 genes (excluding the ER gene) the expression of which correlated highly with clinical ER status. Twenty tumors were used for prospective validation. HER-2 status was correctly identified in all 20 of the cases, based on HER-2 mRNA content detected by TP. ER status was correctly identified in 19 of 20 cases. When the marker set of 105 genes was used to prospectively predict ER status, TP-based classification correctly identified 9 of 10 of the ER-positive and 7 of 10 of the ER-negative tumors. We also explored supervised cluster analysis using various functional categories of genes, and we observed a clear separation between ER-negative and ER-positive tumors when genes involved in signal transduction were used for clustering.

Conclusions: These results demonstrate that comprehensive TP can be performed on FNA biopsies. TPs reliably measure conventional single-gene prognostic markers such as ER and HER-2. A complex pattern of genes (not including ER) can also be used to predict clinical ER status. These results demonstrate that needle biopsy-based diagnostic microarray tests may be developed that could capture conventional prognostic information but may also contain additional clinical information that cannot currently be measured with other methods.

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