Identification of markers of taxane sensitivity using proteomic and genomic analyses of breast tumors from patients receiving neoadjuvant paclitaxel and radiation

Abstract

Purpose: To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies.

Experimental design: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR).

Results: Proteomic and validation immunohistochemical analyses revealed that alpha-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery.

Conclusion: We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane-based therapy.

Fig. 1

Average tumor spectra of pCR (green) and NR (red) from MALDI-MS. Inset, the differentially expressed m/z peaks that were higher (#) or lower (*) in pCR versus NR. Fold change was calculated using significance analysis of microarrays. P values were generated using the linear mixed effects model.

Fig. 2

A, average pCR (green) and NR (red) spectra peaks for DEFA1-3. B, immunohistochemical analysis for DEFAs from two representative pCR and NR tumors. C, MALDI-MS imaging of DEFA1 and DEFA2 was done by overlaying spectral files that were reconstructed into ion density images with an H&E image (left) of an NR and pCR for visualization.

Fig. 3

A, unsupervised hierarchical clustering of a 101-probe set (>3-fold change, P < 0.05) segregates tumors from patients who had a pCR (green) versus those with NR (red). B, MAP2 expression levels were analyzed in NR versus pCR tumors. An FDR-corrected t test showed significant corrected P < 0.03 between NR versus pCR samples for two MAP2 probes (averaged and normalized for absolute fold change).

Fig. 4

A, Western analyses showing MAP2 and GAPDH protein levels in MCF-7 and MDA-MB-468 cells transduced with vector control or MAP2. B, control and MAP2-expressing cells were grown as mammospheres ± paclitaxel. Arrows, nonadherent or apoptotic cells. The number of viable cells was determined after 10 d ± paclitaxel and represented relative to untreated control.