Genome-Wide Profiling of Archived Material from CALGB 9840 and 9342 for Paclitaxel (P) and Trastuzumab (T) Response Biomarkers Using Gene Expression and Copy Number Analysis.

Background :Emerging data suggest that RNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue can produce reliable gene expression profiles. Archived material from two taxane monotherapy studies, CALGB 9342 (comparison of 3 doses of P) and 9840 (weekly vs. q3 week P)were profiled for gene expression and DNA copy number. Methods: A total of 238 patients had primary tumor blocks available from a combined sample size of 680. DNA and RNA was extracted from 1.5mm punch cores and the Ambion Recover-All kit™. quality was measured by spectrophotometric analysis, Bioanalyzer RNA Integrity Number (RIN), and housekeeping genes (RPL13A and Actin). A custom DASL™ array containing 779 genes in two-fold redundancy was designed with genes selected to represent the PAM50 intrinsic subtypes, the Oncotype Dx Score, the Netherlands prognostic signature and the genes most frequently found on recurrent breast cancer amplicons. Several methods for identifying outliers were evaluated, including principal components analysis, pairwise correlations as well as the reproducibility of the platform based on replicate samples. Results: Adequate RNA was obtained from 237/238 of these cases which ranged in age from 12-18 years. Of these, 215/237DASL arrays passed further quality control measures. Adequate DNA for CGH was obtained from 227/238 samples. Analysis of PAM50 intrinsic subtypes showed an excess of basal-like tumors (30%) in the primary tumors of this metastatic cohort compared with expected frequency in an early stage population. Luminal A tumors were less frequent than expected (20%). Patients with basal-like tumors did far worse than other tumor types for both PFS on P (p=0.015) and OS (p=2.7X10-6), which persisted in multivariable analysis (p=0.0047), however the interaction term was not significant (Wald p=0.26). While basal-like tumors had similar PFS and OS on both weekly and q3 week P, luminal A tumors appear to achieve more benefit from weekly P (p= 0.0041).The HER2-enriched expression subtype had a similar prognosis to Luminal A and B tumors. This appeared to be due to the presence of T, as the addition of this agent improved PFS (p=0.026) and OS (p=2.0X10-4). Of note, some centrally confirmed HER2 FISH amplified tumors were classified into luminal A, B, and basal-like subtypes. These tumors have similar prognoses to the overall group, for example the basal-like and HER2 tumors had a poor prognosis despite T (p=0.00086). This suggests that HER2 FISH positive tumors may behave based on the underlying tumor subtype. Sawtooth genomes (45% vs 15%) were more frequent than predicted by an early stage tumor dataset as were simplex genomes (3% vs 24%). HER2 by FISH and CGH were highly concordant suggesting data on gene amplification from this platform is robust. Conclusions: Gene expression and copy number profiling of FFPE material from archived tumor blocks (>10 years) produces quality data for biomarker discovery in CALGB clinical trial datasets. These tools allow discovery of novel patterns of gene expression and genomic aberrations that are associated with differential response to P and T. Further studies using these platforms should be performed. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4032.