Andorfer, Cathy

1037 Background: Folate receptor alpha (FRA the product of the FOLR1 gene) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense expression of FRA in breast tumors and poor prognosis, yet little is known about FOLR1/FRA expression across clinically relevant breast cancer subtypes. Methods: 131 breast cancer tumors including 4 benign, 33 ER+, 26 HER2+, and 68 triple negative (TN) were constructed into tissue microarrays (TMAs). FRA expression was analyzed by immunohistochemistry (IHC) using a high affinity FRA antibody. Tumor membrane staining intensity was scored by a pathologist as negative (0), weak (1+), moderate (2+) and strong (3+). The percent of cells within each tissue core stained at each intensity was recorded to calculate an H-score. The H-score is a weighted score that captured both the proportion of positive staining and intensity for each tumor. H-score values can range from zero (no membrane staining) to a maximum of 300 (100% membrane staining at 3+). H-scores for each patient sample were averaged over 3 TMA cores. The mean H-scores for each tumor subtype and the percentage of 3+ staining in >30% of tumor cells were compared by a Mann-Whitney test. The distribution of FOLR1 mRNA was completed using a TCGA RNA-seq dataset from 691 breast tumors classified as ER+, HER2+ and TN. FOLR1 levels of TN versus ER+ and HER2+ were compared by a Mann-Whitney test. Results: The mean H-score for the benign tumors was 0, ER+ (13.31), HER2+ (39.36), TN (119.02). The median H-score for the benign tumors was 0, ER+ (0), HER2+ (7.5), TN (127.5). The TN tumors mean and median H scores were significantly higher than benign, ER+ or HER2+ (p<0.001). The largest percentage of 3+ staining in >30% of tumor cells was observed in TN tumors (36.7%) and lowest in ER+ tumors (0%) (p<0.001). TN tumors had significantly higher levels of FOLR1 mRNA compared to ER+ and HER2+ subtypes (p<0.0001). Conclusions: Our data indicate that expression of FRA is highly prevalent in TN tumors and is supported by FOLR1 mRNA levels. Anti-FRA therapy may represent an important therapeutic intervention in TNBC who to this point have no active targeted treatment options.

Abstract Epigenetic silencing of tumor suppressing genes by promoter-specific DNA methylation is common in many types of cancer. As an early event, this process has been well shown to promote tumor initiation and progression; however little is known how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes Protein Kinase D1 (PKD1), a serine/threonine kinase expressed in epithelial cells of the normal mammary gland that maintains the epithelial phenotype of normal breast cells and prevents epithelial-to-mesenchymal transition (EMT). PKD1 is also a critical suppressor of tumor cell invasion and is silenced in expression and activity during breast tumor progression. Here, we show that aberrant methylation of PRKD1 promoter region is not only correlated with the silencing of its expression but is also associated with invasiveness of breast cancer cell lines and with aggressiveness of breast tumors. Using the highly invasive MDA-MB-231 cells, we show that the inhibition of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and significantly decreases their invasive abilities in vitro. More importantly, in a tumor xenograft model it dramatically blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion. Our data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors, and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-24.

Abstract Background: The role of fusion genes and associated fusion transcripts has long been recognized in hematopoietic malignancies. Until quite recently it has been difficult to detect such events on a genomic scale in solid tumors. Consequently, little is known about the potential role of fusion genes, transcripts, and proteins as driver mutations, biomarkers, or therapeutic targets in breast cancer. Methods: We have developed a novel analytical pipeline, Snowshoes-FTD, for detection of fusion transcripts in breast cancer cell lines and tumor samples (Asmann, et al. NAR 2011; May 27 ePub ahead of print). Preliminary analyses have been carried out with a panel of 8 each ER+, HER2+, and triple negative (TN) primary breast tumors, 8 primary human mammary epithelial cell (HMEC) lines from biopsy samples, plus 16 normal tissues from the Illumina Body Map dataset. Results: We have identified 120 redundant, tumor-specific fusion transcripts, expressed in two or more tumors and in no non-transformed samples. Sixteen of these represent intrachromosomal fusions and 104 arise from fusion of transcripts that map to two different chromosomes. Every breast tumor expressed one or more fusion transcripts. Twenty-nine fusion transcripts appeared to be tumor subtype specific. Among these, we have identified 2 HER2+, 10 ER+, and 17 triple negative specific redundant transcripts. In general, HER2+ tumors expressed fewer fusion transcripts (range 4 to 28/tumor) compared to TN (range11 to 44/tumor). Chromosomal distribution patterns were also markedly different among the tumor subtypes. For example, ER+ tumors expressed a preponderance of redundant fusion transcripts that involve chr1 and 2, whereas TN tumors had no fusion transcripts that map to either chromosome. Conversely, the predominant locus for TN fusion transcripts was chr19, which contains only one HER2+ fusion and no ER+ fusion transcripts. Conclusions: Primary breast tumors express many chimaeric transcripts, which we presume to arise primarily from genomic rearrangements. The majority of these transcripts are redundant, and a subset are tumor subtype specific. These transcripts may mark regions of chromosomal instability. HER2+ tumors, in general, appear to evidence less chromosomal instability, as inferred from fusion transcripts; although some HER2+ tumors appear to be quite unstable. TN tumors contain many more redundant fusion transcripts, implying increased genomic instability, particularly in chr19. We conclude that these fusion transcripts represent a class of heretofore unrecognized biomarkers that may be used for sub-classification of breast tumors. Some of these transcripts appear to encode proteins that may function as tumor-subtype-specific driver mutations and may have potential as therapeutic targets in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-06-02.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.