Article Figures & Data
Figures
- Figure 1.
MS-PCR targets. MS-PCR primers were designed to amplify regions of known methylation regions in the RARβ2 P2 promoter (14). M3 region (methylated nt −51 to 162; unmethylated nt −49 to 162) includes the RARE, TATAA box, and transcription start region; M4 region (methylated nt 104-251; unmethylated nt 101-250) contains an Sp1 element. •, methylated CpGs were previously identified by sequencing of cell lines and primary tumor samples (14, 15).
- Figure 2.
RPFNA cytology and methylation of RARβ2 P2 promoter. A. Representative RPFNA specimens in high-risk women. Numeric value, Masood cytology index score for this specimen. Presence of RARβ2 P2 promoter methylation at either the M3 or M4 region (Methylated). If neither region is methylated, the specimen is labeled Unmethylated. B. Hypermethylation of RARβ2 P2 promoter M4 region in early-stage breast cancer specimens. To confirm the integrity of the extracted genomic DNA, PCR analysis was used to detect β-actin (actin). C. Hypermethylation of RARβ2 P2 promoter M3 and M4 regions in RPFNA obtained from five representative high-risk women. D. Hypermethylation of RARβ2 P2 promoter M3 region in cellular sensitivity experiment to measure lowest threshold of methylation detection in a sea of unmethylated DNA, as explained in Materials and Methods. M3 and M4, use of primers to identify methylated RARβ2 P2 regions 3 and 4, respectively. U3 and U4, use of primers to identify unmethylated RARβ2 P2 regions 3 and 4, respectively. Methylated positive control in the M3 and M4 gels, HMEC-SR in the U3 gel, and HS578T in the U4 gel (+). Negative control (−).
- Figure 3.
Correlation between RARβ2 P2 promoter methylation in RPFNA with Masood cytology index scores. RPFNA samples were assessed for cytologic atypia using the Masood cytology index. The distribution of RARβ2 P2 promoter methylation is depicted as a function of increased cytologic abnormality. A. Distribution of RFPNA samples with M3 region methylation relative to Masood cytology score. B. Distribution of RFPNA samples with M4 region methylation relative to Masood cytology score. C. RPFNA samples containing methylation at both the M3 and M4 regions relative to Masood cytology score.
- Figure 4.
Correlation between RPFNA cell count and RARβ2 P2 promoter methylation and Masood score. A and B. Presence of RARβ2 P2 promoter methylation at the M3 (A) and M4 (B) region is depicted relative to total cell count of each RPFNA sample. C. RPFNA Masood cytology index scores are reported relative to the total cell count of each sample.
Tables
- Table 1.
MS-PCR primer sequences and reaction conditions
Sequences 1× Buffer (and additives) Annealing temperature (°C) M3 S 5′-GGTTAGTAGTTCGGGTAGGGTTTATC-3′, AS 5′-CCGAATCCTACCCCGACG-3′ 16.6 mmol/L (NH4)2SO4 57 67 mmol/L Tris (pH 9.1) 3.0 mmol/L MgCl2 U3 S 5′-TTAGTAGTTTGGGTAGGGTTTATT-3′, AS 5′-CCAAATCCTACCCCAACA-3′ 15 mmol/L (NH4)2SO4 57 60 mmol/L Tris (pH 8.5) 4.5 mmol/L MgCl2 M4 S 5′-GTCGAGAACGCGAGCGATTC-3′, AS 5′-CGACCAATCCAACCGAAACG-3′ 15 mmol/L (NH4)2SO4 55 60 mmol/L Tris (pH 9.0) 3.5 mmol/L MgCl2 150 mmol/L 2-pyrrolidinone U4 S 5′-GATGTTGAGAATGTGAGTGATTT-3′, AS 5′-AACCAATCCAACCAAAACA-3′ 15 mmol/L (NH4)2SO4 57 60 mmol/L Tris (pH 8.5) 4.5 mmol/L MgCl2 Abbreviations: S, sense; AS, antisense.
- Table 2.
Patient characteristics of early-stage breast cancer
Women enrolled in study 16 No. biopsy samples taken 17 n = 16 (%) Average age and range (y) 57 (34-82) Race Caucasian 14 (88) African American 2 (12) Menopausal status Postmenopausal 11 (69) Premenopausal 5 (31) Stage of breast cancer Stage 0/DCIS 1 (6) Stage I 8 (50) Stage II 7 (44) Average tumor size and range (cm) 3.2 (0.5-5.0) Type of tumor Invasive ductal 12 (75) Invasive lobular 1 (6) Mixed ductal/lobular 2 (12) DCIS 1 (6) Tumor receptor status ER+ 11 (69) PR+ 8 (50) Lymph node–positive disease 5 (31) - Table 3.
Clinical characteristics of patients undergoing RPFNA
A. Patient characteristics for RPFNA Women enrolled in study 38 Bilateral RPFNA 28 Unilateral RPFNA 10 RPFNA samples collected 66 No. RPFNAs with insufficient epithelial cell count 10 No. RPFNAs submitted for analysis 56 n = 38 (%) Average age and range (y) 46 (29-64) Race Caucasian 33 (87) African American 5 (13) Menopausal status Postmenopausal 18 (47) Premenopausal/Perimenopausal 20 (53) Hormone replacement use Current 2 (5) Ever use 9 (24) Never use 27 (71) Antiestrogen therapy (at the time of RPFNA) Tamoxifen 2 (5) Raloxifene 1 (3) Aromatase inhibitor 2 (5) Family history of breast cancer 17 (45) Prior abnormal biopsies LCIS 1 (3) DCIS 5 (13) ADH 10 (26) History of contralateral breast cancer 5 (13) B. Characteristics of patients on prevention therapy at time of RPFNA RPFNA sampling Agent Duration Masood Cell count M3/M4 methylation Unilateral Tamoxifen 2 wk 14 500 +/+ Bilateral (L) Raloxifene 2 y 10 <10 −/− Bilateral (R) 10 10 −/− Unilateral Tamoxifen 2 wk 15 1,000 +/− Unilateral Aromatase inhibitor 1 y NA NA −/− Unilateral Aromatase inhibitor 1 y NA NA −/−
Volume 14, Issue 4
