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Cancer Epidemiology, Biomarkers & Prevention
Cancer Epidemiology, Biomarkers & Prevention
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Retinoic Acid Receptor-β2 Promoter Methylation in Random Periareolar Fine Needle Aspiration

Gregory R. Bean, Victoria Scott, Lisa Yee, Brooke Ratliff-Daniel, Michelle M. Troch, Pearl Seo, Michelle L. Bowie, Paul K. Marcom, Jaimie Slade, Bruce F. Kimler, Carol J. Fabian, Carola M. Zalles, Gloria Broadwater, Joseph C. Baker Jr., Lee G. Wilke and Victoria L. Seewaldt
Gregory R. Bean
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Victoria Scott
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Lisa Yee
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Brooke Ratliff-Daniel
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Michelle M. Troch
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Pearl Seo
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Michelle L. Bowie
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Paul K. Marcom
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Jaimie Slade
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Bruce F. Kimler
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Carol J. Fabian
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Carola M. Zalles
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Gloria Broadwater
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Joseph C. Baker Jr.
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Lee G. Wilke
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Victoria L. Seewaldt
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DOI: 10.1158/1055-9965.EPI-04-0580 Published April 2005
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    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.
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    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 (−).

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    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.

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    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.

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  • Table 1.

    MS-PCR primer sequences and reaction conditions

    Sequences1× Buffer (and additives)Annealing temperature (°C)
    M3S 5′-GGTTAGTAGTTCGGGTAGGGTTTATC-3′, AS 5′-CCGAATCCTACCCCGACG-3′16.6 mmol/L (NH4)2SO457
    67 mmol/L Tris (pH 9.1)
    3.0 mmol/L MgCl2
    U3S 5′-TTAGTAGTTTGGGTAGGGTTTATT-3′, AS 5′-CCAAATCCTACCCCAACA-3′15 mmol/L (NH4)2SO457
    60 mmol/L Tris (pH 8.5)
    4.5 mmol/L MgCl2
    M4S 5′-GTCGAGAACGCGAGCGATTC-3′, AS 5′-CGACCAATCCAACCGAAACG-3′15 mmol/L (NH4)2SO455
    60 mmol/L Tris (pH 9.0)
    3.5 mmol/L MgCl2
    150 mmol/L 2-pyrrolidinone
    U4S 5′-GATGTTGAGAATGTGAGTGATTT-3′, AS 5′-AACCAATCCAACCAAAACA-3′15 mmol/L (NH4)2SO457
    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 study16
    No. biopsy samples taken17
    n = 16 (%)
    Average age and range (y)57 (34-82)
    Race
        Caucasian14 (88)
        African American2 (12)
    Menopausal status
        Postmenopausal11 (69)
        Premenopausal5 (31)
    Stage of breast cancer
        Stage 0/DCIS1 (6)
        Stage I8 (50)
        Stage II7 (44)
    Average tumor size and range (cm)3.2 (0.5-5.0)
    Type of tumor
        Invasive ductal12 (75)
        Invasive lobular1 (6)
        Mixed ductal/lobular2 (12)
        DCIS1 (6)
    Tumor receptor status
        ER+11 (69)
        PR+8 (50)
    Lymph node–positive disease5 (31)
  • Table 3.

    Clinical characteristics of patients undergoing RPFNA

    A. Patient characteristics for RPFNA
    Women enrolled in study38
    Bilateral RPFNA28
    Unilateral RPFNA10
    RPFNA samples collected66
    No. RPFNAs with insufficient epithelial cell count10
    No. RPFNAs submitted for analysis56
    n = 38 (%)
    Average age and range (y)46 (29-64)
    Race
        Caucasian33 (87)
        African American5 (13)
    Menopausal status
        Postmenopausal18 (47)
        Premenopausal/Perimenopausal20 (53)
    Hormone replacement use
        Current2 (5)
        Ever use9 (24)
        Never use27 (71)
    Antiestrogen therapy (at the time of RPFNA)
        Tamoxifen2 (5)
        Raloxifene1 (3)
        Aromatase inhibitor2 (5)
    Family history of breast cancer17 (45)
    Prior abnormal biopsies
        LCIS1 (3)
        DCIS5 (13)
        ADH10 (26)
        History of contralateral breast cancer5 (13)
    B. Characteristics of patients on prevention therapy at time of RPFNA
    RPFNA sampling
    Agent
    Duration
    Masood
    Cell count
    M3/M4 methylation
    UnilateralTamoxifen2 wk14500+/+
    Bilateral (L)Raloxifene2 y10<10−/−
    Bilateral (R)1010−/−
    UnilateralTamoxifen2 wk151,000+/−
    UnilateralAromatase inhibitor1 yNANA−/−
    UnilateralAromatase inhibitor1 yNANA−/−
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Cancer Epidemiology Biomarkers & Prevention: 14 (4)
April 2005
Volume 14, Issue 4
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Retinoic Acid Receptor-β2 Promoter Methylation in Random Periareolar Fine Needle Aspiration
Gregory R. Bean, Victoria Scott, Lisa Yee, Brooke Ratliff-Daniel, Michelle M. Troch, Pearl Seo, Michelle L. Bowie, Paul K. Marcom, Jaimie Slade, Bruce F. Kimler, Carol J. Fabian, Carola M. Zalles, Gloria Broadwater, Joseph C. Baker Jr., Lee G. Wilke and Victoria L. Seewaldt
Cancer Epidemiol Biomarkers Prev April 1 2005 (14) (4) 790-798; DOI: 10.1158/1055-9965.EPI-04-0580

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Retinoic Acid Receptor-β2 Promoter Methylation in Random Periareolar Fine Needle Aspiration
Gregory R. Bean, Victoria Scott, Lisa Yee, Brooke Ratliff-Daniel, Michelle M. Troch, Pearl Seo, Michelle L. Bowie, Paul K. Marcom, Jaimie Slade, Bruce F. Kimler, Carol J. Fabian, Carola M. Zalles, Gloria Broadwater, Joseph C. Baker Jr., Lee G. Wilke and Victoria L. Seewaldt
Cancer Epidemiol Biomarkers Prev April 1 2005 (14) (4) 790-798; DOI: 10.1158/1055-9965.EPI-04-0580
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