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Cancer Epidemiology, Biomarkers & Prevention

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Research Articles

Atypia and DNA Methylation in Nipple Duct Lavage in Relation to Predicted Breast Cancer Risk

David M. Euhus, Dawei Bu, Raheela Ashfaq, Xian-Jin Xie, Aihua Bian, A. Marilyn Leitch and Cheryl M. Lewis
David M. Euhus
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Dawei Bu
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Raheela Ashfaq
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Xian-Jin Xie
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Aihua Bian
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A. Marilyn Leitch
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Cheryl M. Lewis
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DOI: 10.1158/1055-9965.EPI-06-1034 Published September 2007
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  • Figure 1.
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    Figure 1.

    Distribution of study subjects and evaluable samples. Values for “Breasts” include only those were a duct could be cannulated. High risk and lower risk for the unaffected women are defined by the Gail model. ICMD, insufficient cellular material for diagnosis.

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    Figure 2.

    Methylation profiles of FNA samples from primary tumors as compared with NDL samples from ducts ipsilateral to these tumors. Cases are sorted by the degree of methylation of the primary tumor from most methylated to least methylated. Hist is histology of the primary tumor: ductal carcinoma in situ (DCIS; Embedded Image), infiltrating ductal carcinoma (Embedded Image), infiltrating lobular carcinoma (Embedded Image), other (medullary or metaplastic; Embedded Image); any ductal carcinoma in situ: yes (Embedded Image), no (Embedded Image); methylation fraction: 0% (Embedded Image), >10% (Embedded Image), no result (Embedded Image); Cyto is NDL cytology: Masood score ≥15 (Embedded Image), Masood score 11-14 (Embedded Image), Masood score ≤10 (Embedded Image). Ducts classified as cytologically atypical with methylation results that were concordant with the primary tumor for ≥4 genes are marked with asterisk.

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    Figure 3.

    Prevalence of methylation and marked atypia by gene, sample source, and threshold for classifying a duct as positive. Lower 90% thresholds correspond to the 90th percentile methylation values for ducts from unaffected women at lower risk for breast cancer according to the Gail model; Fackler thresholds were calculated to provide the greatest discrimination between ducts with breast cancer and ducts without breast cancer (29). Threshold values for APC are not reported by Fackler. Prevalence of marked atypia or methylation of ≥2 genes is calculated using the 90th percentile of Gail lower-risk ducts. Embedded Image, ducts from unaffected Gail lower-risk women; Embedded Image, ducts from unaffected Gail high-risk women; Embedded Image, ducts contralateral to a breast cancer; Embedded Image, ducts ipsilateral to a breast cancer.

Tables

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  • Table 1.

    Primers and probes for quantitative multiplex methylation-specific real-time PCR

    ForwardReverse
    Cyclin D2
        R1TATTTTTTGTAAAGATAGTTTTGATTACAACTTTCTAAAAAATAACCC
        R2 UMTTAAGGATGTGTTAGAGTATGTGAAACTTTCTCCCTAAAAACCAACTACAAT
        R2 MTTTGATTTAAGGATGCGTTAGAGTACGACTTTCTCCCTAAAAACCGACTACG
        P UMHEX-AATCCACCAACACAATCAACCCTAAC-BHQ1
        P M6FAM-AATCCGCCAACACGATCGACCCTA-BHQ1
    APC
        R1GGGTTAGGGTTAGGTAGGTTGTGAACTACACCAATACAACCACATA
        R2 UMGTGTTTTATTGTGGAGTGTGGGTTCCAATCAACAAACTCCCAACAA
        R2 MTATTGCGGAGTGCGGGTCTCGACGAACTCCCGACGA
        P UM6FAM-AACACCCTAATCCACATCCAACAAAT-BHQ1
        P M6FAM-AACGCCCTAATCCGCATCCAACGA-BHQ1
    HIN1
        R1GTTTGTTAAGAGGAAGTTTTCCGAAACATACAAAACAAAACCAC
        R2 UMAAGTTTTTGAGGTTTGGGTAGGGAACCAACCTCACCCACACTCCTA
        R2 MTAGGGAAGGGGGTACGGGTTTCGCTCACGACCGTACCCTAA
        P UMHEX-CAACTTCCTACTACAACCAACAAACC-BHQ1
        P M6FAM-ACTTCCTACTACGACCGACGAACC-BHQ1
    RASSF1A
        R1GTTTTATAGTTTTTGTATTTAGGAACTCAATAAACTCAAACTCCC
        R2 UMGGTGTTGAAGTTGGGGTTTGCCCATACTTCACTAACTTTAAAC
        R2 MGCGTTGAAGTCGGGGTTCCCCGTACTTCGCTAACTTTAAACG
        P UMHEX-CTAACAAACACAAACCAAACAAAACCA-BHQ1
        P M6FAM-ACAAACGCGAACCGAACGAAACCA-BHQ1
    RAR-β2
        R1GTAGGAGGGTTTATTTTTTGTTAATTACATTTTCCAAACTTACTC
        R2 UMTTGAGAATGTGAGTGATTTGAGTAGTTACAAAAAACCTTCCAAATACATTC
        R2 MAGAACGCGAGCGATTCGAGTAGTACAAAAAACCTTCCGAATACGTT
        P UMHEX-AAATCCTACCCCAACAATACCCAAAC-BHQ1
        P M6FAM-ATCCTACCCCGACGATACCCAAAC-BHQ1
    • Abbreviations: R1, first-round multiplex; R2 UM, second-round uniplex unmethylated; R2 M, second-round uniplex methylated; P UM, probe for unmethylated; P M, probe for methylated.

  • Table 2.

    Characteristics of the study sample

    Patients150
    Mean age (range), y48 (28-93)
    Ethnicity, %
        Caucasian123 (82)
        African American20 (13)
        Hispanic5 (3)
        Asian2 (1)
    Menopausal status, %
        Premenopausal73 (49)
        Perimenopausal8 (5)
        Postmenopausal69 (46)
    Oral contraceptive use (premenopausal)18/73 (38)
    Hormone replacement (peri- and post-menopausal)25/77 (32)
    Risk groups
        Breast cancer patients67 (45)
            Breasts ipsilateral to a breast cancer65*
                Ductal carcinoma in situ only6
                Infiltrating ductal carcinoma50
                Infiltrating lobular carcinoma7
                Medullary carcinoma1
                Metaplastic carcinoma1
                Any associated DCIS53 (82)
            Breasts contralateral to a breast cancer62†
        Unaffected risk assessed patients83 (55)
            History of atypical ductal hyperplasia4 (5)
            BRCA gene mutation5 (6)
            Lower risk by all models‡31 (37)
            High risk by Gail only§12 (15)
            High risk by Claus or BRCAPRO only29 (35)
            High risk by both Gail and a family history model11 (13)
    • ↵* Three bilateral cancers were included; four were excluded because cancer was excised before enrollment; and one was excluded due to inability to cannulate duct.

    • ↵† Three bilateral cancer patients had no contralateral lavage; two were excluded due to inability to cannulate duct.

    • ↵‡ Five-year Gail, Claus, and BRCAPRO risk less than twice the age- and race-matched general population risk.

    • ↵§ High risk is defined as 5-y model probability greater than or equal to twice the age- and race-matched general population risk.

  • Table 3.

    TSG methylation in 34 primary tumors and corresponding ipsilateral duct lavage samples

    GeneMethylation prevalence
    Median methylation fraction
    Correlation*
    Tumor FNA (%)NDL (%)PTumor FNANDLPCoefficientP
    Cyclin D237.18.70.0040.1980.0780.4280.040.846
    APC38.213.00.0190.3790.0410.005-0.140.431
    HIN144.113.30.0050.7300.1140.0040.070.687
    RASSF1A61.88.7<0.00010.4310.0640.045-0.210.238
    RAR-β226.56.50.0310.0280.3100.166-0.060.744
    • ↵* Spearman correlation between paired methylation fractions.

  • Table 4.

    Univariate analysis to identify factors predicting TSG methylation or marked atypia

    FactorMethylation of ≥2 genes
    Marked atypia
    Pos/Neg (%)OR (95% CI)PPos/Neg (%)OR (95% CI)P
    Age (tertiles)
        ≤41.39/93 (10)1—9/114 (8)1—
        41.4-50.112/97 (12)1.32 (0.53-3.29)0.55510/114 (9)1.12 (0.44-2.87)0.811
        >50.113/88 (15)1.62 (0.65-4.00)0.29815/118 (13)1.70 (0.71-4.06)0.232
    Race
        Non-Caucasian6/42 (14)1—10/50 (20)1—
        Caucasian28/236 (12)0.81 (0.31-2.09)0.66024/296 (24)0.35 (0.16-0.79)0.012
    Menopausal status
        Premenopausal18/152 (12)1—15/183 (8)1—
        Perimenopausal6/18 (33)3.72 (1.24-11.15)0.0195/19 (26)3.98 (1.26-12.55)0.019
        Postmenopausal10/108 (9)0.76 (0.34-1.72)0.50914/145 (10)1.19 (0.56-2.55)0.655
    NAF production
        NAF(−)11/87 (13)1—10/123 (8)1—
        NAF(+)23/191 (12)0.95 (0.44-2.05)0.90024/223 (11)1.37 (0.63-2.97)0.425
    Cellularity
        <100 cells5/79 (6)1—5/119 (4)1—
        100-999 cells12/133 (9)1.47 (0.50-4.33)0.48716/157 (10)2.59 (0.92-7.27)0.072
        ≥1,000 cells17/66 (26)5.14 (1.78-14.83)0.00313/70 (19)5.20 (1.77-15.29)0.003
    Risk classification
        Gail lower10/126 (8)1—7/155 (5)1—
        Gail high9/54 (17)2.32 (0.89-6.08)0.0876/61 (10)2.31 (0.74-7.16)0.149
        Contralateral8/47 (17)2.38 (0.88-6.46)0.0899/59 (15)3.81 (1.35-10.75)0.012
        Ipsilateral7/51 (14)1.85 (0.66-5.15)0.24212/71 (17)4.30 (1.61-11.45)0.004
    Mild atypia
        None26/222 (12)1—
        Mild atypia8/56 (14)1.26 (0.54-2.95)0.600
    Marked atypia
        None26/247 (11)1—
        Marked atypia8/31 (26)2.96 (1.20-7.28)0.018
    Any atypia
        None19/204 (9)1—
        Any atypia15/74 (20)2.48 (1.18-5.18)0.016
    Masood score
        <1522/232 (9)1—
        ≥1512/46 (26)3.37 (1.53-7.43)0.003
  • Table 5.

    Multivariate analysis

    FactorMethylation ≥2 genes
    Marked atypia
    OR (95% CI)POR (95% CI)P
    Menopausal status
        Premenopausal1—1—
        Perimenopausal8.40 (2.25-31.36)0.00211.52 (2.84-46.74)0.0006
    Cellularity
        <100 cells1—1—
        ≥1,000 cells5.35 (1.80-15.85)0.0035.72 (1.86-17.60)0.002
    Risk classification
        Gail lower1—1—
        Gail high3.51 (1.16-10.69)0.0273.48 (0.97-12.45)0.056
        Contralateral4.21 (1.28-13.82)0.0186.91 (1.95-24.48)0.003
        Ipsilateral3.70 (1.10-12.42)0.0349.48 (2.79-32.18)0.003
    • NOTE: Factors with P < 0.15 by univariate analysis were combined in multivariate analysis. Only factors with statistically significant results are shown.

  • Table 6.

    Cases with methylation fractions >0.10 for ≥2 genes

    IDClassification*Age (y)Masood scoreGenes†
    002Contralateral4718cyclin D2, APC
    026Lower5217APC, HIN1
    041Lower3715cyclin D2, APC
    054Lower4817cyclin D2, HIN1
    072High (BRCA2)4110HIN1, RASSF1A
    102Lower (BRCA2)3414cyclin D2, APC, HIN1
    113High608cyclin D2, HIN1, RAR-β2
    • NOTE: Outcome: 072 was diagnosed with a breast cancer on this side 29 mo after the lavage; 002 died of breast cancer 42 mo after the lavage; 026, 041, 054, and 102 have had normal breast magnetic resonance imaging scans; 041 was lost to follow-up. With a median follow-up of 39 mo, no other breast cancers have been diagnosed in this group.

    • ↵* Contralateral, breast contralateral to a known breast cancer; High, an unaffected woman with a 5-y Gail risk that is greater than or equal to twice the age- and race-matched general population risk; Lower, an unaffected woman with 5-y Gail risk less than twice the age- and race-matched general population risk. Subject 102 is from a BRCA2-positive ovarian cancer family. She has no family history of breast cancer and was classified as lower risk by the Gail model.

    • ↵† Genes with methylation fractions >0.10.

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Cancer Epidemiology Biomarkers & Prevention: 16 (9)
September 2007
Volume 16, Issue 9
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Atypia and DNA Methylation in Nipple Duct Lavage in Relation to Predicted Breast Cancer Risk
David M. Euhus, Dawei Bu, Raheela Ashfaq, Xian-Jin Xie, Aihua Bian, A. Marilyn Leitch and Cheryl M. Lewis
Cancer Epidemiol Biomarkers Prev September 1 2007 (16) (9) 1812-1821; DOI: 10.1158/1055-9965.EPI-06-1034

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Atypia and DNA Methylation in Nipple Duct Lavage in Relation to Predicted Breast Cancer Risk
David M. Euhus, Dawei Bu, Raheela Ashfaq, Xian-Jin Xie, Aihua Bian, A. Marilyn Leitch and Cheryl M. Lewis
Cancer Epidemiol Biomarkers Prev September 1 2007 (16) (9) 1812-1821; DOI: 10.1158/1055-9965.EPI-06-1034
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