Article Figures & Data
Figures
- 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.
- 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;
), infiltrating ductal carcinoma (), infiltrating lobular carcinoma (), other (medullary or metaplastic; ); any ductal carcinoma in situ: yes (), no (); methylation fraction: 0% (), >10% (), no result (); Cyto is NDL cytology: Masood score ≥15 (), Masood score 11-14 (), Masood score ≤10 (). Ducts classified as cytologically atypical with methylation results that were concordant with the primary tumor for ≥4 genes are marked with asterisk. - 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.
, ducts from unaffected Gail lower-risk women; , ducts from unaffected Gail high-risk women; , ducts contralateral to a breast cancer; , ducts ipsilateral to a breast cancer.
Tables
- Table 1.
Primers and probes for quantitative multiplex methylation-specific real-time PCR
Forward Reverse Cyclin D2 R1 TATTTTTTGTAAAGATAGTTTTGAT TACAACTTTCTAAAAAATAACCC R2 UM TTAAGGATGTGTTAGAGTATGTG AAACTTTCTCCCTAAAAACCAACTACAAT R2 M TTTGATTTAAGGATGCGTTAGAGTACG ACTTTCTCCCTAAAAACCGACTACG P UM HEX-AATCCACCAACACAATCAACCCTAAC-BHQ1 P M 6FAM-AATCCGCCAACACGATCGACCCTA-BHQ1 APC R1 GGGTTAGGGTTAGGTAGGTTGTG AACTACACCAATACAACCACATA R2 UM GTGTTTTATTGTGGAGTGTGGGTT CCAATCAACAAACTCCCAACAA R2 M TATTGCGGAGTGCGGGTC TCGACGAACTCCCGACGA P UM 6FAM-AACACCCTAATCCACATCCAACAAAT-BHQ1 P M 6FAM-AACGCCCTAATCCGCATCCAACGA-BHQ1 HIN1 R1 GTTTGTTAAGAGGAAGTTTT CCGAAACATACAAAACAAAACCAC R2 UM AAGTTTTTGAGGTTTGGGTAGGGA ACCAACCTCACCCACACTCCTA R2 M TAGGGAAGGGGGTACGGGTTT CGCTCACGACCGTACCCTAA P UM HEX-CAACTTCCTACTACAACCAACAAACC-BHQ1 P M 6FAM-ACTTCCTACTACGACCGACGAACC-BHQ1 RASSF1A R1 GTTTTATAGTTTTTGTATTTAGG AACTCAATAAACTCAAACTCCC R2 UM GGTGTTGAAGTTGGGGTTTG CCCATACTTCACTAACTTTAAAC R2 M GCGTTGAAGTCGGGGTTC CCCGTACTTCGCTAACTTTAAACG P UM HEX-CTAACAAACACAAACCAAACAAAACCA-BHQ1 P M 6FAM-ACAAACGCGAACCGAACGAAACCA-BHQ1 RAR-β2 R1 GTAGGAGGGTTTATTTTTTGTT AATTACATTTTCCAAACTTACTC R2 UM TTGAGAATGTGAGTGATTTGAGTAG TTACAAAAAACCTTCCAAATACATTC R2 M AGAACGCGAGCGATTCGAGTAG TACAAAAAACCTTCCGAATACGTT P UM HEX-AAATCCTACCCCAACAATACCCAAAC-BHQ1 P M 6FAM-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
Patients 150 Mean age (range), y 48 (28-93) Ethnicity, % Caucasian 123 (82) African American 20 (13) Hispanic 5 (3) Asian 2 (1) Menopausal status, % Premenopausal 73 (49) Perimenopausal 8 (5) Postmenopausal 69 (46) Oral contraceptive use (premenopausal) 18/73 (38) Hormone replacement (peri- and post-menopausal) 25/77 (32) Risk groups Breast cancer patients 67 (45) Breasts ipsilateral to a breast cancer 65* Ductal carcinoma in situ only 6 Infiltrating ductal carcinoma 50 Infiltrating lobular carcinoma 7 Medullary carcinoma 1 Metaplastic carcinoma 1 Any associated DCIS 53 (82) Breasts contralateral to a breast cancer 62† Unaffected risk assessed patients 83 (55) History of atypical ductal hyperplasia 4 (5) BRCA gene mutation 5 (6) Lower risk by all models‡ 31 (37) High risk by Gail only§ 12 (15) High risk by Claus or BRCAPRO only 29 (35) High risk by both Gail and a family history model 11 (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
Gene Methylation prevalence Median methylation fraction Correlation* Tumor FNA (%) NDL (%) P Tumor FNA NDL P Coefficient P Cyclin D2 37.1 8.7 0.004 0.198 0.078 0.428 0.04 0.846 APC 38.2 13.0 0.019 0.379 0.041 0.005 -0.14 0.431 HIN1 44.1 13.3 0.005 0.730 0.114 0.004 0.07 0.687 RASSF1A 61.8 8.7 <0.0001 0.431 0.064 0.045 -0.21 0.238 RAR-β2 26.5 6.5 0.031 0.028 0.310 0.166 -0.06 0.744 ↵* Spearman correlation between paired methylation fractions.
- Table 4.
Univariate analysis to identify factors predicting TSG methylation or marked atypia
Factor Methylation of ≥2 genes Marked atypia Pos/Neg (%) OR (95% CI) P Pos/Neg (%) OR (95% CI) P Age (tertiles) ≤41.3 9/93 (10) 1 — 9/114 (8) 1 — 41.4-50.1 12/97 (12) 1.32 (0.53-3.29) 0.555 10/114 (9) 1.12 (0.44-2.87) 0.811 >50.1 13/88 (15) 1.62 (0.65-4.00) 0.298 15/118 (13) 1.70 (0.71-4.06) 0.232 Race Non-Caucasian 6/42 (14) 1 — 10/50 (20) 1 — Caucasian 28/236 (12) 0.81 (0.31-2.09) 0.660 24/296 (24) 0.35 (0.16-0.79) 0.012 Menopausal status Premenopausal 18/152 (12) 1 — 15/183 (8) 1 — Perimenopausal 6/18 (33) 3.72 (1.24-11.15) 0.019 5/19 (26) 3.98 (1.26-12.55) 0.019 Postmenopausal 10/108 (9) 0.76 (0.34-1.72) 0.509 14/145 (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) 0.900 24/223 (11) 1.37 (0.63-2.97) 0.425 Cellularity <100 cells 5/79 (6) 1 — 5/119 (4) 1 — 100-999 cells 12/133 (9) 1.47 (0.50-4.33) 0.487 16/157 (10) 2.59 (0.92-7.27) 0.072 ≥1,000 cells 17/66 (26) 5.14 (1.78-14.83) 0.003 13/70 (19) 5.20 (1.77-15.29) 0.003 Risk classification Gail lower 10/126 (8) 1 — 7/155 (5) 1 — Gail high 9/54 (17) 2.32 (0.89-6.08) 0.087 6/61 (10) 2.31 (0.74-7.16) 0.149 Contralateral 8/47 (17) 2.38 (0.88-6.46) 0.089 9/59 (15) 3.81 (1.35-10.75) 0.012 Ipsilateral 7/51 (14) 1.85 (0.66-5.15) 0.242 12/71 (17) 4.30 (1.61-11.45) 0.004 Mild atypia None 26/222 (12) 1 — Mild atypia 8/56 (14) 1.26 (0.54-2.95) 0.600 Marked atypia None 26/247 (11) 1 — Marked atypia 8/31 (26) 2.96 (1.20-7.28) 0.018 Any atypia None 19/204 (9) 1 — Any atypia 15/74 (20) 2.48 (1.18-5.18) 0.016 Masood score <15 22/232 (9) 1 — ≥15 12/46 (26) 3.37 (1.53-7.43) 0.003 - Table 5.
Multivariate analysis
Factor Methylation ≥2 genes Marked atypia OR (95% CI) P OR (95% CI) P Menopausal status Premenopausal 1 — 1 — Perimenopausal 8.40 (2.25-31.36) 0.002 11.52 (2.84-46.74) 0.0006 Cellularity <100 cells 1 — 1 — ≥1,000 cells 5.35 (1.80-15.85) 0.003 5.72 (1.86-17.60) 0.002 Risk classification Gail lower 1 — 1 — Gail high 3.51 (1.16-10.69) 0.027 3.48 (0.97-12.45) 0.056 Contralateral 4.21 (1.28-13.82) 0.018 6.91 (1.95-24.48) 0.003 Ipsilateral 3.70 (1.10-12.42) 0.034 9.48 (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
ID Classification* Age (y) Masood score Genes† 002 Contralateral 47 18 cyclin D2, APC 026 Lower 52 17 APC, HIN1 041 Lower 37 15 cyclin D2, APC 054 Lower 48 17 cyclin D2, HIN1 072 High (BRCA2) 41 10 HIN1, RASSF1A 102 Lower (BRCA2) 34 14 cyclin D2, APC, HIN1 113 High 60 8 cyclin 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.
Volume 16, Issue 9
