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Clinical Cancer Research Vol. 12, 3306-3310, June 1, 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Quantitative Multiplex Methylation-Specific PCR Analysis Doubles Detection of Tumor Cells in Breast Ductal Fluid

Mary Jo Fackler1, Kara Malone1, Zhe Zhang1, Eric Schilling3, Elizabeth Garrett-Mayer1, Theresa Swift-Scanlan1, Julie Lange2, Ritu Nayar4, Nancy E. Davidson1, Seema A. Khan3 and Saraswati Sukumar1

Authors' Affiliations: Departments of 1 Oncology and 2 Surgery, Johns Hopkins University School of Medicine, Baltimore Maryland and Departments of 3 Surgery and 4 Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Requests for reprints: Saraswati Sukumar, Breast Cancer Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Room 410, Bunting/Blaustein Building, 1650 Orleans Street, Baltimore, MD 21231-1000. Phone: 410-614-2479; Fax: 410-614-4073; E-mail: saras{at}jhmi.edu.


   Abstract
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 Abstract
Materials and Methods
 Results and Discussion
 References
 
Purpose: The challenges of cytology for accurate diagnosis of breast cancer are well recognized. We previously showed that normal and tumor tissue can be distinguished using a technique called quantitative multiplex methylation-specific PCR (QM-MSP). We hypothesized that quantitative analysis of methylated genes will provide enhanced detection of cancer cells present in cytologic specimens.

Experimental Design: QM-MSP was done on ductal lavage cells from a set of 37 ductal lavage samples from women undergoing mastectomy (27 with cancer and 3 without). Duct histology information was available for each lavaged duct. QM-MSP data was assessed by measuring cumulative methylation index and by receiver operating characteristic threshold analysis. To determine the baseline level of methylation for each gene in this population, cells from 60 ducts of women at high risk of developing breast cancer were analyzed.

Results: QM-MSP findings on a panel of nine genes were correlated to duct histology and ductal lavage cytology. Cytology detected cancer in 33% (7 of 21 ducts) with a specificity of 99% (92 of 93). QM-MSP detected cancer as calculated by cumulative methylation index with a sensitivity of 62% (13 of 21) and specificity of 82% (62 of 76) and by receiver operating characteristic threshold analysis with a sensitivity of 71% (15 of 21) and specificity of 83% (63 of 76).

Conclusions: Compared with cytology, QM-MSP doubled the sensitivity of detection of cancer. This study provides proof of principle by showing the advantages of using methylation analyses to query cytologic specimens and indicates its potential use in diagnosis and in stratifying risk.

Detection of breast cancer is dependent on sensitive screening methods. Mammography is widely used but is falsely negative in 15% of women due to factors, such as breast density. This has led to a search for improved methods of imaging and sampling of breast tissue for cytologic examination (1–4). Cytology is the current gold standard for the identification of abnormalities typical of cellular transformation. However, recent findings have cast doubt on its effectiveness as a single discriminator of cancer cells. For instance, a recent study documented lack of reliability of cytology in cells collected by ductal lavage in women with biopsy-proven breast cancer (5). Here, similar to previous reports on cells derived through ductoscopy (6) or fine needle aspiration (7), the sensitivity of cytology to detect cancer in ductal lavage was determined to be as low as 43% (5). Additional methods to identify tumor cells, such as those detecting molecular alterations, are clearly needed (8, 9).

Multigene methylation of CpG islands is common in early breast cancer and leads to silencing of genes responsible for tumor suppression (10–13). Recently, we developed a highly sensitive method called quantitative multiplex methylation-specific PCR (QM-MSP) to quantitate cumulative gene promoter hypermethylation in multiple genes in samples where DNA is limiting, such as ductal lavage, nipple aspiration fluids, and fine needle aspirates (12). This method, using a five-gene panel consisting of RASSF1A, RARß, TWIST, HIN1, and Cyclin D2, evaluated percent methylation values in each gene and provided a clear distinction between normal and tumor tissues (12).

A question that is central to efforts aimed at risk assessment and early detection is whether specific molecular alterations typifying tumor can be more sensitive than and precede the appearance of morphologic abnormalities detectable by cytology. Based on the high sensitivity of QM-MSP for detecting small numbers of cancer cells, we addressed the hypothesis that QM-MSP analyses could detect cancer more frequently than ductal fluid cytology. The study was conducted on ductal lavage samples from women undergoing mastectomy. Lavaged ducts were tagged with different colored dyes that permitted the matching of each cytology specimen with duct histology. This study design enabled a direct comparison of results of a molecular assay with that of cytology of the cells collected from ducts of known histology from the diseased breast.


   Materials and Methods
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 Abstract
 Materials and Methods
Results and Discussion
 References
 
DNA extraction. For high-risk women, ductal cells were harvested from ThinPrep vials (Cytyc Corp., Marlborough, MA) and processed for methylation studies. For ductal lavage samples from women undergoing mastectomy, cells were scraped from Papanicolaou-stained slides after cytologic review of ductal cells. DNA was prepared by lysing ductal cells in 30 µL of 10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% SDS, 66 ng/µL salmon sperm DNA containing 40 µg proteinase K for 4 hours at 52°C. Sodium bisulfite treatment was scaled down to a micromethod: DNA (13.5 µL) was heated 10 minutes to 99°C, quick chilled, and incubated with 1.5 µL of 2 mol/L NaOH for 30 minutes at 42°C. Freshly prepared 3.6 mol/L sodium bisulfite containing 1 mmol/L hydroquinone (95 µL) was mixed with the DNA, then overlaid with oil, and incubated at 55°C for 5 hours in the dark. The sample was desalted using ion exchange columns (MicroSpin S200, Amersham, Piscataway, NJ). DNA was precipitated and resuspended in water (5 µL). All of the patients provided informed consent for the research use of their samples and the collection, and use of samples for this study was approved by the appropriate institutional review boards.

QM-MSP analysis. QM-MSP was determined on samples blinded for cytology. QM-MSP was done (12), and the percent methylation for each gene in the panel was calculated as %M = [M / (U + M)] x 100 (6). To determine the cumulative methylation index, the sum of %M for all genes was determined. For example, for nine genes, 100% M x 9 genes = cumulative methylation index of 900. The external primers used were APC1 Ext F(2), 5'-AAAACCCTATACCCCACTAC; APC1 Ext R(2), 5'-GGTTGTATTAATATAGTTATATGT; BRCA1 Ext F, 5'-TATTTTGAGAGGTTGTTGTTTAG; BRCA1 Ext R, 5'-AAACATCACTTAAACCCCCTAT; BRCA2 Ext F, 5'-GTTGGGATGTTTGATAAGGAAT; BRCA2 Ext R, 5'-ATCACAAATCTATCCCCTCAC; p16 Ext F(3), 5'-AAAGAGGAGGGGTTGGTTG; p16 Ext R(5), 5'-AACCCTCTACCCACCTAAAT; HIC1 Ext F, 5'-TTTAGTTGAGGGAAGGGGAA; HIC1 Ext R, 5'-AACTACAACAACAACTACCTAA. The internal primers used were APC1 RT-FUM, 5'-TAAATACAAACCAAAACACTCCC; APC1 RT-RUM, 5'-GTTATATGTTGGTTATGTGTGTTT; APC1 UM probe, 5'-TTCCCATCAAAAACCCACCAATTAAC; APC1 RT-FM, 5'-AATACGAACCAAAACGCTCCC; APC1 RT-RM, 5'-TATGTCGGTTACGTGCGTTTATAT; APC1 M probe, 5'-CCCGTCGAAAACCCGCCGATTA; BRCA1 RT-FUM, 5'-TGGTAATGGAAAAGTGTGGGAA; BRCA1 RT-RUM(4), 5'-CCCATCCAAAAAATCTCAACAAA; BRCA1 UM probe, 5'-CTCACACCACACAATCACAATTTTAAT; BRCA1 RT-FM, 5'-TTTCGTGGTAACGGAAAAGCG; 5'-BRCA1 RT-RM, 5'-CCGTCCAAAAAATCTCAACGAA; BRCA1 M probe, 5'-CTCACGCCGCGCAATCGCAATTT; in the same CpG island described in ref. (14) for BRCA2 RT-FUM, 5'-ATTTTTGGGTGGTGTGTGTGTT; BRCA2 RT-RUM, 5'-TCAAAAACTCACACCACAAACC; BRCA2 UM probe, 5'-AACCACATAACACCATAACACAACAC; BRCA2 RT-FM, 5'-TTTGATTTTCGGGTGGTGCGT; BRCA2 RT-RM, 5'-TCAAAAACTCGCGCCACAAAC; BRCA2 M Probe, 5'-AACCACGTAACGCCGTAACGCGA; p16 RT-FUM(2), 5'-TTATTAGAGGGTGGGGTGGATTGT; p16 RT-RUM, 5'-CAACCCCAAACCACAACCATAA; p16 UM probe, 5'-CTACTCCCCACCACCCACTACCT; p16 RT-FM(2), 5'-TTATTAGAGGGTGGGGCGGATCGC; p16 RT-RM, 5'-GACCCCGAACCGCGACCGTAA; p16 M probe, 5'-AGTAGTATGGAGTCGGCGGCGGG. Primers and probes for RASSF1A, TWIST, Cyclin D2, RARß, and HIN1 were reported previously (12).

Statistical analysis. Cytology was classified as benign, mildly atypical, markedly atypical, and malignant (5). A positive cytologic test was defined as marked atypia or malignant appearance. A side-by-side comparison of the rate of detection of cancer was done. Sensitivity was defined as the number of true-positive results divided by the number of true-positive results plus false-negative results. Specificity was defined as the number of true-negative results divided by the number of true-negative results plus false-positive results. Accuracy was defined as the number of true-positive results plus true-negative results divided by the total of all samples. A positive methylation test was defined using QM-MSP by two different methods: (a) cumulative methylation index above the normal threshold of 2.0; the threshold was obtained by determining the 90th percentile rank among normal controls and (b) by receiver operating characteristic threshold analysis. The performance of a single gene was assessed by constructing a receiver operating characteristic threshold. A receiver operating characteristic curve is a graphical representation obtained by plotting the sensitivity (true-positive rate) against 1 – specificity (false-positive rate), where the different points on the curve correspond to different cutoff points used to determine if the test results were positive. For each cutoff point, the sensitivity, specificity, and classification accuracy were calculated, and the associated area under the curve for the single gene test was also reported. An optimal cutoff was chosen by specifying the minimum threshold for classification accuracy as 80% and then maximizing the sensitivity. If there were multiple cutoff points in the receiver operating characteristics satisfying the criteria, the optimal threshold was the one with specificity maximized. Five genes were then combined to improve the performance of the QM-MSP test in differentiating cancer from normal ductal samples. A positive panel was defined as the % methylation of at least one gene exceeding its corresponding optimal threshold. A similar analysis was done on a larger set of nine genes. The sensitivity, specificity, and classification accuracy were calculated for the gene panel along with exact 95% confidence intervals. A two-sided z test was used to compare sensitivity, specificity, and classification accuracy of QM-MSP relative to cytology. Statistical tests were considered statistically significant at P < 0.05. Statistical analyses were done using SAS (version 9.1) and STATA (version 8) software packages.


   Results and Discussion
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 Abstract
 Materials and Methods
 Results and Discussion
References
 
Methylation in ductal cells from high-risk women. QM-MSP allows the establishment of a laboratory cutoff based on the reference range of low level methylation in samples. To establish the reference range of methylation in ductal lavage samples, QM-MSP was done for the five-gene panel consisting of RASSF1A, RARß, TWIST, HIN1, and Cyclin D2, in 60 samples of ductal lavage cells from 34 women at a high risk of developing breast cancer (≥1.7% chance of developing invasive breast cancer after 5 years by the Gail model). A cutoff for cumulative methylation index in ductal cells was defined as 2.0, such that 13.3% of the samples exhibited positive signals. Thus, ductal cells from most mammographically and clinically normal high-risk women displayed little to no methylation in the five-gene panel.

Methylation in ductal cells from women with cancer. Next, we examined 37 archival ductal lavage samples from 30 women before mastectomy: 27 women (33 ducts) had biopsy proven carcinoma in situ or invasive cancer, and three women (4 ducts) had no known cancer (underwent prophylactic mastectomy; one of the four ducts had occult cancer; ref. 5). QM-MSP analysis was done blinded to cytology. Both lavage cytology and duct histology information were available for each sample, as tracking dye was infused into the duct after lavage, so that correlations could be made with the results of the molecular analysis.

In the mastectomy group, a three-way comparison among duct histology, lavage cell cytology, and methylation showed the following. Cytology of ductal lavage fluid detected cancer in ducts proven by histology to have ductal carcinoma in situ or invasive cancer in 33% of the cases (7 of 21 ducts; Tables 1 and 2 ), with a specificity of 98.6% (72 of 73) and accuracy of 84.0% (79 of 94; Table 1). Thus, cytology was found relatively insensitive but highly specific in the ductal lavage setting, confirming earlier findings (5). Although 3 of the 37 ductal lavage samples had insufficient cellular material for diagnosis by cytology, methylation results were informative. QM-MSP analysis was positive for methylation in ductal lavage with a sensitivity of 57.1% (12 of 21), specificity of 82.9% (63 of 76), and accuracy of 77.3% (75 of 97; Tables 1-3 ; Fig. 1 ).


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  		Table 1. Comparison of cytology versus QM-MSP for ductal lavage studies

 

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  		Table 2. Distribution of ductal lavage samples by QM-MSP, histology, and cytology

 

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  		Table 3. Single gene and cumulative methylation index of QM-MSP for the nine-gene panel

 

Figure 1
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  		Fig. 1. Gene promoter methylation analysis in ductal lavage fluid from cancer-free women at high risk for breast cancer and in women undergoing mastectomy for breast cancer. Based on the 90th percentile rank of 60 high-risk control women (A, scale 0-20 cumulative methylation index units), an upper threshold of normal was defined (horizontal line in A and B; for 9 genes = 2.00 cumulative methylation units). For ductal lavage samples from women undergoing mastectomy, cumulative methylation index values are shown in B (scale 0-20 cumulative methylation index units). Inset, full scale (0-300 cumulative methylation index units). Top, duct histology: ductal or lobular carcinoma in situ (CIS), invasive carcinoma (INV), hyperplasia of usual type (HUT), atypical hyperplasia (AH), normal (N), and unknown (UNK). Bottom, ductal lavage cytology: benign (Ben); mild atypia (Mild); marked atypia (Mark); malignant (Malig); insufficient cellular material for diagnosis (ICMD).

 
We speculated that expansion of the panel by four more genes (APC, BRCA1, BRCA2, and p16) implicated as markers of risk (15–19) could improve the sensitivity and specificity of detection of cancer cells in ductal lavage. For the new nine-gene panel, a cutoff value of 2.0 for cumulative methylation was again established using the ductal samples from high-risk individuals (Fig. 1A). QM-MSP detected cancer by cumulative methylation index with a sensitivity of 61.9% (13 of 21), specificity of 81.6% (62 of 76), and accuracy of 77.3% (75 of 97; Fig. 1B; Tables 1-3). Receiver operating characteristic threshold analysis was also done on the data for each individual gene (Table 4 ) and then the gene panel (Table 1). With the five-gene panel, QM-MSP detected cancer with a sensitivity of 71.4% (15 of 21), specificity of 88.2% (67 of 76), and accuracy of 84.5% (82 of 97; Table 1). Analysis of the nine-gene panel did not provide a significant advantage over the five-gene panel (Table 1). These data suggest the ability of QM-MSP-based analysis of methylated genes to double the rate of detection of cancer cells compared with cytology (33% versus 71.4%, P = 0.013), with some loss of specificity (98.6% versus 88%, P = 0.01) but with no significant difference in accuracy.


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  		Table 4. Single gene receiver operating characteristic threshold analyses

 
There was complete concordance between cases with positive cytology and QM-MSP. QM-MSP detected all cytologically positive cancer, and methylation levels were highest in these samples (Fig. 1B; Table 3). In six cases, cytology was negative, and QM-MSP was positive in ducts with tumor. Thus, QM-MSP successfully detected tumor cells that were not recognized as abnormal by cytopathologists. Six ducts showed hyperplasia by histology. Among these, five were positive by QM-MSP (Fig. 1B). These results suggest that QM-MSP may detect premalignant changes not visible by histology or cytology, a concept that needs further investigation.

In summary, these data show the ability of QM-MSP-based analysis of methylated genes to double the rate of detection of cancer cells compared with cytologic examination alone. These findings provide proof of principle for the advantages of using methylation analyses to query cytologic specimens, although the limitations of ductal lavage still remain to be solved. The work also suggests that molecular alterations may occur before the appearance of the morphologic features associated with malignancy, indicating the potential use of QM-MSP in diagnosis and in stratifying risk. Furthermore, any assay that is using cytology as an end point would benefit from this molecular test.


   Footnotes
 
Grant support: National Cancer Institute Specialized Programs of Research Excellence grant P50 CA88843 (S. Sukumar), American Breast Cancer Foundation (S. Sukumar), and AVON/National Cancer Institute PFP award CA88843-AV-14P1 (S. Sukumar and S.A. Khan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/13/05; revised 2/ 7/06; accepted 2/14/06.


   References
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