Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR

doi:10.1016/S0140-6736(00)04501-3

The Lancet

Volume 357, Issue 9265, 28 April 2001, Pages 1335-1336

https://doi.org/10.1016/S0140-6736(00)04501-3 Get rights and content

Summary

If detected early, breast cancer is curable. We tested cells collected from the breast ducts by methylation-specific PCR (MSP). Methylated alleles of Cyclin D2, RAR-β, and Twist genes were frequently detected in fluid from mammary ducts containing endoscopically visualised carcinomas (17 cases of 20), and ductal carcinoma in situ (two of seven), but rarely in ductal lavage fluid from healthy ducts (five of 45). Two of the women with healthy mammograms whose ductal lavage fluid contained methylated markers and cytologically abnormal cells were subsequently diagnosed with breast cancer. Carrying out MSP in these fluid samples may provide a sensitive and powerful addition to mammographic screening for early detection of breast cancer.

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Epigenetics is a connection between genotype and phenotype, where the phenotypic changes are observed without hampering genomic sequences of an organism. Among the various epigenetic alterations, DNA methylation is relatively stable and well-established form of covalent modification, where a methyl group is attached to the 5th carbon of the cytosine ring in a gene, whereby altering the function of that gene. It directly affects gene transcription by regulating the accessibility of the gene promoter to transcription factors (TFs) toward their binding sites (TFBSs) that leads to changes in chromatin structure, thereby restricting access of TFs to the gene promoter. In order to detect the pattern of DNA methylation, bi-sulfite conversion-based method is generally considered as the “gold-standard,” which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (5mC) unchanged. The methylation-specific PCR (MSP) is the most commonly used technique that relies on this conversion method, where modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers, which has been developed as the most commonly used minimally invasive method for early diagnosis or prognosis of different types of cancer. This chapter presents a comprehensive review on the principles of bisulfite conversion of dsDNA along with its certain modifications for improving the conversion rate of C to U with a moderate degree of DNA degradation and various applications of MSP in biological research.
Ductal Lavage for Patients With Nonlactational Mastitis: A Single-Arm, Proof-of-Concept Trial
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Surgery, steroids, and/or observations alone have been proposed for patients with nonlactational mastitis (NLM), but most of these studies were retrospective. The optimal treatment for these patients remains unclear. This prospective, single-arm, proof-of-concept trial aimed to evaluate the feasibility and safety of ductal lavage as a novel treatment for patients with NLM.
Eligible patients with NLM received an intraductal infusion of corticosteroids and antimicrobial agents and returned the next day for a breast massage. This cycle was repeated for 2 wk, and we followed up these patients for 1 y. Patients did not receive surgery or steroids after ductal lavage. The primary endpoint was the time to complete response (CR).
This trial included 32 patients with a median (range) age of 32 (20-53). Skin erythema and tenderness were the major symptoms. The median (range) visual analog score was 5 (0-9). There were 21 (65.6%), 4 (12.5%), and 7 (21.9%) patients diagnosed as idiopathic granulomatous mastitis, periductal mastitis, and unspecific NLM, respectively. During the ductal lavage, the median (range) number of cannulated ducts at first attempt was 5 (3-8). Ductal lavage significantly reduced the visual analog score and mastitis score (M-score) (P < 0.01). Within a median follow-up of 15.6 mo, 93.8% (30/32) of patients achieved CR. The median (range) time to CR was 6 (0.5-21) mo. Three patients (10.0%) relapsed. No adverse events associated with ductal lavage were observed.
Ductal lavage for patients with NLM is feasible and safe, and a definitive randomized controlled trial for further investigation is warranted.
ClinicalTrials.gov, NCT02794688.
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Citation Excerpt :
DNA methylation in future may serve as a marker of breast tumor cell lineage, thereby reflecting the cell type from which a cancer originates and perhaps explaining the correlations of histological heterogeneity and prognosis of breast cancers with the DNA methylation profiles in them [19]. Past and present research studies showed that methylation of various genes such as cyclin D2, retinoic acid receptor beta (RARβ), Twist, glutathione S-transferase P1 (GSTP1), p16, p14, Ras-associated domain family member 1A (RASSF1A), and death-associated protein kinase (DAPK) were reported in ductal lavage fluid and nipple aspirate of breast cancer patients [20, 21]. Besides, detection of methylated DNA in plasma/serum of breast cancer patients is more convenient compared to detection of specific mutations for the breast cancer in whole genome.
Epigenetic alterations are clearly involved in cancer initiation and progression as recent epigenetic studies of genomic DNA, histone modifications and micro-RNA alterations suggest that these are playing an important role in the incidence of breast cancer. Epigenetic information has recently gained the attention of researchers because epigenetic modification of the genome in breast cancer is still an evolving area for researchers. Several active compounds present in foods, poisons, drugs, and industrial chemicals may as a result of epigenetic mechanisms increase or decrease the risk of breast cancer. Epigenetic regulation is critical in normal growth and development and closely conditions the transcriptional potential of genes. Epigenetic mechanisms convey genomic adaption to an environment thereby ultimately contributing towards given phenotype. In addition to the use of epigenetic alterations as a means of screening, epigenetic alterations in a tumor or adjacent tissues or peripheral blood may also help clinicians in determining prognosis and treatment of breast cancer. As we understand specific epigenetic alterations contributing to breast tumorigenesis and prognosis, these discoveries will lead to significant advances for breast cancer treatment, like in therapeutics that target methylation and histone modifications in breast cancer and the newer versions of the drugs are likely to play an important role in future clinical treatment.
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Since cytomorphological analysis of these specimens is often unsatisfactory, previous studies evaluated the possibility to detect tumour in ductal lavages by analysing promoter methylation of several genes. However, promoter methylation was also detected in healthy individuals and was unable to distinguish between pre-invasive and invasive lesions [86,87]. It is now well established that serum and plasma carry significant amount of cell-free DNA that can be analysed to detect genetic abnormalities present in the tumour.
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Fast track — Research LettersDetection of breast cancer cells in ductal lavage fluid by methylation-specific PCR

Summary

Ten-year risk of false positive screening mammograms and clinical breast examinations

N Engl J Med

Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation

Cancer Res

Fast track — Research Letters
Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR