I.M. Sechenov First Moscow State Medical University MOSCOW 2011

1. Non-microarray DNA differential methylation screening in breast cancer. Investigation of new markers of carcinogenesis I.M. SechenovFirstMoscowStateMedicalUniversity MOSCOW 2011 Rudenko V.V.

2. «We are more than the sum of our genes». (Klar 1998) «You can inherit something beyond the DNA sequence. That’s where the real excitement in genetics is new». (Watson 2003)

3. Genetics vs. Epigenetics Genetics: mutations of the DNA template are heritable somatically and through the germ line. Epigenetics: variations in chromatin structure modulate the use of the genome by 1. histone modifications 2. chromatin remodeling 3. histone variant composition 4. DNA methylation, and noncoding RNAs. Marks on the chromatin template may be heritable through cell division and collectively contribute to determining cellular phenotype.

5. Epigenetic and Cancer Accumulation of aberrant epigenetic modifications is associated with tumor cells. The total hypomethylation of DNA was, in fact, the first type of epigenetic transition to be associated with cancer (Feinberg and Vogelstein, 1983). Conversely, DNA hypermethylation is concentrated at the promoter regions of CpG islands in many cancers.

6. TECHNOLOGICAL ADVANTAGES OF DETERMINING THE METHYLATION MARKERS IN CANCER VERSUS DETECTION OF MUTATIONS / DELETIONS OF THE DNA • Disturbancesin DNA methylationare amongtheearliestprocessesofcarcinogenesis. • Frequenciesofabnormalmethylationofa numberofgenesconsiderablyexceedsfrequenciesofstructuraldamagesofthesamegenesincancer. • Identificationofabnormallymethylated DNA moleculesinexcessofthenormalbiologicalmaterialdoesnotcauseproblems, whiledetectionofmutations / deletionsinthissituationispracticallyimpossible. • Analysisofmethylationofonegeneistechnicallysimpleandinmostcasesitcomesdownto PCR withonetothreepairsofprimers. Searchformutationsinonegenerequirestheuseofuptoseveraldozenpairsofprimers, inaddition, satisfactoryperformanceisachievedonlyafterthesequencingofthe PCR fragment.

7. TECHNOLOGICAL ADVANTAGES OF THE DEFINITION OF MARKERS OF DNA METHYLATION IN CANCER VERSUS DETECTION OF EXPRESSION MARKERS AT THE RNA LEVEL • Thestabilityof DNA ismuchhigher. • Abnormallymethylated DNA canbedetectedinbloodplasmaandotherbiologicalfluids (non-invasive / low-invasivetests). • Abnormalmethylationisaqualitativeratherthanquantitativetrait, sothereliabilityofitsdeterminationisnotaffectedbythepresenceofnucleicacidsfromnormaltissues.

10. A value of an IM (methylation index) in group T1 (<2sm) 0.46±0.14, in group T2 0.68±0.22 (2÷5см), p<0.01; r=0.48 (p<0.001).

13. Silencing of tumor suppressor genes through aberrant DNA hypermethylation is particularly critical in cancer progression. There is considerable cross talk between chromatin modifications and DNA methylation, demonstrating that more than one epigenetic mechanism can be involved in the silencing of a tumor supressor gene.

14. Cliniko-geneticassociations

19. Conclusions 1. The developed algorithm, laboratory protocols, and software have allowed us to reveal 27 novel loci prone to abnormal methylation in breast cancer, as well as a number of constantly methylated and constantly unmethylated genome loci. 2. Analysis of methylation of the genomic regions can enter the system of epigenetic classification of breast cancer on the grounds of: histological type, stage, grade and expression of estrogen and progesterone receptors. 3. Tumor size in this system has the only epigenomic qualifier, which is the index of metylation. 4. The system of classification of breast tumors based on the features of epigenomic pathology does not cover the whole spectrum of clinical and morphological characteristics of tumors taken into consideration. This suggest that for a satisfactory complete portrait of breast cancer epigenetic panel should include more methylation markers.

20. Acknowledgements TanasA.S. KuznetsovaE.B. Strelnikov V.V. Zaletaev D.V.