Current and emerging technologies for DNA methyla7on analysis. What a pathologist needs to know.

Transcription

1 Current and emerging technologies for DNA methyla7on analysis. What a pathologist needs to know. RCPA Short Course in Medical Gene6cs and Gene6c Pathology 20 June 2011 Alex Dobrovic Molecular Pathology Research and Development Laboratory Peter MacCallum Cancer Centre

2 Epigene6c Diagnos6cs DNA methyla6on as a marker of response to therapy diagnosis of a condi6on or predisposi6on to a condi6on diagnos6c use requires appropriately robust sensi6ve and specific methodology

3 What are we measuring?

4 PCR based methods capable of detec6ng methyla6on at all sites from limi6ng amounts of material some may be used with degraded material eg formalin fixed paraffin sec6ons PCR amplifica6on loses methyla6on informa6on!

5 Bisulfite modifica6on converts non- methylated cytosine residues of single stranded DNA to uracil uracil converted to thymine by PCR any Cs correspond to methylated Cs GACAT CG!-> GATATTG! GACAT m CG!-> GATATCG!

6 Bisulfite sequencing

7 Methyla6on specific PCR (MSP) rapid screening specifically amplifies methylated sequences by using 3 mismatches at Cs of CpG sites - only assays a few sites sensi6ve to approximately 0.1% not quan6ta6ve

8 MSP detection of APC methylation in breast cancer cell lines Marker T24 T42D ZR75 MCF7 MB 468 HS578t BT20 MB 435 MB453 MB231 + control

9 When do we want to sensi6vely measure methyla6on? cancer biomarker for early detec6on cancer biomarker for monitoring disease aver therapy (mrd) plasma DNA marker of disease predisposi6on in normal 6ssues (cons6tu6onal soma6c methyla6on)

10 MethyLight FAM TAMRA FAM

11 MethyLight Quan6ta6ve adapta6on of MSP based on TaqMan Real 6me analysis of amplifica6on lends itself to high throughput analysis Poten6ally sensi6ve to 1/10,000 Allows discrimina6on of signal from background Suitable for monitoring treatment using methyla6on as a tumour marker Eads et al. (2000)

12 Methodologies based on high resolution melting (HRM) fluorescent dyes dyes fluoresce only when intercalated into ds DNA heat PCR product strands separate (melt) monitor fluorescence derive melting profile melt temperature dependent on sequence closed tube analysis of the PCR product

13 Methyla6on- sensi6ve HRM(MS- HRM) MGMT

14 BRCA1 methyla6on from FFPE A B

15 no BRCA1 methylation in NSCLC No methylation detected in 56 N1 NSCLC samples 18% - Wang % - Lee 2007

16 SMART- MSP uses dye instead of probe

17 MGMT methyla6on - a predic6ve marker

18 Heterogeneous MGMT methyla6on Patient MDA-MB % 10% WGA

19 MGMT methyla6on - Tm curves WGA 100% Patient 10%

20 MGMT methyla6on associated with the T allele of a promoter SNP in normal 6ssues Methylated Not methylated CC (3) CT and TT p <

21 MGMT are we measuring tumour specific methyla6on tumour MGMT methyla6on frequency es6mates by MSP are exaggerated Is the tumour heterozygously or homozygously methylated?

22 What does quan6fica6on of methyla6on mean? Are we quan6fying CpG sites? Are we quan6fying methylated alleles ( epialleles )

23 Overall readout between primers HRM SSCA DGGE

24 C D Average reading at each position between primers Direct bisulfite sequencing Bisulfite pyrosequencing MassARRAY ->Quantitation at individual CpGs

25 Sequenom MassArray

26 Pyrosequencing

27 DAPK1 MS- HRM

28 Pyrosequencing reveals heterogeneity Sample S S S S S

29 Reading of epialleles Cloned from undiluted templates: Bisulfite genomic Sequencing Limiting dilution Digital MS-HRM & sequencing Massively parallel sequencing ->epiallele quantification

30 Digital MS- HRM eliminates heteroduplexes eliminates bias will dis6nguish PCR amplifica6on posi6ve wells from primer- dimers counts the methylated alleles assesses extent of methyla6on of each allele only methylated alleles need to sequenced

31 DAPK1 dms- HRM

32 13,14,23,18 15

33 Future prospects deep massively parallel sequencing methylbeaming cheap digital nanotechnology approaches bisulfite modifica6on free methodologies Array based methodologies.

34 Infinium: CDH1 methyla6on

35 Take home messages appropriate choice of methodology relates to ques6on asked diagnos6c needs to be FFPE friendly needs to be quan6ta6ve/ related to tumour purity most methods perform well for homogeneous methyla6on heterogeneous methyla6on is difficult to quan6fy without digital methologies much of the current literature is unreliable