Issues rela)ng to detec)on of soma)c muta)ons What a pathologist needs to know RCPA Short Course in Medical Gene)cs and Gene)c Pathology 21 June 2011 Why do we want to characterise soma6c muta6ons? •  Prognos6c biomarkers –  TP53 muta6on •  Early detec6on biomarkers –  need to be high frequency early changes –  KRAS muta6on in pancrea6c •  Residual disease biomarkers –  need to be early changes •  Predic6ve biomarkers (response to therapy) –  EGFR TKD muta6on, KRAS muta6on Soma6c muta6ons & cancer therapy Posi6ve predic6ve markers –  therapeu6c index –  small molecule inhibitors/an6bodies –  targets cancer specific changes –  oncogenic addic6on •  EGFR TKD muta6ons –  gefi6nib (Iressa) –  erlo6nib (Tarceva) Nega6ve predic6ve markers •  KRAS codon muta6ons –  cetuximab (Erbitux) 2004: EGFR muta6ons underlie sensi6vity to gefi6nib 2009: IPASS trial Phase III, randomized, first-­‐line study of gefi6nib vs carbopla6n/paclitaxel in selected pts with advanced NSCLC Issues (6ssue) •
•
•
•
•
•
Tissue availability (if and when) Tissue fixa6on Tissue amount Tissue purity Mul6ple tes6ng Appropriate and 6mely methodology Tissue is the issue •  surgically removed 6ssue normally FFPE –  small biopsies –  low propor6on of tumour –  macrodissec)on is key •  if not surgically resected –  core biopsies –  fine needle aspirates –  bronchial washings –  plasma DNA, CTCs Muta6ons in tumour samples •  May be present in variable propor6on –  Purity of tumour sample –  Gene6c heterogeneity •  Need appropriate muta6on detec6on protocol –  Direct sequencing –  High resolu6on mel6ng/sequencing –  Commercial direct iden6fica6on (subset) •  DxS real-­‐6me PCR (ARMS/Scorpions) (Therascreen) •  SNAPShot •  CAST PCR The “gold standard” Sanger sequencing 2235_2249del15 E746_A750del
Pyrosequencing •  ideal for short fragments –  target muta6ons must be rela6vely localised To sequence or to scan •  Frequency of muta6on •  Loca6on of muta6ons in hotspots •  Turnaround 6me Scanning methodologies Principles of HRM
•
heat PCR product
•
•
dyes fluoresce only when
intercalated into ds DNA
•
•
strands separate (melt)
monitor fluorescence
melt temperature dependent on sequence
•
•
mutations will have different melt temperature
heteroduplexes aid mutation detection
Muta6on detec6on by HRM Heteroduplex formation
normalised melting curve
EGFR HRM -­‐ normalised melt PC9 cell line: 15bp
deletion, 2235-2249
Patient 9: deletion
present at low %,
from 2235 onwards
Patient 8: 15bp
deletion, 2235-2249
wt
EGFR HRM – Difference plot PC9 cell line: 15bp
deletion, 2235-2249
Patient 9: deletion
present at low %
Patient 8: 15bp
deletion, 2235-2249
wt
Patient 16:
EGFR Sequencing Wild type sequence
Patient 8: deletion,
predominant
Patient 9: deletion
minor
HRM •  Cost effec6ve •  Sensi6ve •  Works with FFPE •  Must be sequencing validated –  Pyrosequencing •  Can sequence directly from PCR product •  2 amplifica6on replicates minimum •  HRM acts as QC for sequencing Working from FFPE •  extensive valida6on of HRM screening from FFPE 6ssues •  200 pa6ents sent for EGFR sequencing •  -­‐ 73 EGFR muta6ons •  -­‐ 25 KRAS muta6ons •  HRM led to a decrease of sequencing by 80% Sensi6vity:HRM vs. Sanger sequencing WT
1%
5%
10%
20%
KRAS c.38G>A mutation
HRM posi6ve -­‐ sequencing nega6ve •  need to validate HRM posi6ves •  limi6ng dilu6on >limited copy number templates •  “find the needle by making the haystack smaller” 1. LCN-HRM set-up
4. Sanger sequencing
2. PCR amplification
3. Melting curve analysis
•  Measurement of the
amount of templates using
qPCR.
•  An average of 3-7 copies of
templates per reaction.
•  Stochastic mutant allele
enrichment.
5% allele freq cell lines LCN-­‐HRM detects false posi6ves False posi6ves in the literature False posi6ves in the literature False posi6ves from FFPE •  exacerbated by low copy numbers •  can be more than 10% damaged templates in FFPE •  short amplicons increase the success rate •  badly damaged samples will give false posi6ves across mul6ple exons •  sequencing iden)fica)on of independent PCR products necessary The future •  The death of companion diagnos6cs •  Next genera6on sequencing tsunami –  detec6on of mul6ple muta6ons at high resolu6on (deep sequencing) Take home points •  Sanger sequencing is not the gold standard •  Methodology must work with FFPE •  Tumour enrichment is ohen necessary –  essen6al role of anatomical pathologist •  HRM screening amplicons prior to sequencing reduces costs –  Pyrosequencing of HRM products –  Hongdo Do False posi6ves Direct readout PCR methods •  rapid turnaround of results •  allele specific-­‐ only screen for common muta6ons –  DxS –  Sequenom Oncocarta •  Sequencing based –  Sanger Sequencing (HRM) –  Pyrosequencing (HRM) –  Next Gen sequencing DxS Therascreen EGFR29 muta6on test •
•
•
•
•
•
•
19 Dele6ons in exon 19 (does not dis6nguish) T790M L858R L861Q G719X ( G719S, G719A or G719C) S768I 3 inser6ons in exon 20 (does not dis6nguish) •  cost!!! •  sensi6vity DxS -­‐ ARMS & Scorpions