Molecular testing for mutations –
E pluribus Unum
Dr. Harriet Feilotter
Queen’s University
Oct 19 2016
Taken from http://cancer.global-summit.com/europe/index.php
The many?
DNA
RNA
Epigenetic
SNV/indel
mRNA, rRNA, tRNA, miRNA
methylation,
CNV
piRNA, snRNA, snoRNA, Y RNA
hydroxymethyl
translocations TERT, lncRNA, gRNA, RNaseP, siRNA,
histone
shRNA, eRNA
Taken from BGI website
metabolites
lipids
Protein
localization
function
expression
modification
Why measure biomarkers?
Clinical Use
Translation
Discovery
Biomarkers in Cancer Medicine
Source: Biomarkers in Cancer- An Introductory Guide for Advocates, Research Advocacy Network, 2010.
Single Biomarkers
Sethi et al Int. J. Mol. Sci. 2013, 14(7), 14771-14784
One biomarker- one assay
Methods to detect EGFR mutations
NSCLC SNVs of interest
Pao & Hutchinson, Nature Medicine (2012)
One assay- multiple biomarkers
A “generic” approach to solid tumours
~50 gene
>2800 COSMIC variants
Target panel to a tumour type
Content developed in collaboration with the
OncoNetwork consortia
Gene List
ALK
BRAF
EGFR
ERBB2
KRAS
MAP2K1
MET
NRAS
PIK3CA
ROS1
TP53
Assays designed to detect
• Primary tumor drivers
• Resistance mutations
169 hotspot mutations like
EGFR: T790M, L858R, Exon19 del, C797S
KRAS: G12X, G13X, Q61X
ALK: 1151Tins, L1152R, C1156Y
BRAF: V600E
Detect rare variants present down to 0.1% with
Mean Sensitivity – 90%
Mean Specificity – 98%
Identification of key variants in
cfDNA
40,000
35,000
35
cfDNA
Limit of Detection
1 ng
0.6% LOD
5 ng
0.25% LOD
10 ng
0.15% LOD
10
20 ng
0.1% LOD
5
30 ng
0.05% LOD
0.05%
30
30,000
25,000
25
0.09%
20
20,000
0.11%
15
15,000
0.15%
10,000
0.25%
5,000
0.40%
0.60%
0.00%
0.10%
0.20%
0.30%
0.40%
0.50%
0.60%
0
0.70%
NSCLC fusions of interest
Fusion Transcript Design Strategy
ALK-positives
ALK-negatives
3’-5’ assays to supplement
identification of fusion products
RET-positives
RET-negatives
Expect the ratio to deviate from
1 if fusion is present and
being expressed
Specific value determined for each
gene
ROS1-positives
ROS1-negatives
One assay- multiple biomarker
types
DNA analysis
Lung AmpliSeq ™
panelv2
DNA mutations
Tumor
Sample
Ion Reporter ™ Software
Ion PGM ™ System
RNA lung fusion research panel
semiconductor
sequencing
ALK, ROS1,RET, NTKR1 fusions & expression
RNA Analysis
automated analysis & reporting
NSCLC CNVs of interest
Cancers 2014, 6(3), 1540-1552; doi:10.3390/cancers6031540
CNVs by NGS
The Journal of Molecular Diagnostics, Vol. 17, No. 1, January 2015
Oncomine Comprehensive
Development
>8,000 Exomes + COSMIC
Hotspot mutations
>30,000 arrays
Copy number regions
>6,000 transcriptomes + COSMIC
Gene fusion events
Current and near term
clinically relevant
targets
73 oncogenes
49 copy number alterations
26 TSG
22 fusion drivers
>2500 amplicons (DNA)
148 gene fusion isoforms (RNA)
Oncomine Comprehensive
Full circle- from one to many? (ex
uno plures)
Clinical Use
Translation
Discovery