Treating cancer effectively requires an understanding of the molecular alterations driving each patient’s tumor. Targeted sequencing efforts that characterize prevalent somatic alterations and require limited sample input may provide an effective diagnostic approach. Herein, we describe the design and characterization of the Oncomine™ Cancer Research Panel (OCP) that includes recurrent somatic alterations in solid tumors derived from the Oncomine™ cancer database. Using Ion AmpliSeq™ technology, we designed a DNA panel that includes assays for 73 oncogenes with 1,826 recurrent hotspot mutations, 26 tumor suppressor genes enriched for deleterious mutations, as well as 75 genes subject to recurrent focal copy gain or loss. A complementary RNA panel includes 183 assays for relevant gene fusions involving 22 fusion driver genes. Recommended sample inputs were 10 ng of nucleic acid per pool. Sequencing libraries were analyzed on an Ion Torrent™ Personal Genome Machine™. Initial testing revealed an average read depth of > 1,500X with > 95% uniformity and on target frequency. The panel was shown to reliably detect known hotspots, insertions/deletions, gene copy changes, and gene fusions in molecular standards, cell lines and formalin-fixed paraffin embedded samples. Retrospective analysis of large sample cohorts has been completed and the results of analysis of 100 lung cancer and 100 prostate cancer cases will be summarized. In addition, a prospective cohort of 100 samples from the University of Michigan Molecular Diagnostics laboratory was profiled with OCP. Overall, we achieved >95% sensitivity and specificity for detection of KRAS, EGFR and BRAF mutations and ALK gene fusions.

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All products presented here are for Research Use Only.
Development and validation of the Oncomine™ Cancer Research
Panel (OCP), a scalable next-generation sequencing system for
assessing recurrent somatic alterations in solid tumors.
Kate Rhodes2, Andi Cani1, Dan Hovelson1, Geoff Bien2, Sophie Rozenzhak2, Cristina Van Loy2, Denise Topacio2, Natalia Jun2, Andrew McDaniel1, Albert Liu1, Paul
Choppa2, Jeoffrey Schageman2, Guoying Liu2, Fiona Hyland2, Rajesh Gottimukkala2, Jim Veitch2, Santhoshi Bandla2, Paul Williams2, Bryan Johnson2, Melvin
Wei2, Miroslav Dudas2, Adam Broomer2, Peter Wyngaard2, Seth Sadis2, Dan Rhodes2, Scott Tomlins1,
1Department of Pathology, University of Michigan, Ann Arbor, MI, USA; 2, Life Sciences Solutions, Thermo Fisher Scientific
Figure 2: Workflow from FFPE to Sequence Data
Day One Day Two Day Three
Fig. 2: DNA and RNA is isolated from FFPE slices using various methods. Ion AmpliSeq
library preparation is performed on 10 ng of DNA and 10 ng of RNA per primer pool.
Libraries are quantified using the Ion Library Quant Kit and templated on the Ion
OneTouch™ 2 system. Samples are sequenced on the Ion Torrent™ PGM instrument.
Total turn around time from FFPE to data is 3 days.
ALK
AKT1
BRAF
CTNNB1
DDR2
EGFR
ERBB2
ERBB4
FBXW7
FGFR1
FGFR2
FGFR3
KRAS
MAP2K1
MET
NOTCH1
NRAS
PIK3CA
PTEN
SMAD4
STK11
TP53
ABL1
AKT1
ALK
AR
ARAF
BRAF
BTK
CBL
CDK4
CHEK2
CSF1R
CTNNB1
DDR2
DNMT3A
EGFR
ERBB2
ERBB3
ERBB4
ESR1
EZH2
FGFR1
FGFR2
FGFR3
FLT3
FOXL2
GATA2
GNA11
GNAQ
GNAS
HNF1A
HRAS
IDH1
IDH2
IFITM1
IFITM3
JAK1
JAK2
JAK3
KDR
KIT
KNSTRN
KRAS
MAGOH
MAP2K1
MAP2K2
MAPK1
MAX
MED12
MET
MLH1
MPL
MTOR
MYD88
NFE2L2
NPM1
NRAS
PAX5
PDGFRA
PIK3CA
PPP2R1A
PTPN11
RAC1
RAF1
RET
RHEB
RHOA
SF3B1
SMO
SPOP
SRC
STAT3
U2AF1
XPO1
AKT1
ALK
AR
ARAF
BRAF
CDK4
CTNNB1
DDR2
EGFR
ERBB2
ERBB3
ERBB4
ESR1
FGFR2
FGFR3
GNA11
GNAQ
HRAS
IDH1
IDH2
JAK1
JAK2
JAK3
KIT
KRAS
MAP2K1
MAP2K2
MET
MTOR
NRAS
PDGFRA
PIK3CA
RAF1
RET
ROS1
SMO
OCP
Oncomine™
Focus
Oncomine ™
Solid Tumor
Figure 1: Three Oncomine Panels Offer Flexibility to Test Gene Panels of
Different Size Depending on the Your Research Interests
Fig. 1: The OCP, Oncomine Focus and Oncomine Solid Tumor panels provide primers
that capture regions of human somatic variants (deletions, insertions, inversions, and
substitutions) present in the genes shown above for analysis using next generation
sequencing technology. All three panels are designed to work with formalin fixed
paraffin-embedded (FFPE) tissues.
Fig. 3: Application of OCP to a prostate cancer cohort identifies variable alterations
across histologic and treatment subtypes and confirms isoform-specific gene fusion
detection. (A) Alterations found in a retrospective cohort of aggressive FFPE prostate
cancers. (B) The RNA component of the OCP contains forward primers in known 5′
fusion partners and reverse primers in known 3′ fusion partners for recurrent gene
fusions in prostate cancer. Normalized log2 read counts for indicated gene fusion
isoforms are indicated in each cell according to the color scale, with individual fusions
indicated by the color blocks (right) and fusion isoforms named by the exon junctions of
the involved genes (e.g., T2:ERGT1E4 indicates a fusion junction ofTMPRSS2 exon
1 and ERG exon 4). qRT-PCR was previously performed on a subset of these cases, as
indicated in qPCR type. T2:ERG T1E4 status (including low expression), and ERG outlier
expression without T1E4 isoform detection (ERG+), ETV1 (ETV1+), ETV4 (ETV4+), or ETV5
(ETV5+) are indicated in the header. Samples without any of these alterations (Neg) or
not tested (N/A) by qPCR are indicated.
Figure 3: Validation of OCP with a Prostate Cancer Cohort
Figure 4: Validation of Oncomine Focus for Copy Number Alterations
Fig. 4: Twenty tumor samples containing copy number amplifications and twenty tumor
samples without copy number alterations were sequenced with the Oncomine Focus
Panel in four different labs. The copy number of all forty of the genes were assessed
with FISH. The R2 correlation between the two methods was 0.93%.
Treating cancer effectively requires an understanding of the molecular alterations driving
each patient’s tumor. Targeted sequencing efforts that characterize prevalent somatic
alterations and require limited sample input may provide an effective diagnostic
approach. Herein, we describe the design and characterization of the Oncomine™ Cancer
Research Panel (OCP) that includes recurrent somatic alterations in solid tumors derived
from the Oncomine™ cancer database. Using Ion AmpliSeq™ technology, we designed a
DNA panel that includes assays for 73 oncogenes with 1,826 recurrent hotspot mutations,
26 tumor suppressor genes enriched for deleterious mutations, as well as 75 genes
subject to recurrent focal copy gain or loss. A complementary RNA panel includes 183
assays for relevant gene fusions involving 22 fusion driver genes. Recommended sample
inputs were 10 ng of nucleic acid per pool. Sequencing libraries were analyzed on an Ion
Torrent™ Personal Genome Machine™. Initial testing revealed an average read depth of >
1,500X with > 95% uniformity and on target frequency. The panel was shown to reliably
detect known hotspots, insertions/deletions, gene copy changes, and gene fusions in
molecular standards, cell lines and formalin-fixed paraffin embedded samples.
Retrospective analysis of large sample cohorts has been completed and the results of
analysis of 100 lung cancer and 100 prostate cancer cases will be summarized. In addition,
a prospective cohort of 100 samples from the University of Michigan Molecular
Diagnostics laboratory was profiled with OCP. Overall, we achieved >95% sensitivity and
specificity for detection of KRAS, EGFR and BRAF mutations and ALK gene fusions.
CopyNumberfromOncomineFocusSequencing
Copy Number from FISH
Lab 1
Lab 2
Lab 3
Lab 4