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3D Biology view of cancer: Simultaneous Detection of Somatic DNA Mutations and Expression Profiling of Genes and Signaling
Proteins From Melanoma Tumor FFPE Samples
Jinho Lee1, Christopher P. Vellano1, Gavin Meredith2, Jill McKay-Fleisch2, P. Martin Ross2, Michael Tetzlaff3, Alexandre Reuben4, Courtney Hudgens5, Jennifer Wargo4, Jessica Garber2, Andrew White2, Joseph Phan2, Mike Krouse2, Mekala Pansalawatta2, Lucas Dennis2, Anisha Kharkia2, Erin Piazza2, Afshin Mashadi-Hossein2, Rich Boykin2, Nathan Elliott2, Brian Filanoski2, Gokhan Demirkan2, Sara Warren2, Gary Geiss2, Dae Kim2, Joseph Beechem2, Gordon Mills1
1Department of Systems Biology, MD Anderson, Texas; 2NanoString Technologies, Inc., Seattle, WA; 3Department of Pathology, MD Anderson; 4Department of Surgical Oncology, MD Anderson; 5Department of Translational Molecular Pathology, MD Anderson
Background Information and Data for Synchronous Melanoma Metastases
Table 1. Patient sample description. Synchronous metastases resected from melanoma patients were examined. A total of 11 samples from 5
patients were analyzed which were treatment-naïve or treated with either targeted therapy (BRAFi + MEKi) or immune checkpoint blockade (PD-1 or
CTLA-4). SD = stable disease, PR = partial response, PD = progressive disease, NA = Not available.
Patient #4
As proof of concept demonstrating the utility of this 3D Biology platform, we have simultaneously analyzed DNA
variants, RNA expression, and protein expression using NanoString’s nCounter ® Vantage 3D™ Solid Tumor Panel on 10
FFPE melanoma tumor samples and one adjacent normal tissue from five patients. This sample set included two
metastatic tumors from each patient in order to assess tumor heterogeneity within the same patient. Importantly, this
sample set has associated mRNA expression measured using the nCounter® PanCancer Pathways for Human panel as
well as whole exome sequencing (WES) data. Somatic variants detected by WES were compared with the results from
the DNA SNV Solid Tumor Panel. Further, all samples were subjected to T200.1 deep sequencing analysis for validation.
Overall, we show that this multiplex and multi-omic platform has the potential for rapid and sensitive assessment of
patient samples that will impact clinical care.
Patient #5
43 (M)
None
49 (M)
BRAFi + MEKi
55 (M)
None
76 (F)
CTLA-4 Blockade
Metastasis 1
Metastasis 2
Metastasis 1
Metastasis 2
Metastasis 1
Metastasis 2
Metastasis 1
Metastasis 2
normal tissue
Metastasis 1
Metastasis 2
Left abdominal mass
Right abdominal mass
Left axillary mass
Left axillary mass
Right axillary lymph node
Right neck lymph node
Left axillary mass
Right supraclavicular mass
Spleen
Spleen
Right abdominal wall
73% / 50%
80% / 63%
70% / 71%
76% / 73%
62% / 20%
70% / 47%
70% / 73%
96% / 87%
NA
70-85% / 69-88%
85-90% / 60-75%
a
RNA Probe Design
MUT
T
WT
A
Endogenous RNA
Photocleavable Linker
86B (BRAF V600K)
120A (NRAS Q61R)
120B (NRAS Q61R)
92B (BRAF V600E)
70.1 (negative mutation call)
92C (BRAF V600E)
Pt. 3
Treatment-naïve
Targeted therapy
Allele Frequency
0%
Mutation
120
120A
120B
86A
86B
Normal
tissue
NRAS
Q61R
NRAS
Q61R
BRAF
V600K
BRAF
V600K
120A1
120B1
120
JAK-STAT pathway-related genes
86B
86A
Immunotherapy
Protein Probe Design
Unique
ssDNA Tag
NA
NA
NA
NA
-0.5
0.15
NA
NA
NA
0.072
0.24
b
3D BiologyTM Technology on the nCounter Platform
SNV Probe Design
NA
NA
NA
NA
PR
SD
NA
NA
NA
SD
PD
DNA
Mutation
PD-1 Blockade
Change in
Tumor Size
(mm3)
BRAFi + MEKi
100%
Log2
Patient #3
35 (M)
Tumor Sites
RECIST
Class
CTLA-4 blockade
Protein
Phosphorylation
Patient #2
51AL
51BR
70.1
70.2
86A
86B
92B
92C
120
120A
120B
Tumor Types
Tumor Content
(Pathological/
Bioinformatic)
3D Biology Data Analysis
86A (BRAF V600K)
120 (adjacent normal tissue)
Pt. 4
Patient #1
Prior
Therapy
Pt. 2
In order to integrate the strengths of different molecular platforms, we have modularized NanoString Technologies’
molecular barcoding technology to permit simultaneous digital measurement of cancer-associated DNA mutation
variants, mRNA expression, and protein expression in one assay from the same sample (3D Biology). Novel nucleotide
variant probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection
of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Gene expression is measured via unique digital barcoding
technology to measure mRNA transcripts, and protein expression and activity (via phosphorylation) is measured by
DNA-labeled antibodies. The multi-omic workflow requires only two 5-10 micron sections of FFPE tissue, whereby DNA
and RNA are extracted from one section and multiplex digital protein profiling is conducted on the second.
Age
(Gender)
Pt. 5
Patient #
Sample
name
Pt. 1
Prognosis is favorable in patients with primary localized melanoma but poor in patients with metastatic disease. With
more than 76,000 cases expected to be diagnosed in 2016, more precise prognostic technologies and new therapies are
needed. Although targeted treatment regimens have been approved in recent years, resistance has emerged in large
part due to adaptive response mechanisms and intratumoral heterogeneity. Analysis of tumor samples across multiple
molecular platforms will help elucidate the complexities within and across tumors which may underlie response to
therapy as well as assist in identifying predictive biomarkers; however, these approaches require significant amounts of
sample, time, and resources.
Nucleotide Variant Detection Using NanoString Solid Tumor Mutation Panel
Gene Expression
Abstract
Figure 4. Heterogeneity across metastases of the same patient. Differential RNA expression and phospho-protein expression
levels were detected between metastases of the same patient even though the same genomic variants were detected by nCounter.
For instance, NRAS-mutated 120A and 120B samples treated with CTLA-4 blockade showed that differential JAK-STAT pathway
score related genes and protein, p-AKT. In addition, 86A sample showed relatively higher gene expression related in PI3K pathway
(red) although both carried the same BRAF mutation. 86A and 86B exhibited differential expression levels in phospho-proteins.
Primary
Antibody
Perfect match
Methods
MUT
Vantage 3D Solid
Tumor RNA Assay-770
mRNAs
Or
Vantage 3D Solid
Tumor Signaling RNA
Assay-192 mRNAs
Single mis-match
WT
Vantage 3D Solid
Tumor SNV Assay104 SNVs
DNA
Vantage 3D Solid
Tumor Protein
Assay-~30 proteins
Figure 1. Molecular and immune profiling of synchronous melanoma samples from a previous study (1). (a) Two different metastatic samples from the same
patient were collected and analyzed for molecular and immune profiling. (b) Whole exome sequencing (WES) identified genomic drivers in these melanoma
tumors.
a
b
RNA Pathway Score
high
92C
92B
70.2
70.1
51BR
51AL
120B1
120A1
120
86A
86B
low
DNA Damage - Repair
TGF-beta
Hedgehog
Wnt
Ras
MAPK
JAK-STAT
Transcriptional Misregulation
Cell Cycle - Apoptosis
PI3K
Driver Gene
Notch
Chromatin Modification
5ng
25ng
0.25-2.5mg
or
FFPE slide
The nCounter platform can be used to digitally count DNA, RNA, and proteins from a single sample by using fluorescent optical
barcodes that attach to a probe, which then binds directly to the analyte of interest. Probes have been adapted to allow single
base pair resolution and protein detection. For the experimental procedure, DNA and RNA samples were separately collected from
FFPE slides. The antibody mix for the protein panel was added onto the whole tumor area of the FFPE slide and DNA barcodes
were collected by UV cleavage. RNA and protein codesets* were put together for the analysis after hybridization. SNV analysis
was performed in a different cartridge independently from RNA:protein analysis.
* Codeset: A set of unique nucleotide sequences to capture the target of interest. It consists of a bipartied-50 base capture
probe and reporter probe which carries the fluorescent signal
For Research Use Only. Not for use in diagnostic procedures. © 2016 NanoString Technologies, Inc. and MDACC All rights reserved.
NanoString, NanoString Technologies, the NanoString logo, nCounter, nSolver, 3D Biology and Vantage 3D are trademarks or registered trademarks of NanoString Technologies, Inc.,
in the United States and/or other countries.
Treatment:
Immune
Naïve
Targeted
Pt#4 Pt#2 Pt#1 Pt#5 Pt#3
Protein Expression
Patient #
low
• nSolver 4.0 was used for nCounter used for analysis.
Pt#2 Pt#3 Pt#5 Pt#4 Pt#1 Pt#5
Figure 2. RNA:protein analysis in a set of metastatic melanoma tumors. RNA and protein samples were prepared from FFPE slides and analyzed with the
RNA:protein solid tumor pathway panel. (a) Pathway score heatmap was generated from RNA analysis. Advanced analysis tool of nSolver 3.0 was used for
calculating pathway scores. Targeted- or immunotherapy-treated samples primarily clustered together, with the treatment naïve samples exhibiting marked
expression differences compared with treated samples. For example, yellow boxes indicate the differential pathway scores between two mets or compared to
normal tissue. (b) Unsupervised hierarchical clustering heatmap of protein panel data shows clustering associated with patient and treatment. For example, patient
#1 samples (51AL and BR) showed up-regulation of PI3K pathway-related proteins (blue box), which might be due to PTEN activity decrease by PD-1 inhibition (2) .
pt = patient.
Patient #1
Patient #2
Variant Allele
Frequency by
T200.1
DNA Quality
(DIN)
NA
Y/Y
NA
2.5
No detection
NA
Y/Y
NA
2.2
70.1
70.2
No detection
No detection
NA
NA
Y/Y
Y/Y
NA
NA
6
6.2
• The NRAS Q61R variant in both patient #5 metastases (120A1, B1) was not detected by WES; however, all variants detected by
nCounter were concordant with T200.1.
86A
BRAF V600K
(1798_1799GT>AA)
COSM473
Y/Y
20.6% / 20.6%*
4.8
• nCounter showed high specificity of DNV detection of BRAF V600K (1798_1799 GT>AA) while WES / T200.1 called it as
individual variants BRAF V600E or V600M (SNVs).
86B
BRAF V600K
(1798_1799GT>AA)
COSM473
Y/ Y
36.0% / 35.8%*
4.4
• Multiplex RNA expression data showed differential pathway scores between metastatic regions within the same patient.
92B
BRAF V600E
COSM476
Y/Y
62.4%
3.2
92C
BRAF V600E
COSM476
Y/Y
58.1%
4.2
• Phospho-protein level changes indicative of pathway activity were well-captured with responses to targeted and immune
checkpoint therapies.
120
No detection
NA
NA
NA
4.1
120A1
NRAS Q61R
COSM584
N/Y
25.8%
4.3
120B1
NRAS Q61R
COSM584
N/Y
37.8%
4.4
COSMIC ID
51AL
No detection
51BR
Patient #3
Patient #4
Patient #5
Conclusions
Concordance with
detection by
WES / T200.1
Mutation call by
nCounter
Sample ID
high
p53p53
p-AMPK
(T172)
p-AMPK
S6
S6
HER2
HER2
p-TSC2
(T1462)
p-TSC2
4E-BP1
4E-BP1
Ki-67
Ki-67
p-EGFR (Tyr1068)
p-EGFR
Histone H3
Histone H3
PR PR
EGFR
EGFR
Pan-Keratin
Pan-Keratin
p-MEK1
(S217/221)
p-MEK1
p-S6
(S235/236)
p-S6
p-GSK3b
(S9)
p-GSK3b
p-4E-BP1
(T37/46)
p-4E-BP1
p-PRAS40
(T246)
p-PRAS40
Pan-Akt
Pan-Akt
ERK1/2
ERK1/2
p-PDK1
(S241)
p-PDK1
TSC2
TSC2
p-c-Raf
(S259)
p-c-Raf
p-H3
(S10)
p-H3
p-AKT
(S473)
p-AKTS473
GSK3b
GSK-3B
p-ERK1/2
(T202/204)
p-ERK1/2
MetMet
• We performed simultaneous detection of DNA variants (104-plex of 25 genes), RNA expression levels (770 genes) and protein
expression levels (29 targets) on FFPE samples from synchronous metastases from 5 melanoma patients using the NanoString
3D Biology nCounter platform.
• 5 ng of DNA and 100 ng of RNA from each FFPE slide were10 mm each) were used for protein panel assessment using the
whole area of the tumor
Cross-Platform Genomic Variant Comparison
Protein and RNA Clustering Analysis of Melanoma Samples
70.2A
70.1A
86B2
86A
120B1
120A1
92C3
92B3
51BR-11
51AL-1
120
RNA
Figure 3. Nucleotide variant detection from SNV analysis. DNA samples were collected from FFPE patient slides and analyzed with nCounter using 104-plex
variant probes. The representative plots show the variants detected from metastatic samples, as well as the adjacent normal spleen sample (120). Fold-change
to reference DNA showing greater than 2-fold (log2) was marked for the mutation call (p-value < 0.01).
Table 2. Genomic variant concordance is shown between data generated from the nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel and WES (Illumina
HiSeq 2000) and T200.1(3) next generation sequencing (NGS) platforms. T200.1 is a deep candidate gene sequencing approach. WES read depth were 100x
for normal and 200x for tumor samples. nCounter detected the same variants in all metastases of a given patient. Importantly, the BRAF V600K dinucleotide
variant (DNV; GT>AA) is readily distinguished from BRAF V600E (T1799A) and BRAF V600M (G1798A) SNVs in patient #3 samples (86A,B). BRAF V600E
variants were detected in all patient #4 samples (92B,C). In addition, nCounter detected a NRAS Q61R mutation in both patient #5 metastases (120A1,B1),
which was not called by WES. Sequence validation by T200.1 indeed confirmed the presence of this mutation in these samples.
* T200.1 detected each single nucleotide variant of BRAF V600E and V600M. The 2 SNV calls at same residue with similar AF suggests the presence of DNV.
• 3D Biology platform integrates the features of RNA and protein expression levels along with mutation status in one multiplex,
multi-omics assay to capture the molecular heterogeneity across synchronous melanoma metastases from the same patient,
which may potentially underlie differential response to therapies.
References and Acknowledgments
1) Reuben A et al. (2017) Genomic and Immune Heterogeneity are Associated with Differential Responses to Therapy in Melanoma. npg
Genomic Med. In press
2) Patsoukis N et al. (2013) PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2.
Mol. Cell Biol. 33, 3091-98.
3) Chen K et al. (2015) Clinical Actionability Enhanced through DeepTargeted Sequencing of Solid Tumors. Clin. Chem. 61, 544-553.
Funding: Data and results cited in this study were funded, in part, as follows: The Sheikh Bin ZayedAl NahyanFoundation (1U01 CA180964),
NCATS grant UL1 TR000371 (Center for Clinical and Translational Sciences). The BosargeFoundation, The MD Anderson Cancer Center Support
grant (NIH/NCI P30 CA016672), the MD Anderson Moon Shot Program, NanoStringTechnologies also supports ongoing work supervised by G.B.
Mills at The University of Texas MD Anderson Cancer Center.