Poster # LBT-057
Detection of increasing numbers of point mutations and fusions in thyroid nodules
using next-generation sequencing dramatically reduces marker panel specificity
Kennedy, G.C., Pagan, M., Walsh, P.S., Matsuzaki, H., Huang, J., Travers, K., Tom, E., Wong, M., Mathison, J., Kim, S.Y. and Kloos, R.T.
FNA material was prospectively collected (n=81)
and post-surgically diagnosed by a panel of
experts as malignant (PTC, mPTC, FVPTC, PTC-TCV,
MTC, WDC-NOS, HCC, FC) or benign (BFN, FA, HCA,
HTA, LCT). We also evaluated surgical tissues
(n=57) with histopathology truth, and a
consecutive series of indeterminate FNAs without
histopathology from our CLIA lab (n=101).
Samples were subjected to NGS and 14 genes
were evaluated, with increasing numbers of
interrogated genomic sites and fusion pairs.
Mutation Panel
Genomic Sites
Fusion Pairs
Panel 1
9
3
Panel 2
19
25
Panel 3
208
25
Panel 4
929
25
Panel 5
3670
25
AKT1
BRAF
NRAS**
PIK3CA
CTNNB1 EIF1AX
PTEN
RET
GNAS
HRAS
KRAS
TERT***
TSHR***
TP53
**
**
* Panels 2-5 include these genes and fusions as reported by Nikiforov et al.
** Panel 1 Evaluates only a subset of genes and fusions as reported by Beaudenon et al.
*** Included only in Panel 5
Test Performance Across Five Mutation Panels
in VERA FNA (n=81)
Test Performance Across Five Mutation Panels
in Tissue (n=38)*
VERA FNA Mutation Frequency
1.0
1.00
0.93
The sensitivity gained by detecting increasingly larger numbers of point mutations and fusions
comes at the cost of specificity, and runs the risk
of overcalling malignancy in truly benign samples.
A further understanding of the significance of
point mutations and fusions detected in benign
nodules is warranted.
0.95
1.00
1.0
1.0
0.89
LCT
0.6
0.53
1.0
HCA
BFN
0.4
0.43
0.6
0.6
0.4
0.45
0.45
0.4
Histo M
Mutation Frequency
Panel 3: 21/40 (53%)
Panel 4: 40/40 (100%)
FA
0.8
0.78
0.45
Overall
Mutation Frequency
Panel 3: 24/81 (30%)
Panel 4: 76/81 (94%)
BFN/HN
0.89
0.8
Specificity
0.6
0.4
1.00
HTA
FVPTC
0.8
0.53
1.00
0.93
0.8
MTC
FC
Histo B
Mutation Frequency
Panel 3: 3/41 (7%)
Panel 4: 36/41 (88%)
WDC-NOS
HCC
PTC
PTC-TCV
Mutation positive nodules (Panel 3) are indicated in dark gray
Nodules are depicted size-proportional. Smallest nodule = 1 cm.
0.2
0.2
0.0
Panel 1
9 sites
3 fusions
Panel 2
19 sites
25 fusions
Panel 3
208 sites
25 fusions
0.2
Detected with Panel 3
0.2
Gene
0.12
0.10
Panel 4
929 sites
25 fusions
Panel 5
3670 sites
25 fusions
0.07
0.0
0.0
Panel 1
9 sites
3 fusions
Panel 2
19 sites
25 fusions
Panel 3
208 sites
25 fusions
Panel 4
929 sites
25 fusions
0.04
Panel 5
3670 sites
25 fusions
0.0
* While a total of 57 tissues were evaluated only 38 had definitive
Histo B or Histo M pathology to be used in test performance calculations.
Mutation Performance by Cytology in Panel 3
Bethesda
Cytology
Category
Cyto B
Total No.
Samples
28
Histo B Mut Histo M Mut
(+)/total
(+)/total
2/22
3/6
Sensitivity
(95% CI)
0.50 (0.12-0.88)
12
1/11
0/1
0 (0-0.98)
0.91 (0.59-1.00)
FN/SFN
9
0/7
1/2
0.50 (0.01-0.99)
1.00 (0.59-1.00)
All Samples
12
20
81
0/1
0/0
3/41
4/11
13/20
21/40
0.36 (0.11-0.69)
0.65 (0.41-0.85)
0.53 (0.36-0.68)
AUS/FLUS
Afirma B
FN/SFN
Afirma B
Afirma B (n=31)
Afirma S (n=9)
Afirma B (n=4)
FN/SFN
Afirma S
AUS/FLUS
Afirma S
SFM
Afirma S
NA
V600E
HRAS
Q61R
KRAS
G12D
NRAS
Q61K
NRAS
Q61R
2
TP53
R248Q
1
Fusions
PAX8/PPARG
2
15
1
1
1
1
1
1
2
0
Gene
Mutation
Afirma B (n=54)
AUS/FLUS (n=31)
Tissue Mutation Frequency
Overall
Mutation Frequency
Panel 3: 22/101 (22%)
Panel 4: 89/101 (88%)
Overall
Mutation Frequency
Panel 3: 18/57 (32%)
Panel 4: 36/57 (63%)
CN
FA
Histo M
Mutation Frequency
Panel 3: 5/11 (45%)
Panel 4: 11/11 (100%)
NHP
FN/SFN
Mutation Frequency
Panel 3: 5/22 (23%)
Panel 4: 20/22 (91%)
Histo U
HCA
BFN
BRAF
K601E
GNAS
R201H
1
HRAS
Q61K
1
HRAS
Q61R
KRAS
G12D
1
KRAS
Q61R
1
NRAS
G12V
1
NRAS
Q61R
1
TP53
P152L
1
TP53
R213*
Fusions
PAX8/PPARG
Mutation Total
FN/SFN (n=16)
Mutation positive samples (Panel 3) are indicated in dark gray
GNAS+ nodules are indicated in light gray
NA (n=16)
1
Detected with Panel 3
Gene
1
2
1
1
4
1
3
2
8
HCC
PTC
4
7
Histo B
Mutation Frequency
Panel 3: 6/27 (22%)
Panel 4: 25/27 (93%)
Histo U
Mutation Frequency
Panel 3: 5/19 (26%)
Panel 4: 18/19 (95%)
FVPTC
Afirma S (n=47)
AUS/FLUS (n=15)
22
Figure 5.
Mutation positive samples (Panel 3) are indicated in dark gray
GNAS+ nodules are indicated in light gray
0.93 (0.80-0.98)
Afirma S (n=36)
BRAF
ANT
Cyto NA
Afirma S
1.00 (0.03-1.00)
1.Filicori F, Keutgen XM, Buitrago D, et al. Risk stratification of indeterminate thyroid fine-needle
aspiration biopsy specimens based on mutation analysis. Surgery 2011; 150(6): 1085-91.
2.Nikiforova MN, Nikiforov YE. Molecular diagnostics and predictors in thyroid cancer. Thyroid
2009; 19(12): 1351-61.
3.Nikiforov YE, Carty SE, Chiosea SI, et al. Highly accurate diagnosis of cancer in thyroid nodules
with follicular neoplasm/suspicious for a follicular neoplasm cytology by ThyroSeq v2 nextgeneration sequencing assay. Cancer 2014; 120(23): 3627-34.
4.Beaudenon-Huibregtse S, Alexander EK, Guttler RB, et al. Centralized molecular testing for
oncogenic gene mutations complements the local cytopathologic diagnosis of thyroid nodules.
Thyroid 2014; 24(10): 1479-87.
5.Cancer Genome Atlas Research N. Integrated genomic characterization of papillary thyroid
carcinoma. Cell 2014; 159(3): 676-90.
Mutation
Histo M (n=40)
AUS/FLUS
Mutation Frequency
Panel 3: 10/46 (22%)
Panel 4: 41/46 (89%)
Detected with Panel 3
REFERENCES
Cyto B
Afirma B
Cyto NA
Afirma B
0.91 (0.71-0.99)
AUS/FLUS
SFM
CLIA FNA Mutation Frequency
Specificity
(95% CI)
Histo B (n=41)
Mutation Total
Figure 3.
Figure 1b.
Cyto M
CONCLUSIONS
Genes Targeted in Mutation Panels*
**
Sensitivity to detect malignancy improved in all
cohorts with increasing number of loci, while specificity showed the opposite trend, falling dramatically. In FNAs, the smallest 9 site panel rendered
a sensitivity of 53% and a specificity of 93%; while
the largest panel renders a sensitivity of 100%
and a specificity of 10%. In surgical tissues, a similar trend was observed: in the smallest panel 89%
specificity was associated with 45% sensitivity; in
the densest panel a sensitivity of 100% was associated with 0% specificity. Overall, the two larger
panels wrongly called 87-90% of histology benign
FNAs as malignant, while the two smaller panels
missed 48-58% of known cancers. The frequency
of mutations and fusions in CLIA samples across
the five panels was 13%, 4%, 21%, 89% and 92%,
respectively.
Figure 4.
Sensitivity
METHODS
RESULTS
Figure 2.
Sensitivity
Recent advances in next-generation sequencing
(NGS) technology have vastly increased the rate
of detection of cancer-associated mutations
and fusions. In thyroid nodules, some mutations
are highly specific for detecting cancer, while
less is known about the accuracy of others. The
number of thyroid cancer-associated genomic
aberrations will continue to grow as more data
emerges using this sensitive technology. Yet, the
value of ever-increasing numbers of loci in the
preoperative diagnosis of thyroid FNA remains
unclear. Here we assess the accuracy of five point
mutation and fusion panels.
Figure 1a.
Specificity
INTRODUCTION
Several stringency filters were applied to score
the data. Samples were scored negative when
no fusions or point mutations were present,
and scored positive if at least one fusion or
point mutation was detected, except for GNAS
mutations which are considered to be a markers
of benignity.
Mutation
Histo B (n=27)
Afirma B (n=8)
Afirma S (n=18)
Afirma NA (n=1)
Histo M (n=11)
Histo U (n=19)
Afirma S (n=11)
Afirma S (n=16)
BRAF
V600E
GNAS
Q227H
1
NRAS
Q61K
1
3
NRAS
Q61R
1
2
TSHR
D633Y
TSHR
I486F
Fusions
PAX8/PPARG
Fusions
CCDC6/RET
Mutation Total
5
2
1
1
1
2
4
1
5
6
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