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Distinct biomarker features in triple-negative breast cancer metastases to the brain, liver and bone
Joanne Xiu1, Zoran Gatalica1, Sandeep Reddy1, James Waisman2, John Link3
1Caris Life Sciences, Phoenix, AZ, United States, 85040; 2City of Hope Medical Oncology, Duarte, CA, United States, 91010 and 3Breastlink Medical Group, Orange, CA, United States, 92868.
Abstract
Background: Triple-negative breast cancer (TNBC) is characterized by its
aggressive nature and accounts for a disproportionate number of metastatic
disease cases and breast cancer-related deaths. Despite recent improvements,
TNBC patients who develop metastatic diseases have limited treatment options.
We investigated biomarkers from brain, liver and bone metastases collected from
TNBC patients to identify therapeutic options and to examine molecular
differences between the metastatic sites.
Method: Triple-negative breast cancer tumors referred to Caris Life Sciences
(Phoenix, AZ) between 2009 and 2015 were tested with a combination of
immunohistochemistry (IHC), fluorescent/chromogenic in-situ hybridization
(FISH/CISH) and sequencing (Next-generation sequencing, or NGS and Sanger).
Result: 1570 TNBC tumors were analyzed, including 1297 tumors taken from
breast, 54 from brain, 172 from liver and 47 from bone. Select biomarker
frequencies of protein overexpression (IHC), gene amplification (ISH) and
mutations (SEQ) are summarized in Table 1. Brain metastases showed the highest
protein expression of TOPO2A and PDL1; liver metastases showed the highest
expression of AR and SPARC, as well as the highest mutation rate of PIK3CA. Bone
metastases showed the lowest expression of TS, RRM1 and ERCC1. BRCA1 and
BRCA2 mutation rates ranged from 0-11% in various specimen sites.
Table 1
Biomarker
And Method
TOPO2A IHC
PDL1 IHC
AR IHC
SPARC IHC
PIK3CA SEQ
TS IHC*
RRM1 IHC*
ERCC1 IHC*
BRCA1 SEQ
BRCA2 SEQ
Breast
specimens
(%)
76
15
15
17
16
49
39
35
7
11
Brain
Metastases
(%)
100
40
10
30
5.3
54
43
55
0
11
Liver
Metastases
(%)
73
8
36
40
29
24
32
48
8
4
Bone
Metastases
(%)
39
17
26
15
25
15
16
16
0
0
P value$
<0.0001
0.03
0.0005
0.0027
0.036
<0.0001
0.006
0.002
ns
ns
Results
Background
TNBC is known for its highly aggressive behavior and resistance to current
standard therapies. It’s is a well-defined subtype of breast cancer
immunohistochemically however it remains a molecularly heterogeneous group
(1). Clinical data suggest that TNBC patients with metastases to different organs
experience different survival times however different responses to therapies
haven’t been systematically explored (2). Multiplex tumor-profiling has shown to
effectively prolong patients’ progression-free survival in metastatic heavily pretreated breast cancer (3), therefore we investigated the profiles in TNBC tumors
with different metastases.
Methods
Results:
60%
Brain Mets TNBC
Breast specimen - TNBC
50%
100.0%
N (NGS)
Av. Age
Age
InterQuartile
Range
TNBC
7.9%
IHC-PD-L1
p=0.0266
Immune checkpoint inhibitors
40.0%
15.3%
50.3%
Low IHC-PTEN
p=0.0382
Breast specimens
45.5%
(N=1297)
484
54.6 yrs
47-64 yrs
(N=54)
(N=172)
(N=47)
10
52.3 yrs
45-59 yrs
62
55.6 yrs
48-63 yrs
9
57.7 yrs
51-65 yrs
10%
15%
p=0.0438
15%
13%
8%
8%
9%
6%
3%
8%
5%
5%
2%
2%
2%
3%
0%
Irinotecan
74.5%
IHC-TOPO1
p=0.0004
66.0%
Most prevalent in
TNBC-brain mets
56.3%
14.6%
43.0%
IHC-TLE3
p=0.0009
28.3%
33.6%
15.0%
Taxanes
Agents
most likely
to benefit
TNBC liver
mets
Taxanes
40.0%
IHC-SPARCp
29.5%
17.1%
p=0.0027
26.1%
Bicalutamide, enzalutamide
36.5%
IHC-AR
p=0.0005
10.4%
15.3%
85.1%
76.4%
Low IHC-TS
p<0.0001
Fluropyrimidines
45.8%
51.0%
68.4%
Gemcitabine
57.4%
61.0%
52.4%
45.5%
Platinums
65.0%
50.0%
Low IHC-TUBB3
p=0.0009
Brain metastases
20%
PI3K/Akt/mTor inhibitors
67.3%
29%
58.0%
81.3%
TNBC Bone
metastases
Agents most
likely to
benefit TNBC
brain mets
61.7%
Low IHC-ERCC1
p=0.0018
TNBC Liver
metastases
Bone mets -TNBC
p=0.0355
30%
76.6%
40.0%
37.8%
5.6%
10.2%
5.6%
7.2%
IHC-cMET
TNBC
Liver mets - TNBC
Top2A inhibitors
73.4%
83.9%
(N=1570)
Brain mets -TNBC
40%
39.3%
Low IHC-RRM1
p=0.0059
TNBC
Breast specimens-TNBC
Liver Mets - TNBC
84.4%
Figure 1: Patient Characteristics
85%
80%
Bone Mets -TNBC
16.7%
IHCs were performed on formalin-fixed paraffin-embedded (FFPE) tumor samples
using commercially available detection kits, automated staining techniques
(Benchmark XT, Ventana, and AutostainerLink 48, Dako), and commercially
available antibodies. FISH was used for evaluation of the HER-2/neu [HER2/CEP17 probe] and EGFR [EGFR/CEP7 probe] (Abbott Molecular/Vysis). CISH was
also used (INFORM HER-2 Dual ISH DNA Probe Cocktail;EGFR DNP probe,
Ventana). NGS was performed using the Illumina MiSeq platform. Specific regions
of 47 genes of the genome were amplified using the Illumina TruSeq Amplicon
Cancer Hotspot panel. Sanger sequencing included selected regions of BRAF,
KRAS, NRAS, c-KIT, EGFR, and PIK3CA genes and was performed by using M13linked PCR primers designed to amplify targeted sequences.
TNBC was defined as ER IHC negative(Ab: SP1; =0+ or <10%); PR IHC negative (Ab:
1E2; =0+ or <10%), Her2 IHC negative (Ab:4B5) and Her2 ISH negative (Her2
results and interpretation follow the ASCO/CAP scoring criteria. Wolff, AC. et al.
(2013) J Clin Oncol: 31 (31):3997-4013).
90%
70%
IHC-TOP2A
p<0.0001
n/a: data not available due to low N. * low levels predict response to associated
drugs. $p values are calculated from comparing the group with the highest
frequency with the lowest frequency. ns: non-significant, i.e., p>0.05
Conclusion: Distinct biomarker features identified in different metastatic sites in
TNBC present the rationale to investigate differential treatment strategies. Based
on biomarker results, etoposide, immune-modulatory agents may seem
promising for brain metastases; anti-androgen therapies and nab-paclitaxel may
be promising in treating liver metastases; while fluoropyrimidines, gemcitabine
and platinum may be considered for TNBC patients with bone metastases.
Figure 2: IHC and ISH marker comparison of TNBC tumors taken from the breast, bone, liver
and the brain. Bars represent the frequency of the biomarker measurement observed.
Therapeutic agents are listed for corresponding biomarkers that show significantly different
frequencies. A star indicates statistical significance when the highest mutation rate is
compared to the lowest.
Figure 3: Comparison of gene mutations rates in primary TNBC and metastases to the
brain, liver and bone. No statistical significance was seen with the exception of ERBB4 and
PIK3CA with borderline significant p values.
Most
prevalent in
TNBCliver
mets
Conclusions
• The investigation of biomarkers by IHC, ISH and NGS shows molecular
heterogeneity of TNBC tumors metastasized to the brain, liver and bone.
• These results suggest agents that can potentially deliver increased benefit
for TNBC patients with metastases to different organs, including TOP2A
inhibitors and immune checkpoint inhibitors for brain metastases, taxanes
and anti-androgen agents for liver metastases and fluoropyrimidine,
gemcitabine and platinum agents for bone metastases.
• The mutation data show promising results including high mutation rates of
ERBB4 in brain mets and PIK3CA in liver mets, however, this portion of the
study is from a limited NGS dataset.
• Distinct molecular profiles identified in different metastases support the
rationale of tailoring treatments of TNBC cancers to the biomarker features
of individual patients.
75.0%
43.5%
37.5%
IHC-EGFR
Agents
most likely
to benefit
TNBC bone
mets
Most prevalent in TNBC-liver mets
65.9%
50.0%
15.8%
ISH-EGFR
References
26.7%
22.1%
1.
50.0%
41.7%
41.7%
42.8%
Low IHC-MGMT
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
120.0%
2.
3.
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