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-3
-2
-1
0
1
2
3
4
log fo
ld ch
ange
The whole transcriptional landscape of circulating tumor cells compared to metastases in stage IV breast cancerAlexander Ring, Tania B. Porras, Daniel Campo, Pushpinder K. Bains, Victoria Forte, Debu Tripathy, Janice Lu, Gabriel Zada, Naveed Wagle, Julie E. Lang
Background
Methods
Conclusion
−60
−40
−20
0
20
40
−50 0 50 100PC1: 46% variance
PC2:
7%
var
ianc
e
groupCTC
Met
PB
Figure 1: Principal component (PC) analysis of CTCs, Metsand PB. The results show separation of the majority of CTCsversus mets and PB in PC1, and separation of CTCs and metsfrom PB in PC2.
Table 1: SNVs common to all CTC/met pairs in ESR1and ERBB2: Using IGV genome viewer, six SNVs inESR1 (n=4 patients) were identified that are sharedbetween CTCs and distant metastases. Five are listed indbSNP. From those listed, rs3798577 hasbeen associated with increased and decreased breastcancer risk in White and Asian women, respectively.rs2228480 is linked to decreased BC risk in whitewomen. rs2747648 occurs in the miR-453 bindingsite resulting in higher ESR1 protein expression. Knownand novel SNVs were identified with RNA Seq.
Patient ID SNVs in ESR1 Rs number in dbSNP
78536_CTCT>C chr6:152,097,179 not found
78536_MET79412_CTC
T>C chr6:152,099,995 dbSNP: rs379857779412_MET
101738_CTC_FOLLOWUP G>T chr6:152,101,052C>T chr6:152,101,200
dbSNP: rs72993667dbSNP: rs2747648101738_MET
36541_CTC C>A chr6:152,098,960C>T chr6:152,101,200
dbSNP: rs2228480dbSNP: rs274764836541_MET_BREAST
PIK3CARPTORNF1AKT1AKT3FBXW7
EGFRGRB7MAP3K1MAP2K1BRAFKRAS
CDKN2ACCND1CCND2CCND3CCNE1CDK4CDK6MYBL2PTTG1
CXXC5SFRP1CXCR4FGF1WNT1NOTCH1NOTCH4ALDH1A1TBX3MET
TCGB1IL4IL6IL12IL15IL23AGATA3CXCL9CSCL13CD3D
BRCA1BRCA2ATMPALB2BARD1
ESR1ESR2PGRHER2HER3
4
2
0
-2
-4
3
1
-3
-1fold
cha
nge
p=0.94 p=0.41
CTCs metsp=0.80p=0.29 p=0.33p=0.80 p=0.66
EGFR
GRB7
MAP3K1
MAP2K1
BRAF
KRAS
-20
-15
-10
-5
0
5
10
15
20
25
19065
28089
36541
68185
78536
78908
79388
79412
79555
79644
80541
81103
101738
112165
112370
113059
113166
113457
113488
C M
C M C M
C M
C MC M
fold
cha
nge
Figure 5: Analysis of potentially clinically actionable genes in breastcancer. 5A: Comparison of overall gene expression in different druggablepathways between CTCs and mets. We queried for n=66 potentially clinicallyactionable genes and on paired T tests for n=9 (7 shown here) pathways therewas no significant difference in mean gene expression between CTCs andmetastases. 5B: Expression of clinically actionable target genes in CTCs andmets per patient for the EGFR/RAF/MEK pathway (C – CTCs, M – mets).
(E) Clinically actionable genes/ signaling pathways
carboplatin/paclitaxel
6/2014
doxorubicin10/2016
2/7/2017
disease progression
CTCs 1st collection12/13/2016
fulvestrant/palbociclib
8/2015
Met collection10/24/2016
gemcitabine1/2017
gemcitabineexemastane/everolimus
2/2017
Sample collectionCTCs
2nd collection3/16/2017
3/23/20178/6/2015
Figure 6: Intra-patient (n=1) two time-point comparison: 6A: Clinical data (includingtreatment and imaging studies) and sample collection are shown. 6B: differentiallyexpressed breast cancer genes (KEGG pathway) in met (ascites), 1st CTC and 2nd CTCsample were analyzed using IPA pathway analysis tool, demonstrating differential geneexpression and pathway activation.
(F) Intra-patient analysis
Met (ascites) vs. PB 1st time point CTCs vs. PB 2nd time point CTCs vs. PB
Results (A) Whole transcriptome RNA Seq gene expression - group analysis
CTCs from 21 MBC patients were enumerated and captured from 10mL peripheral blood(PB) via the ANGLE Parsortix system. RNA Seq was performed on fresh metastatic tumorbiopsies (mets), CTCs and peripheral blood (PB) from all patients. Biopsy sites included:skin (n=1), lung (n=1), pleural effusion (n=5), pericardial effusion (n=1), breast (n=3),lymph node (n=2), brain (n=4), liver (n=1), ascites (n=3), cerebrospinal fluid (n=2), bonetissue (n=1). 19/21 patients were included in subsequent data analysis. (A) Groupcomparison of biologically relevant gene expression patterns in CTCs, mets and PB wasperformed. (B) Differential expression of genes of interest (oncogenes, breastcancer related genes, mesenchymal and cancer stem cell (CSC) genes) betweenCTCs, mets and PB was investigated. (C) Survival analysis based on gene expressionin CTCs and mets compared to PB was performed using data from The Cancer GenomeAtlas (TCGA). (D) Single nucleotide variants (SNV) analysis using IGV was performedin corresponding CTCs/mets pairs. (E) Clinically actionable gene (n=66) expressionand molecular signaling pathways (n=7) for each patient were explored. (F) Intra-patient serial time points were analyzed, and detailed clinical-pathological andtreatment data was evaluated.
(D) Single nucleotide variants (SNV) analysis
Metastasis is responsible for the vast majority of breast cancer related deaths. Metastaticbreast cancer (MBC) is inherently different than primary breast cancer (BC), evolvingunder selection pressure at different organ sites or during systemic therapy. The currentASCO guidelines call for biopsy of a metastatic site to guide decision making for systemictherapy. Yet, biopsies of macro metastasis are oftentimes not feasible in the clinicalsetting. Circulating tumor cells (CTCs) have been shown to be prognostic in MBC, buttheir use as clinical biomarker beyond CTC enumeration has been limited. A betterunderstanding of CTC-biology compared to metastasis may shed light on treatmentopportunities and help advance the application of CTCs as liquid biopsies in clinicalpractice. The ANGLE Parsortix system is a microfluidics device that separates CTCsbased on size and deformability, without the need for cell-surface marker selection. Ourlab has previously demonstrated the feasibility of gene expression profiling of rare CTCs.Here, we evaluated whether whole transcriptome sequencing (RNA Seq) geneexpression profiling of ANGLE Parsortix isolated CTCs may serve as a surrogatefor biopsies of macro metastases.
CTC
s vs
PB
Met
s vs
PB
CTC
s+M
ets
vs P
B
CTC
s vs
Met
s
AKT1CCND1CCNE1CCNE2
PLK1BIRC5
CENPFCENPAAURKAAURKB
CDC25ACDC25C
JUNMYCNMTORABL1ABL2BCR
EGFRFGFR2FGFR1MYBL2
BCL6ELK4
HMGA1PIK3CA
-1 0 1 2 3 4
CTC
s vs
PB
Met
s vs
PB
CTC
s+M
ets
vs P
B
CTC
s vs
Met
s
SHHGLI1GLI2GLI3SMO
RARGSOX3SOX7SOX8
SOX11SOX12SOX13SOX18SOX30
WNT10AWNT3AWNT5A
WNT10BCD133OCT4
KITABCG2
MDR1NESID2
ALDH1A2ALDH1A3
CD44
-1 0 1 2 3 4
CTC
s vs
PB
Met
s vs
PB
CTC
s+M
ets
vs P
B
CTC
s vs
Met
s
SCGB2A2
KRT7
KRT6B
KRT6C
KRT8
KRT3
KRT4
KRT9
KRT13
KRT16
ESR1
PGR
CDH1
KRT14
KRT18
KRT19
-3 -2 -1 0 1 2 3 4
CTC
s vs
PB
Met
s vs
PB
CTC
s+M
ets
vs P
B
CTC
s vs
Met
s
SNAI1TWIST1
FN1CDH2
-2 -1 0 1 2 3 4Figure 3: Expression of genes of interestin CTCs or mets compared to PB: CTCsas a group showed much stronger geneexpression of oncogenes, stem cell genes,keratins and mesenchymal markers than didmets from the same patients.
(B) Differential expression of genes of interest
Oncogenes Mesenchymal genesCSC genes Breast epithelial genes
0 20 40 600%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Months
Perc
ent s
urvi
val
50_gene_coding_CTCsANDMetsGENESinBOTH_vs_PB
Signature expressed
Signature not expressedp = 0.0099
CTCs/Mets common vs. PB
0 20 40 600%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Months
Per
cent
sur
viva
l
50_gene_coding_Cancer_vs_PB
Alteration
No alteration p = 0.0099
CTCs or Mets combined vs. PB
0 20 40 600%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Months
Per
cent
sur
viva
l
50_gene_coding_CTCs_vs_PB
Alteration
No alteration p = 0.8749
0 20 40 600%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Months
Per
cent
sur
viva
l
50_gene_coding_Mets_vs_PB
Alteration
No alteration p = 0.0476
Figure 4: TCGA BC (n=817) overall survival (OS) based on 50-geneexpression signatures. The top 50 highest expressed genes in fourcomparison conditions are shown. The 50-gene signature expressedCTCs and/or mets was superior to all other comparisons in predictingpoor OS (p=0.01).
CTCs vs. PB Mets vs. PB
Signature expressedSignature not expressed
(C) Survival analysis
p=0.84
p=0.87 p=0.048
p=0.01
*
**
Log
(RPK
M+1
)
fold
cha
nge
Figure 2: Differential gene expression analysis of CTCs, mets and PB.Two dimensional hierarchical clustering of all samples based on a 253gene signaturRe that distinguishes CTCs (blue), mets (grey) and PB (red)(FDR adjusted p<0.05).
We present the whole transcriptomic landscape of CTCs with comparison to metastasesand peripheral blood all acquired prior to treatment of Stage IV breast cancer. Multiplegenes, including oncogenes, breast epithelial, mesenchymal genes and CSC genes, werefound with higher expression in CTCs versus metastases. When focusing on 66 knownpotentially clinically actionable genes in breast cancer, represented by 7 molecularsignaling pathways, CTCs did not show significantly different patterns of expressionversus mets compared to PB. Longitudinal analysis of 4 patients over time who had serialCTC assessments showed changing biological characteristics of CTCs isolated atdifferent time points during treatment and disease progression. RNA Seq of CTCs may beutilized to identify molecular alterations in MBC patients that are potentially clinicallyactionable.
Metastatic site profiled: acsites, ER/PR+,HER2-. Other sites ofmetastatic disease: liver, lung, pleural effusion and bone.Molecular profiling with RNA Seq was done to evaluate for potentialtreatment targets in CTCs as a liquid biopsy as well as metastaticdisease.