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RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
• Alterations in KRAS, BRAF and EGFR as well as pan-Ras classifier score (data not shown)strongly predict high expression of the adenosine fingerprint in pan-cancer TCGA.
• In KRAS, BRAF and EGFR wild-type patients, high CD73 expression is significantly associatedwith poor prognosis. High CD73 expression is strongly associated with poor prognosis in EGFRand KRAS mutant patients, and good prognosis in BRAF mutant patients.
• Adenosine fingerprint exhibits higher expression in KRAS mutant versus wild-type in CRC andNSCLC patients as well as cell lines at the gene and protein level.
• EGFR mutant NSCLC patients have significantly poor prognosis associated with lower rate ofdurable clinical benefit with pembrolizumab treatment (data not shown).
• High expression of adenosine fingerprint and PD1 in T cells is associated with resistance to anti-PD1 therapy in mutant KRAS and EGFR murine NSCLC.
Udyavar AR, DiRenzo D, Piovesan D, Ashok D, Anderson AE, Young SW, Walters MJ, Tan JBL Arcus Biosciences, Inc. 3928 Point Eden Way, Hayward, CA 94545 (USA)
Altered pan-Ras pathway and activating mutations in EGFR result in elevated CD73 in multiple cancers
• AB928, a dual A2aR/A2bR antagonist, andAB680, a potent selective inhibitor of CD73, canrescue the immunosuppressive effects ofadenosine in experimental cell culture andtumor models.
• Oncogene-driven cancers tend to be non-responsive to PD(L)-1 inhibition.
• Here we show that pan-RAS, BRAF and EGFRalterations drive the expression of CD73, whichmay contribute to suppressed anti-tumorimmunity.
• Regression analysis: We used linear models adjusted for individual tumor types to assess if theexpression of CD73 and other genes in the adenosine fingerprint can be predicted by alterations in299 consensus cancer driver genes (Bailey et.al Cell (2018)) in pan-cancer TCGA dataset (GDCCommons). For regression models, multiple testing correction was performed using Benjamini-Hochberg method.
• Survival analysis: Kaplan-Meier curves were plotted in R showing log-rank p-values. Cox-regression analysis were used to compute hazard ratios and p-values.
• Histology: CD73 (Cell Signaling, D7F9A) immuno-histochemistry (IHC) was performed onsections of formalin fixed paraffin embedded (FFPE) tumor tissue and quantified using QuPathsoftware.
• Cancer cell lines: RNAseq data was obtained from Broad’s Cancer Cell Line Encyclopedia(CCLE) database. RNA from human cancer cell lines was extracted and converted into cDNA.Real-time qPCR was performed using Taqman probes. HPRT1 was used as the housekeepinggene and raw data was analyzed using the 2-ΔCT method. CD73, CD39 and CD26 proteinexpression on human cancer cell lines was determined using flow cytometry.
Introduction
KRAS, BRAF and EGFR strongly predict adenosine fingerprint expression in pan-cancer TCGA
Results
Materials and Methods
Conclusions
Interested in a career at Arcus? Visit www.arcusbio.com
AACR 2019 National Meeting; Atlanta Abstract # 2526
Figure 1. (A) CD73 and TNAP expression was derived from pan-cancer TCGA dataset. Numbers onY-axis indicate ratio of log2 CPM values for CD73 and TNAP. Tumors on left are high in CD73 andlow in TNAP whereas tumors on right are high in TNAP and low in CD73. (B) Linear model estimatesadjusted for tumor type of alterations in cancer driver genes that predict CD73 expression. (C) X-axisdenotes the positive (left) and negative (right) regulators of CD73 from panel B. Y-axis shows linearmodel estimates adjusted for tumor type for each gene in the adenosine fingerprint. Stars indicatesignificant FDR (*** < 0.001,** <0.01,* < 0.2).
CD73 expression is prognostic in KRAS, BRAF and EGFR mutant patients in pan-cancer TCGA
Figure 2. (A) shows frequency and type of alterations in KRAS, BRAF and EGFR from cBioPortal.Inset shows number of mutations in a given gene. (B) Kaplan-Meier curves show impact of CD73expression on the individual cancer drivers with wild-type and altered status on overall survival.High protein expression of CD73 in KRAS mutated patients compared
to wild-type in CRC and NSCLC
Figure 4. (A) Heatmap of adenosine fingerprint in Broad’s CCLE RNAseq data for representativeCRC and NSCLC cell lines. (B) Correlation scatterplot of CD73 expression in cancer cell lines frompanel A and qPCR. (C) Flow cytometry proteomic validation of CD73, CD39 and CD26 expression inKRAS WT and ALT CRC and NSCLC cell lines.
High expression of adenosine fingerprint in KRAS altered versus wild-type in CRC and NSCLC cell lines
Figure 3. (A) Representative CD73 IHC images for KRAS wild-type and altered NSCLC and CRCpatients. (B) Quantification of percent CD73 positive area in CRC and NSCLC patients.
High expression of adenosine fingerprint and PD1 in T cells correlates with resistance to anti-PD1 in KRAS and EGFR mutant NSCLC
Figure 5. Left to right – RNAseq gene expression (dataset from Koyama et.al Nat. Comm. (2015)) ofCD73 (Nt5e), CD39 (Entpd1), A2aR (Adora2a) and PD1 (Pdcd1) in T cells derived from either EGFRL858R/T790M mutant or KRAS G12D mutant mouse tumors that were either untreated (Untrt) orresistant to anti-PD1 (aPD1-R). P-values denote significance by Wilcoxon ranked-sum test.
A
0.0
2.5
5.0
7.5
10.0
WT ALT
KRAS alterations
% C
D73
pos
itive
sta
inin
g
N=9 N=13
KRAS alterationsBWT ALT
NSCLC
CRC
A
p = 0.23
n=3 n=5
p = 0.037
n=5 n=54
5
6
CD39 expression in T-cells
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p = 0.14
n=3 n=5
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p = 0.4
n=5 n=5
EGFRmut KRASmut
Untrt aPD1-R
7.0
7.5
8.0
8.5
Log2
CPM
CD73 expression in T-cells
p = 0.14
n=3 n=5
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p = 0.037
n=5 n=54.5
5.0
5.5
6.0
A2aR expression in T-cells
p = 0.037
n=3 n=5
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p = 0.022
n=5 n=5
6
7
8
9
PD1 expression in Tcells
Untrt aPD1-RUntrt aPD1-R Untrt aPD1-R Untrt aPD1-R Untrt aPD1-R Untrt aPD1-R Untrt aPD1-R
EGFRmut KRASmut EGFRmut KRASmut EGFRmut KRASmut
CD73 CD39 A2aR PD1
Log2
CPM
Alte
ratio
n Fr
eque
ncy
10%
20%
30%
40%
50%
GBM LUADESCAHNSCLGG STADLUSCSKCMUCSBLCACESCSARCCOADACC HCCBRCACHOLOV READPAADKIRPPRADTGCTPCPG KIRCKICHMESOTHCAUCECUVM
Mutation Fusion Amplification Deep Deletion
A289V/T/D
Recep_L_domain Furin-like Recep_.. GF_recep.. Pkinase_Tyr
0 1210aa200 400 600 800 1000
0
35
# EG
FR M
utat
ions
Alte
ratio
n Fr
eque
ncy
10%
20%
30%
40%
50%
60%
PAADCOADREADLUADUCSUCECSTADTGCTOV ESCABLCACHOLCESCLUSCSARCSKCMLGG MESOHCCBRCAGBM KIRPHNSCPRADACCTHCAKIRCKICHPCPG UVM
Mutation Fusion AmplificationDeep Deletion
G12D/V/C and 4 more
Ras
0 189aa100
0
560
# KR
AS M
utat
ions
Alte
ratio
n Fr
eque
ncy
10%
20%
30%
40%
50%
60%
THCASKCMCOADOV LUADUCSBLCALUSCSTADSARCREADUCECGBM PRADCHOLESCAPAADHNSCACC LGG KIRPBRCAMESOPCPG CESCKIRCHCCKICHTGCTUVM
Mutation Fusion Amplification Deep Deletion Multiple Alterations
V600E/M/G and 2 more
RBD C1_1 Pkinase_Tyr
0 766aa200 400 600
0
589
# BR
AF M
utat
ions
G598V/A/E L858R
BRAF
KRAS
EGFR
A2aR
A2aR
A2aRA2bR
ATP AMP Adenosine
CD39 CD73
NK Cells
T cells
DC, TAM, MDSC
PI3Kγ
PI3Kδ
PI3Kγ/δ
IPI-549
AB928
AB928
AB928
AB680
CD38 CD203a
NAD+ ADPR
Mutation
Alteration type
Fusion
Amplification
Deep deletion
Multiple alterations
KRAS EGFRBRAFB+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++ ++++++ +
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++ + + +++
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++ +
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p < 0.0001
0.00
0.25
0.50
0.75
1.00
0 5 10 15 20 25Time (years)
Ove
rall
Surv
ival
(OS)
++++
KRAS:ALT; CD73:HighKRAS:ALT; CD73:LowKRAS:WT; CD73:HighKRAS:WT; CD73:Low ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++ + + +
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++
+++ + ++ ++
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++ +
++ +
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p < 0.0001
0.00
0.25
0.50
0.75
1.00
0 5 10 15 20 25
++++
BRAF:ALT; CD73:HighBRAF:ALT; CD73:LowBRAF:WT; CD73:HighBRAF:WT; CD73:Low ++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++ ++
+
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++++
++++++ ++ +
+ +
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++ +
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p < 0.0001
0.00
0.25
0.50
0.75
1.00
0 5 10 15 20 25
++++
EGFR:ALT; CD73:HighEGFR:ALT; CD73:LowEGFR:WT; CD73:HighEGFR:WT; CD73:Low
Time (years) Time (years)
IsotypeStain
H651 (WT)
CD39
CD73
H441 (ALT)
CD26
H1395 (WT)
H650 (ALT)
NSCLC
SKCO1 (ALT)
RKO (WT)
SW1116 (ALT)
CRCC
A B
●
●●NCI−H1395
NCI−H1651
NCI−H441
NCI−H650
RKO
SKCO1
SW1116
0.0
2.5
5.0
7.5
2 4 6 8CD73 (CCLE RNAseq)
CD
73 (q
tPC
R)
Tissue● CRC
NSCLC
Mutation●●
ALTWT
●
●●NCI−H1395
NCI−H1651
NCI−H441
NCI−H650
RKO
SKCO1
SW1116
0.0
2.5
5.0
7.5
2 4 6 8CD73 (CCLE RNAseq)
CD
73 (q
tPC
R)
Tissue● CRC
NSCLC
Mutation●●ALTWT●
●●NCI−H1395
NCI−H1651
NCI−H441
NCI−H650
RKO
SKCO1
SW1116
0.0
2.5
5.0
7.5
2 4 6 8CD73 (CCLE RNAseq)
CD
73 (q
tPC
R)
Tissue● CRC
NSCLC
Mutation●●ALTWT
CCLE RNAseq
Aden
osin
e pa
thw
ay g
enes
CD39
CD73
ADA
CD26
ADORA1
ADORA2A
ADORA2B
ADORA3
NCI−H1395
RKO
NCI−H1651
SKCO
1
SW11
16
NCI−H441
NCI−H650
KRAS_StatusTissue
Z−score
−2−10123
KRAS StatusALTWT
TissueNSCLCCRC
CCLE RNAseq
Aden
osin
e pa
thw
ay g
enes
CD39
CD73
ADA
CD26
ADORA1
ADORA2A
ADORA2B
ADORA3
NCI−H1395
RKO
NCI−H1651
SKCO
1
SW11
16
NCI−H441
NCI−H650
KRAS_StatusTissue
Z−score
−2−10123
KRAS StatusALTWT
TissueNSCLCCRC
CCLE RNAseq
Aden
osin
e pa
thw
ay g
enes
CD39
CD73
ADA
CD26
ADORA1
ADORA2A
ADORA2B
ADORA3
NCI−H1395
RKO
NCI−H1651
SKCO
1
SW11
16
NCI−H441
NCI−H650
KRAS_StatusTissue
Z−score
−2−10123
KRAS StatusALTWT
TissueNSCLCCRC
CCLE RNAseq
Aden
osin
e pa
thw
ay g
enes
CD39
CD73
ADA
CD26
ADORA1
ADORA2A
ADORA2B
ADORA3
NCI−H1
395
RKO
NCI−H1
651
SKCO
1
SW11
16
NCI−H4
41
NCI−H6
50
KRAS_StatusTissue
Z−score
−2−10123
KRAS StatusALTWT
TissueNSCLCCRCA1R
A2AR
A2BR
A3R
CD73
/TNA
P (L
og2)
ratio
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CD73CD39CD38
ENPP1CD73/TNAP
TNAPADA
CD26A1R
A2aRA2bR
KRASBRAF
METFUBP1
RAC1EGFR
CDK4CTCF
PGRRET
RASA1JA
K1PHF6
NF1CIC
ARID1AZFHX3
ZCCHC12
GNA11
SMAD4
USP9X
CDKN2AFA
T1
PIK3R1
SCAF4PMS2
RNF43
SMC1ABCOR
FGFR2
COL5A1ATMKMT2B
CTNNB1MYC
RAD21PTEN
Positive regulators of CD73
Aden
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thw
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*
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CD73CD39CD38
ENPP1CD73/TNAP
TNAPADA
CD26
RHOB
TBL1XR1
KEAP1
ZFP36L2
FGFR3
FOXA1FLT
3
TRAF3
RNF111
PPP2R1ATXNIP
STAG2
RIT1TGIF1
FOXQ1ATR
CYSLTR2
PCBP1
PIK3R2
ASXL1
HIST1H1CKLF5
PIK3CBSPOP
MECOM
CACNA1A
CTNND1
DACH1XPO1
ZNF750
FBXW7MUC6
KDM6AGATA
3
ZBTB20
PIK3CARB1SOX17
SMARCA4KITCHD8
CHD4APOB
Negative regulators of CD73
Increased expression in Mutants
−0.50
0.00
Model estimates0.25
Decreased expression in Mutants
A1RA2aRA2bR
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CD73CD39CD38
ENPP1CD73/TNAP
TNAPADA
CD26A1R
A2aRA2bR
KRASBRAF
METFUBP1
RAC1EGFR
CDK4CTCF
PGRRET
RASA1JA
K1PHF6
NF1CIC
ARID1AZFHX3
ZCCHC12
GNA11
SMAD4
USP9X
CDKN2AFA
T1
PIK3R1
SCAF4PMS2
RNF43
SMC1ABCOR
FGFR2
COL5A1ATMKMT2B
CTNNB1MYC
RAD21PTEN
Positive regulators of CD73
Aden
osin
e pa
thw
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CD73CD39CD38
ENPP1CD73/TNAP
TNAPADA
CD26
RHOB
TBL1XR1
KEAP1
ZFP36L2
FGFR3
FOXA1FLT
3
TRAF3
RNF111
PPP2R1ATXNIP
STAG2
RIT1TGIF1
FOXQ1ATR
CYSLTR2
PCBP1
PIK3R2
ASXL1
HIST1H1CKLF5
PIK3CBSPOP
MECOM
CACNA1A
CTNND1
DACH1XPO1
ZNF750
FBXW7MUC6
KDM6AGATA
3
ZBTB20
PIK3CARB1SOX17
SMARCA4KITCHD8
CHD4APOB
Negative regulators of CD73
Increased expression in Mutants
−0.50
0.00
Model estimates0.25
Decreased expression in Mutants
A1RA2aRA2bR
A B
C
CD73 higher in Altered vs WT
CD73 lower in Altered vs WT
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*
*
**
**
*
**
*
*
*
*
*
*
**
*
**
*
*
*
*
*
**
**
**
***
******
***
*
***
*
*
******
*
*
****
*
*
*
**
**
**
*
***
**
***
*
*
**
**
*
*
*
*
*
*
****
**
*
*
***
**
*
*
*
*
**
*
*
*
*
*
***
*
*
*
*
*
*
*
*
*
***
**
*
*
*
*
*
**
*
*
**
*
***
*
*
*
****
**
****
CD73CD39CD38
ENPP1CD73/TNAP
TNAPADA
CD26A1R
A2aRA2bR
KRASBRAF
METFUBP1
RAC1EGFR
CDK4CTCF
PGRRET
RASA1JA
K1PHF6
NF1CIC
ARID1AZFHX3
ZCCHC12
GNA11
SMAD4
USP9X
CDKN2AFA
T1
PIK3R1
SCAF4PMS2
RNF43
SMC1ABCOR
FGFR2
COL5A1ATMKMT2B
CTNNB1MYC
RAD21PTEN
Positive regulators of CD73
Aden
osin
e pa
thw
ay g
enes
*
*
*
***
*
*
**
***
*
*
*
*
**
*
**
***
*
*
*
*
**
****
**
*
*
*
**
***
*
***
*
*
*****
**
*
********
*
*
**
**
**
*
***
***
***
*
****
*
****
***
***
*****
*
**
*
*
*****
**
*
*
*
*
*
*
*
***
**
*
*
**
**
*
*
*
*
*
**
*
*
**
*
*
*
*
***
*
*
***
*
**
*
*
*
*
*
*
***
*
**
***
**
*
*
*
*
*
*
*
* ***
*
*
*
*
*
*
*
*
*
*
**
*
*
*
*
CD73CD39CD38
ENPP1CD73/TNAP
TNAPADA
CD26A1R
A2aRA2bR
RHOB
TBL1XR1
KEAP1
ZFP36L2
FGFR3
FOXA1FLT
3
TRAF3
RNF111
PPP2R1ATXNIP
STAG2
RIT1TGIF1
FOXQ1ATR
CYSLTR2
PCBP1
PIK3R2
ASXL1
HIST1H1CKLF5
PIK3CBSPOP
MECOM
CACNA1A
CTNND1
DACH1XPO1
ZNF750
FBXW7MUC6
KDM6AGATA
3
ZBTB20
PIK3CARB1SOX17
SMARCA4KITCHD8
CHD4APOB
Negative regulators of CD73
Increased expression in Mutants
−0.50
0.00
Model estimates0.25
Decreased expression in Mutants
Positive regulators of CD73 expression Negative regulators of CD73 expression
Oncogenic drivers of CD73 expression
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n=63
5n=
502n=
161n=
375n=
178n=
177n=
155n=
259n=
500n=
86n=
288n=
36n=
103n=
171n=
411n=
511n=
304n=
371n=
80n=
528n=
498n=
257n=
524n=
141n=
65n=
534n=
501n=
547n=
56n=
374n=
79n=
150
**** * ** **** ****
****
****
****
****
****
**** *** ****
****** ****
****
****
****** *****
****
****
****
****
−10
−5
0
5
10
CRCTHCA
ESCASTA
DPCPG
PAAD
GBMSARC
HNSCMESO
KIRPCHOL
SKCM
HER2+ BRCA
BLCALGG
CESCLIHC
UVM
ER/PR+ BRCA
PRADBRCA
LUAD
TNBC BRCAKICH
KIRCLUSC
UCECUCS OV
ACCTGCT
log2
ratio
(CD7
3/TN
AP)
4 8CD73 (log2 CPM) TNAP (log2 CPM) ● ● ●0 4 8
CD73 vs TNAP
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n=63
5n=
502n=
161n=
375n=
178n=
177n=
155n=
259n=
500n=
86n=
288n=
36n=
103n=
171n=
411n=
511n=
304n=
371n=
80n=
528n=
498n=
257n=
524n=
141n=
65n=
534n=
501n=
547n=
56n=
374n=
79n=
150
**** * ** **** ****
****
****
****
****
****
**** *** ****
****** ****
****
****
****** *****
****
****
****
****
−10
−5
0
5
10
CRCTHCA
ESCASTA
DPCPG
PAAD
GBMSARC
HNSCMESO
KIRPCHOL
SKCM
HER2+ BRCA
BLCALGG
CESCLIHC
UVM
ER/PR+ BRCA
PRADBRCA
LUAD
TNBC BRCAKICH
KIRCLUSC
UCECUCS OV
ACCTGCT
log2
ratio
(CD7
3/TN
AP)
4 8CD73 (log2 CPM) TNAP (log2 CPM) ● ● ●0 4 8
CD73 vs TNAP
CD73 (log2 CPM)
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n=63
5n=
502n=
161n=
375n=
178n=
177n=
155n=
259n=
500n=
86n=
288n=
36n=
103n=
171n=
411n=
511n=
304n=
371n=
80n=
528n=
498n=
257n=
524n=
141n=
65n=
534n=
501n=
547n=
56n=
374n=
79n=
150
**** * ** **** ****
****
****
****
****
****
**** *** ****
****** ****
****
****
****** *****
****
****
****
****
−10
−5
0
5
10
CRCTHCA
ESCASTA
DPCPG
PAAD
GBMSARC
HNSCMESO
KIRPCHOL
SKCM
HER2+ BRCA
BLCALGG
CESCLIHC
UVM
ER/PR+ BRCA
PRADBRCA
LUAD
TNBC BRCAKICH
KIRCLUSC
UCECUCS OV
ACCTGCT
log2
ratio
(CD7
3/TN
AP)
4 8CD73 (log2 CPM) TNAP (log2 CPM) ● ● ●0 4 8
CD73 vs TNAP
TNAP (log2 CPM)
CD73/TNAP ratio
-log 1
0FD
R
CD73
/TNA
P (lo
g 2ra
tio)
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