(a) Baseline and emergent single nucleotide variants (SNVs) detected by CAPP-Seq in the plasma of patients treated with rociletinib. Only patients in whom ...
b Baseline Baseline Alterations Emergent Patient ID Genomic Present at Progression Alterations Alterations CO3 CDKN2A D74A None CO4 EGFR only EGFR only CDKN2A D74A PIK3CA E542K CO7 EGFR only EGFR only PIK3CA E545K CO9 EGFR only EGFR only ALK R1061Q CO10 EGFR S768I EGFR S768I CO11 EGFR only EGFR only CDKN2A D74A CO16 CO19 CO22 CO24 CO27 CO29 CO31
RB1 G587* PIK3CA N345K EGFR G724S RB1 S485C PIK3CA E545K EGFR A750P RB1 V654M PIK3CA C420R PIK3CA E545K RB1 R251* PIK3CA N345S
15
PIK3CA EGFR KRAS CDKN2A RB1 ALK KIT
5 10
4 3 2
5
1
1
1
RB1 G587* PIK3CA N345K EGFR G724S
0
RB1 R787Q KRAS G12A
Emergent SNVs
c
None
15
PIK3CA EGFR KRAS CDKN2A RB1 ALK KIT MET
EGFR A750P RB1 V654M PIK3CA E545K RB1 R251* PIK3CA N345S
CO33
EGFR only
EGFR only
CO34 CO37 CO39 CO43
EGFR only EGFR only RB1 Y606* EGFR only
EGFR only EGFR only RB1 Y606* EGFR only
CO44
EGFR only
EGFR only
CO45
MET D1304H
MET D1304H
CO46
PTEN K125T
PTEN K125T
PIK3CA E542K PIK3CA E545K PIK3CA E542K PIK3CA E545K EGFR L798I EGFR L692V EGFR C797S KRAS A146T KIT L576P KRAS Q61H PIK3CA E81K EGFR E709K
Fraction of Patients (%)
CO15 #
Fraction of Patients (%)
a
5
5
10 3 5
3 2 1
1
1
0 All Putative SNVs
Supplementary Figure 1. Summary of Baseline and emergent SNV’s in rociletinib treated patients. (a) Baseline and emergent single nucleotide variants (SNVs) detected by CAPP-Seq in the plasma of patients treated with rociletinib. Only patients in whom non-EGFR activating and T790M mutations were detected are listed and mutations in TP53 are excluded (* indicates baseline mutations were observed in a pre-treatment tissue biopsy but not plasma due to undetectable levels of ctDNA). (b) The fraction of patients in whom an emergent (absent at baseline and detected following treatment) SNV was detected in the genes listed. (c) The fraction of patients in whom any SNV was identified as a putative mechanism of resistance in the genes listed. This includes emergent SNVs as well as SNVs that were present at baseline and increased in relative abundance following treatment with rociletinib.
a 100,000
EGFR L858R EGFR T790M
CO29
Mutant copies/mL
10,000
RB1 V654M
1,000
TP53 V272L PIK3CA C420R PIK3CA E545K PIK3CA E542K ND
100 10
15 0
12 5
10 0
75
50
25
0
ND Day of Study 10,000
Mutant copies/mL
b
CO31 EGFR Ex19Del EGFR T790M
1,000
RB1 R251*
100
PIK3CA N345S PIK3CA E545K ND
10
20 0
15 0
10 0
50
1
ND Day of Study
Supplementary Figure 2. Vignettes of patients with emergent PIK3CA mutations. (a-b) Representative vignettes of patients with emergent PIK3CA mutations. Baseline and emergent SNVs detected by CAPP-Seq in the plasma of patients treated with rociletinib are displayed. ND=“not detected”.
a parental 1uM rociletinib
-
b
wtEGFR lentivirus
+
-
+
parental parental
kDa 175
pEGFR (Y1068)
1uM rociletinib rociletinib 1uM
-
+
wtERRB2 lentivirus -
+
kDa
pERBB2 (Y1248) (Y1248) pERBB2
185 175
pAKT (S473)
60
pEGFR (Y1068) (Y1068) pEGFR
pMAPK (T202/Y204)
44 42
pAKT (S473) (S473) pAKT
60
pMAPK (T202/Y204) (T202/Y204) pMAPK
44 42
175
EGFR AKT
60
ERBB2 HER2
185
MAPK
44 42
EGFR EGFR
175
55
AKT AKT
60
MAPK MAPK
44 42
tubulin tubulin
55
tubulin
c Treatment
NCI-H1975
NCI-H1975 wtEGFR
NCI-H1975 wtHER2
rociletinib
47 ± 26
102 ± 33*
176 ± 57***
fold over parental
NA
2.2
3.7
Supplementary Figure 3. Wild-type EGFR and ERBB2 overexpression decreases rociletinib sensitivity. The NCI-H1975 (L858R/T790M EGFR) NSCLC cell line was transduced with lentiviral vectors expressing full-length wild-type (wt) EGFR or ERBB2, and cell populations were selected for infected cells using puromycin and blasticidin, respectively. The expression of EGFR (a) and ERBB2 (b) and markers of downstream signaling were evaluated by Western blot analysis in the presence and absence of 1µM rociletinib for 1 hour in full media. (c) Cell viability was evaluated in rociletinib treated parental, EGFR, and ERBB2 overexpressing cell lines after 72 hrs of treatment. Experiments were performed in triplicate, repeated three times, and data are reported as the mean percentage viability ± SD relative to control. Both EGFR and ERBB2 overexpression significantly reduced the potency of rociletinib, by 2.2- and 3.7-fold, respectively (*P < 0.05 for EGFR, ***P < 0.0005 for ERBB2, Wilcoxon rank-sum test).
a
b Vehicle tumor
Vehicle tumor
pEGFR
RR tumor (crossover)
pMET RR tumor (monotherapy)
RR tumor (monotherapy)
pMET
Supplementary Figure 4. MET amplification and pathway activation is associated with acquired resistance to rociletinib. (a) Tumors were collected at endpoint from vehicle and rociletinib resistant (RR) treated animals. Tumor lysates were prepared and exposed to receptor tyrosine kinase (RTK) profiler arrays, which simultaneously detect 42 different phosphorylated RTKs. The spots not labeled with an arrow in at least one array are reference spots used to align the template. (b) Fluorescence in-situ hybridization (FISH) analysis was performed on vehicle and RR tumors. The MET/CEP7 ratio for vehicle and RR tumors are 1.98 and 5.79, respectively. MET staining is in red and CEP7 staining is in green. Images were taken at a magnification of 100X.
Mean body weight (g ± SEM)
30
Vehicle Rociletinib (100 mg/kg BID) Crizotinib (50 mg/kg QD) Combination
25 20 15 10
0
5
10
15
20
25
Days of dosing
Supplementary Figure 5. Combined treatment with rociletinib and crizotinib does not lead to weight loss in mice. LU0858 patient-derived xenograft-bearing animals were orally administered rociletinib, crizotinib, or the combination using the dose and schedules indicated (n = 10 mice/group) and body weights were measured twice weekly (the average weight of the mice is plotted and error bars represent ± standard error).
a
b
c Sensitivity Prediction
Threshold Determination
60
60 40 20
40
0
1
2
3
4
5
6
0
7
0.1
0.3
Copy Number Index (t)
2
ME T
MET ME T MET
100
3
4
Sensitivity (%) 5
6
7
Copy Number Index (t)
Specificity (%)
t= 3.045
80
2
2.5
5
10
0
1
2
3
4
5
6
7
20
15
15 10 10 5 0
0
2
4
6
8
0
Observed copy number index Normalized copy number ND
% ctDNA
60
MET
40
20
20 0
5
t = 2.58
80
0.1
0.3
0.5
0.75
1
1.25
1.5
1.75
2
2.5
5
10
20
3.37% ctDNA 15
15
10
10
5
5
0
60 40
1.75
Amount of H1573 into Healthy Plasma (%)
ERBB2 ERBB2
100
1.5
Amount of H1573 into Healthy Plasma (%)
60
1
1.25
MET
80
0
1
MET MET
t= 3.09
40
0.75
25
0.95% ctDNA
t = 3.09
0
2
4
6
8
0
Normalized Copy Number
Specificity (%)
100
0.5
Copy Number index (t)
80
Sensitivity (%)
Specificity (%)
80
EGFR
20
Normalized Copy Number
t= 2.58
EGFR
E GFR EGFR
100
Copy Number index (t)
EGFR EGFR
100
Empirical Spike
Observed copy number index Normalized copy number ND
% ctDNA
Copy Number Index (t)
Supplementary Figure 6. Copy number index threshold determination and validation for somatic copy number alteration assessment. (a) Copy number index threshold (t) determination for EGFR, MET, and ERBB2. The threshold (t) at which a copy number gain was considered significant in the plasma was defined as the copy number index value at which 95% specificity was achieved when analyzing 27 healthy control plasma samples. (b) In-silico spiking analysis to predict the sensitivity of detection for EGFR and MET copy number gains. Sequencing reads from the NCI-H1573 cancer cell line, which harbors ~20 copies of EGFR and ~13 copies of MET, were “spiked” into sequencing data from the cfDNA of 27 healthy individuals at varying ratios (0.1-10%). (c) Empirical spike experiment to determine the sensitivity of somatic copy number alteration (SCNA) detection in plasma. Fragmented genomic DNA from NCI-H1573 cells was spiked into cfDNA from a healthy control individual at 9 different concentrations ranging from 0.2-7%. Samples were sequenced using CAPP-Seq and SCNA analysis was performed. Copy number index thresholds (t) were determined in (a) and are indicated with a horizontal red dashed line. EGFR and MET copy number gains were detected at concentrations as low as 0.95% and 3.37% ctDNA, respectively (vertical black dashed line), congruent with the predictions from (b). The normalized copy number in samples in which a SCNA was detected ranged from 9.4-9.6 and 10.1-15 for MET and EGFR, respectively.
Figure 7b 700nm channel
a
Erlotinib-resistant (ER) population
PC-9 parental
MW (kDa)
1µM erlotinib
!
+
!
!
!
+
!
1µM CO-1686
!
!
0.2µM crizotinib
!
!
+
!
!
!
!
+
!
!
Figure 7b 800nm channel
!
+
!
+
!
!
!
+
!
+
!
!
!
!
Erlotinib-resistant (ER) population
PC-9 parental
CO-1686-resistant (RR) population
!
+
!
!
!
+
+
+
+
MW (kDa)
260
!
+
!
!
!
+
!
1µM CO-1686
!
!
0.2µM crizotinib
!
!
+
!
!
!
!
+
!
!
CO-1686-resistant (RR) population
!
!
+
!
+
!
+
!
!
!
+
!
!
+
!
+
!
!
!
+
+
+
260
EGFR 175 kDa
160 125
1µM erlotinib
!
pEGFR (Y1068) 175kDa
125
90
Blot #1
non-specific
70
AKT 60 kDa
50
70
Blot #1
MAPK 42,44 kDa
38
pAKT (S473) 60kDa pMAPK (T202/Y204) 42,44 kDa
38
30 25
25
260
260
MET 145 kDa
160 125
pMET (Y1234/1235) 145kDa
125 90
Blot #2
90
70
70
Blot #2
50
tubulin 55kDa
38
38
30
pS6RP (S235/236) (unused in manuscript)
Figure 7b 700/800nm merged Erlotinib-resistant (ER) population
PC-9 parental
MW (kDa) 260 160 125
CO-1686-resistant (RR) population
1µM erlotinib
!
+
!
!
!
+
!
!
!
+
!
!
+
!
1µM CO-1686
!
!
+
!
!
!
+
!
!
!
+
!
!
+
0.2µM crizotinib
!
!
!
+
!
!
!
+
!
!
!
+
+
+
EGFR merged 175kDa
90 70
Blot #1 50 38
non-specific AKT merged 60kDa MAPK merged 42,44 kDa
30 25 260 160 125
MET merged 145kDa
90
Blot #2
70 50 38 30
tubulin 55kDa pS6RP (S235/236) (unused in manuscript)
Supplementary Figure 7. Complete scans of western blots depicted in the manuscript. (a) Annotated raw and merged scans of the western blot depicted in Figure 7b.
Figure 7d 800nm channel
Figure 7d 700nm channel
b
Figure 7d 700/800nm merged
Supplementary Figure 7 continued. Complete scans of western blots depicted in the manuscript. (b) Annotated raw and merged scans of the western blot depicted in Figure 7d. In these blots PC-9 parental and PC-9 RR cells were infected at a MOI of 0.5 or 1.5 as indicated with lentivirus harboring constructs to overexpress shRNA targeting MET or a scrambled control sequence. The results were comparable using both concentrations of lentivirus, and Figure 7d only shows the results from the infections at a MOI of 0.5.
Supplementary Figure 3a 700nm channel
c MW$ 1uM# (kDa)$ rocileEnib# 260$ 160# 125# NCI-H1975 parental vs. wtEGFR lentivirus
parental#
wtEGFR# lenEvirus#
4# +# 4# +#
parental# 1uM# rocileEnib#
Supplementary Figure 3a 800nm channel
wtEGFR# lenEvirus#
MW# 1uM& (kDa)# rocileEnib&
4# +# 4# +#
50# 38# 30#
wtEGFR& lenEvirus&
H& +& H& +&
260# EGFR# 175kDa#
non4specific#AKT# AKT# 60kDa#
tubulin# 55kDa#
MAPK# 44,#42kDa#
NCI-H1975 parental vs. wtEGFR lentivirus
parental& 1uM& rocileEnib&
wtEGFR& lenEvirus&
H& +& H& +&
pEGFR&(Y1068)& 175kDa&
125#
90# 70#
parental&
70#
pAKT&(S473)& 60kDa& pMAPK&(T202/Y204)& 44,&42kDa&
38#
Supplementary Figure 3a 700/800nm merged MW# 1uM& (kDa)# rocileEnib& 260# 160# 125# NCI-H1975 parental vs. wtEGFR lentivirus
parental&
wtEGFR& lenEvirus&
;& +& ;& +&
parental& 1uM& rocileEnib&
wtEGFR& lenEvirus&
;& +& ;& +&
Merged&EGFR& 175kDa&
90# 70# 50# 38#
merged&AKT& 60kDa& merged&MAPK& 44,&42kDa&
tubulin& 55kDa&
Supplementary Figure 7 continued. Complete scans of western blots depicted in the manuscript. (c) Annotated raw and merged scans of the western blot depicted in Supplementary Figure 3a. Blots were sectioned into strips for incubation with different primary antibodies before being reassembled prior to imaging. The background intensity is different in some strips based on the primary antibodies used.
d Supplementary Figure 3b 700nm channel
Supplementary Figure 3b 800nm channel
Supplementary Figure 3b 700/800nm merged
Supplementary Figure 7 continued. Complete scans of western blots depicted in the manuscript. (d) Annotated raw and merged scans of the western blot depicted in Supplementary Figure 3b. Blots were sectioned into strips for incubation with different primary antibodies before being reassembled prior to imaging. The background intensity is different in some strips based on the primary antibodies used. Unrelated western blots scanned in parallel were cropped from the left and right of each panel shown.
Supplementary Table 1. Characteristics of the 43 rociletinib-treated patients profiled by CAPP-Seq
a
Patient ID
Smoking Status
Gender
Age
Race
CO3 CO4 CO5 CO6 CO7 CO8 CO9 CO10 CO11 CO14 CO15 CO16 CO17 CO18 CO19 CO20 CO21 CO22 CO23 CO24 CO25 CO26 CO27 CO28 CO29 CO30 CO31 CO32 CO33 CO34 CO35 CO36 CO37 CO39
Never Smoked Former Smoker Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Never Smoked Former Smoker Never Smoked Never Smoked Never Smoked Never Smoked Former Smoker Former Smoker Former Smoker Never Smoked Never Smoked
F M F F M F M F F M F F F M F F M F F F F M F F F F F M F M M M F M
53 71 53 51 48 53 74 55 30 64 48 48 64 53 66 60 58 55 62 57 47 47 67 47 72 37 84 40 56 46 66 72 54 52
CO40
Never Smoked
M
59
CO41 CO43 CO44 CO45 CO46 CO47 CO48
Former Smoker Never Smoked Former Smoker Never Smoked Never Smoked Former Smoker Never Smoked
M F F F F F F
68 45 66 65 81 66 51
CO50
Never Smoked
M
65
WHITE WHITE WHITE WHITE WHITE WHITE WHITE WHITE WHITE WHITE ASIAN ASIAN WHITE WHITE ASIAN WHITE WHITE WHITE WHITE WHITE WHITE WHITE ASIAN WHITE WHITE NOT PROVIDED WHITE NOT PROVIDED WHITE ASIAN WHITE WHITE WHITE WHITE BLACK OR AFRICAN AMERICAN WHITE WHITE WHITE WHITE WHITE ASIAN WHITE BLACK OR AFRICAN AMERICAN
Immediately Prior History Prior Lines of Baseline Prior EGFR of CNS Therapy (EGFR ECOG Status TKI Disease Directed)
Starting Dose
Duration on Best Response PFS by Duration on Rociletinib after by Investigator Investigator Rociletinib Progression (%) (month) (month) (month) a
NA erlotinib erlotinib erlotinib erlotinib erlotinib afatinib erlotinib NA NA erlotinib erlotinib erlotinib NA NA NA afatinib erlotinib erlotinib NA erlotinib erlotinib erlotinib afatinib afatinib NA erlotinib erlotinib erlotinib erlotinib erlotinib erlotinib erlotinib erlotinib
N N Y Y Y N N N N Y N Y Y N Y Y N Y Y Y N Y Y Y N Y Y N N N N N Y Y
4(1) 6(3) 5(4) 7(7) 3(1) 1(1) 4(2) 3(1) 4(1) 2(1) 4(1) 6(1) 5(2) 5(2) 2(1) 4(2) 3(2) 3(2) 5(2) 2(1) 6(1) 7(2) 6(3) 5(2) 6(3) 1(1) 1(1) 1(1) 6(2) 3(2) 4(3) 2(1) 3(2) 1(1)
1 1 1 1 1 0 1 0 1 0 1 1 1 0 1 1 1 0 1 0 1 1 0 1 1 1 1 1 1 0 1 0 1 1
900 mg BID FB 900 mg BID FB 900 mg BID FB 900 mg BID FB 500 mg BID HBr 500 mg BID HBr 1000 mg BID HBr 500 mg BID HBr 750 mg BID HBr 400 mg TID FB 900 mg BID FB 625 mg BID HBr 750 mg BID HBr 200 mg QD FB 750 mg BID HBr 625 mg BID HBr 625 mg BID HBr 625 mg BID HBr 750 mg BID HBr 625 mg BID HBr 500 mg BID HBr 750 mg BID HBr 750 mg BID HBr 625 mg BID HBr 500 mg BID HBr 750 mg BID HBr 750 mg BID HBr 625 mg BID HBr 750 mg BID HBr 625 mg BID HBr 500 mg BID HBr 625 mg BID HBr 750 mg BID HBr 750 mg BID HBr
-56 32 -10 -31 26 41 30 -32 -71 -24 -71 8 -58 -22 -41 8 15 0 -5 -47 -42 -39 -14 -67 -47 -47 -53 -60 -33 -33 -9 -11 -35 -40
4.6 1 1.4 4 1.3 1 1.3 3.9 2.7 4 12.2 2.8 2.9 13.5 4.1 1.3 5 1.1 3.8 2.8 6 6.1 4.2 2.6 3.9 1.4 6.1 6.1 8.3 4.1 12.7 2.6 7 4.3
15.1 1.2 6.2 5.5 2.1 1.2 1.4 7.3 6.4 11.4 13.3 2.8 2.9 14.9 4.6 2.5 0.7 1.1 12.4 5.8 8.8 6.1 4.2 7 4.3 3.5 5.5 6.2 11.3 9.5 12.7 5.3 9.6 8.5
10.5
erlotinib
N
1(1)
1
500 mg BID HBr
-27
4.2
erlotinib NA erlotinib erlotinib erlotinib afatinib NA
N N N N N Y Y
1(1) 2(1) 4(1) 3(1) 3(2) 8(3) 2(1)
1 0 1 1 1 1 1
750 mg BID HBr 625 mg BID HBr 625 mg BID HBr 500 mg BID HBr 625 mg BID HBr 500 mg BID HBr 500 mg BID HBr
-28 -40 -31 -50 -4 -30 7
5.6 6.1 5.2 3.9 3.9 3.7 2.8
4.9 12 10.7 9 13.1 4.1 3.5 9.7
0.7 6.4 4.6 3.8 9.2
erlotinib
N
5(3)
0
750 mg BID HBr
-46
6.2
5.8
Values are only listed for patients that remained on rociletinib ≥ 0.5 months after progression
Abbreviations: M = male; F = female; NA = not applicable; ECOG = Eastern Cooperative Oncology Group.
4.8 1.5 0.8
3.4 3.7 7.4 1.1
1.4 0.5 1.2
8.6 3 2.8
4.4 2.1
3 5.4 2.7 2.6 4.2
6.9
ABCC9 ABL1 ABR ADAMTS12 AKT1 ALK AMOT ANK2 APOB ASB18 ASTN1 ASTN2 ASXL3 ATP11B BAGE5 BCL11B BCL2 BRAF BRINP3 C14orf177 C22orf34 C6orf118 C9 CA10 CASC11 CASC8 CCND1 CD226 CDH10 CDH12 CDH18 CDH7 CDH9 CDKN2A CHEK2 CLDN11 CNTNAP2 CNTNAP5 COL22A1 CSMD1 CSMD3 CTNNA2
CTNNB1 CTSS CUL3 DCAF12L1 DCAF12L2 DCAF4L2 DCC DDR2 DDX1 DENND4B DMD DUSP27 EGFLAM EGFR EPHA3 ERBB2 ERBB4 ERICH3 FAM135B FAT3 FBN2 FBXL7 FBXW7 FGD1 FGF3 FGFR1 FGFR2 FGFR3 FKBP9P1 FLJ26245 FOXP1 FRYL GABRA2 GABRA6 GPR112 GPR158 GRID1 GRIK3 GRM8 HAPLN1 HAX1 HCN1
HDAC9 HRAS HTR1A HTR3E IGFL3 IL6R IQCJ-SCHIP1 KCNA4 KCND2 KCNJ3 KCNT2 KDR KEAP1 KIF2B KIT KLHL1 KLHL6 KMT2C KMT2D KRAS LANCL2 LELP1 LINC00441 LMNTD1 LOC349160 LPL LPPR4 LRFN5 LRP1B LRRC4C LRRIQ3 LRRTM4 MACF1 MAP2K1 MAP2K2 MAPK1 MARCH1 MCCC1 MCF2L2 MCL1 MDM2 MECOM
MET MKRN3 MPHOSPH8 MRGPRD MS4A3 MYC MYCL MYCN MYD88 MYEOV MYNN NAV3 NCAM1 NETO1 NF2 NFE2L2 NKD2 NLRP3 NLRP5 NOTCH1 NRAS NT5C1A NTM NUP155 PABPC4 PACRG PAK7 PAPPA2 PARK2 PCDH10 PCDH17 PDGFRA PDGFRB PDHA2 PDYN PDZRN3 PEG3 PHC3 PIK3CA PKLR POLDIP2 POM121L12
PRIM2 PSPC1 PTEN PTPRD PXDNL RAF1 RB1 REG3A RET RFX5 RIT2 RLF ROBO1 ROS1 RP1L1 RPS4Y1 RYR2 S100A7 SALL1 SAMD7 SEMA6C SERPINB3 SETBP1 SF3B1 SFTA3 SGCZ SKP2 SLC12A7 SLC14A2 SLC1A3 SLC2A2 SLC45A2 SLC6A19 SLC8A1 SLIT3 SLITRK1 SLITRK2 SLITRK3 SMAD4 SOX2-OT SPHKAP SPTA1
ST6GAL2 STK11 SYT4 TARS2 TERT TG TGFBR3 TLR4 TNN TNR TP53 TP63 TPCN2 TPTE2 TRDMT1 TRIM58 TRIP13 TRPC5 TSHZ3 TUBA3C U2AF1 UGT3A2 VGLL4 VSTM2A WDR7 WHSC1L1 XIRP2 ZAN ZFHX4 ZFY ZIC1 ZIC4 ZMYM5 ZNF236 ZNF423 ZNF521 ZNF536 ZNF713 ZNF804A ZNF804B ZNF831 !!
Supplementary Table 2. NSCLC-focused CAPP-Seq Selector summary. This table lists the 252 genes that were either partially or fully covered by the 302kb CAPP-Seq selector used in this study.
