Supplemental Table. I Algorithm for Treatment Selection
Order of Precedence Methodology
1
IHC/FISH, and DNA Microarray, or RPPA and DNA Microarray
2
IHC/FISH, or RPPA
3
IHC/FISH, DNA Microarray
4
RPPA alone
5
IHC/FISH alone
6
RPPA and DNA Microarray
7
DNA Microarray alone
The above algorithm was used in addition to the patient’s past medical and treatment
history, current AE’s from prior therapies e.g. peripheral neuropathy, etc., in selecting
the MP recommended treatment.
Supplemental Material II. MP Analysis Methods
DNA MA Analysis: 0.25 mg of FF tissue containing at least 20% tumor was required.
IHC/FISH analysis, required one FFPE block or 30 unstained, unbaked, positive
charged slides. A complete list of the genes and IHC/FISH targets is included as
Supplemental Tables II and III.
RPPA analysis: 8 micron sections obtained from optimal cutting temperature Compound
(OCT)-embedded FF tissue was required. Using laser capture microdissection (LCM)
approximately 5,000-20,000 tumor cells were isolated from each sample and derived
lysates subjected to RPPA analysis as previously described and summarized in
Supplemental Figure 11-4. Samples were printed in triplicate along with calibrators
spanning the dynamic range of the analytes of interest.
Inter and intra-assay
performance of the platform was monitored over time using high and low controls that
were developed and implemented. The RPPA assay was run at least on a weekly basis
in order to maintain proficiency during the entire course of the trial, regardless of accrual
frequency. A total of 11 proteins and phosphoproteins, including drug targets (e.g.
phospho HER2, phospho EGFR) and downstream substrates (e.g. phospho AKT,
phospho ERK), were analyzed as part of this study (Supplemental Table IV). Predefined cut-points, based on population data from a large number of LCM human
tumors, were used to identify patients whose tumor protein activation levels were in the
top-quartile of the population data. Pathway activation, used as a final determination of
a positive reported finding, was defined in this study as a tumor profile that had
activation of at least one drug target, listed in the FDA-approved package insert
(Mechanism of Action) and activation of at least one known downstream substrate of
that protein drug target, both of which were required to be in the top quartile of the
population data.
1. Espina V, Heiby M, Pierobon M, Liotta LA. Lasers capture microdissection
technology. Expert Rev Mol Diagn. 2007 Sep;7(5):647-57. Review.
2. Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH,
Petricoin EF 3rd, Liotta LA. Laser-capture microdissection. Nat Protoc. 2006;
1(2):586-603.
3. VanMeter A, Signore M, Pierobon M, Espina V, Liotta LA, Petricoin EF 3rd.
Reverse-phase protein microarrays: application to biomarker discovery and
translational medicine. Expert Rev Mol Diagn. 2007 Sep;7(5):625-33. Review.
4. Pierobon M, Vanmeter AJ, Moroni N, Galdi F, Petricoin EF 3rd. Reverse-phase
protein microarrays. Methods Mol Biol. 2012; 823:215-35. doi: 10.1007/978-160327-216-2-14.
Supplemental Table II. DNA Microarray Genes
ABCC1
GSTP1
PTGS2
ABCG2
HCK
RAF1
ADA
HDAC1
RARA
AR
HIF1A
RRM1
ASNS
HSP90AA1
RRM2
BCL2
IGFBP3
RRM2B
BIRC5
IGFBP4
RXRB
BRCA1
IGFBP5
RXRG
BRCA2
IL2RA
SIK2
CD33
KDR
SPARC
CD52
KIT
SRC
CDA
LCK
SSTR1
CES2
LYN
SSTR2
DCK
MET
SSTR3
DHFR
MGMT
SSTR4
DNMT1
MLH1
SSTR5
DNMT3A
MS4A1
TK1
DNMT3B
MSH2
TNF
ECGF1
NFKB1
TOP1
EGFR
NFKB2
TOP2A
EPHA2
NFKBIA
TOP2B
ERBB2
OGFR
TXNRD1
ERCC1
PARP1
TYMS
ERCC3
PDGFC
VDR
ESR1
PDGFRA
VEGFA
FLT1
PDGFRB
VHL
FOLR2
PGP
YES1
FYN
PGR
ZAP70
GART
POLA1
GNRH1
PTEN
Supplemental Material Table III. IHC /FISH Protein Expression
IHC Targets
Androgen Receptor
BCRP
CAV-1
CK 5/6
CK14
CK17
c-KIT
Cyclin D1
ECAD
EGFR
ER
ERCC1
Her2/Neu
Ki67
MGMT
MRP1
p53
PDGFR
PGP
PR
PTEN
RRM1
SPARC
SPARC Monoclonal
SPARC Polyclonal
TLE3
TOP2A
TOPO1
TS
FISH Targets
Cmyc
EGFR
Her2/neu
TOP2A
Supplemental Material Table IV. Antibody and phosphorylation site used for the RPPA analysis
Vendor
Catalog number
Antibody
AKT (S473)
Cell Signaling
9271
c-Abl (T735)
Cell Signaling
2864
c-Kit (Y719)
Cell Signaling
3391
EGFR (Y1173)
Invitrogen
44-794
ErbB2/HER2 (Y1248)
Imgenex
90189-1
ERK (T202/Y204)
Cell Signaling
9101
mTOR (S2481)
Cell Signaling
2974
P70S6 Kinase (T389)
Cell Signaling
9205
PDGF Receptor beta (Y751)
Cell Signaling
3161
PTEN
Cell Signaling
9552
VEGFR 2 (Y996)
Cell Signaling
2474
Supplemental Material Table V. Selected Targets and Treatments
Physician
Subject
Recommended
Profiling Recommended
ID
Treatment if Not on
Treatment
Study
gemcitabine +
A - irinotecan + lapatinib
100
trastuzumab
B - lapatinib + capecitabine
A - irinotecan
101
ixabepilone
B - nab-paclitaxel
A - irinotecan + lapatinib
102
ixabepilone
B - lapatinib
A - AI + erlotinib
B - AI + sorafenib
C -(1) irinotecan + erlotinib
(2) irinotecan + cetuximab
103
VP-16 (etoposide)
D - AI alone
A - capecitabine
B - temozolomide
104
ixabepilone
C - nab-paclitaxel
A - pemetrexed
105
navelbine
B - irinotecan
A - FOLFIRI
107
navelbine
B - XELIRI
A - liposomal doxorubicin
108
navelbine
B - temozolamide
A - trastuzumab + irinotecan
109
ixabeilone
B - trastuzumab + capecitabine
A – liposomal doxorubicin + nabpaclitaxel
Phase I clinical trial
with calcitriol
110
LHRH antagonist
B - FOLFIRI with calcitriol
A - liposomal doxorubicin
111
gemcitabine
B - FOLFIRI
112
gemcitabine
A - FOLFIRI
A - irinotecan + AI
B - irinotecan single agent
113
navelbine
C - liposomal doxorubicin
A - AI
B - fulvestrant
C - liposomal doxorubicin
114
eribulin or Phase I Study D - irinotecan
ixabepilone or P13K
A - FOLFIRI
115
Inhibitor
B - fulvestrant or AI
A - nab-paclitaxel
B - irinotecan +/- cetuximab
116
navelbine
C - erlotinib
A - irinotecan
B - liposomal doxorubicin or
117
navelbine
doxorubicin
Delivered Treatment
lapatinib + capecitabine
irinotecan
lapatinib + trastuzumab*
erlotinib + letrozole
capecitabine
irinotecan
FOLFIRI
liposomal doxorubicin
irinotecan + trastuzumab
liposomal doxorubicin
→doxorubicin → FOLFIRI
liposomal doxorubicin
FOLFIRI
exemestane + irinotecan
letrozole
letrozole → irinotecan+
fluorouracil
nab-paclitaxel
irinotecan
119
carboplatin +
gemcitabine
trastuzumab + Lapatinib
or
gemcitabine
120
eribulin mesylate
121
capecitabine
124
paclitaxel
125
navelbine
126
capecitabine
118
128
liposomal doxorubicin
A – capecitabine + irinotecan
B - gemcitabine + carboplatin
C - erlotinib
FOLFIRI
A - gemcitabine + trastuzumab
B - irinotecan + trastuzumab
A - flutamide
B - erlotinib
C - megestrol acetate
A - erlotinib
B - nab-paclitaxel
C - FOLFIRI
D - fulvestrant
A - erlotinib
B - nab-paclitaxel
A - lapatinib + modified FOLFIRI
B - lapatinib + nab-paclitaxel
A - XELIRI
B - irinotecan
C - capecitabine
A - capecitabine
B - irinotecan
*Treating physician added Herceptin
Abbreviations: AI, Aromatase
capecitabine+irinotecan
Inhibitor;
FOLFIRI,
gemcitabine + trastuzumab
flutamide
erlotinib
erlotinib + letrozole
lapatinib + paclitaxel
irinotecan
XELIRI
Irinotecan+5FU+Folinic
Acid;
XELIRI,
Supplemental Figure I. Reverse Phase Protein Microarray
Work-flow
Tumor cells were isolated using Laser
Capture Microdissection technology.
Samples, calibrators and controls were
printed in triplicate onto nitrocellulose slides.
Calibrator
Controls
Patients (up to 900)
Arrays were
tested with 11
antibodies
against FDAapproved drug
targets and
downstream
substrates.
Supplemental Figure II .Kaplan-Meier Plot - Overall Survival from Start of Study
Treatment
Supplemental Figure III. Kaplan-Meier Plot – Comparison of Overall Survival from
Start of Study Treatment, GMI Evaluable Patients