Download Molecular Profiling of Patients with Colorectal Cancer and Matched

Published OnlineFirst June 21, 2012; DOI: 10.1158/1535-7163.MCT-12-0290
Molecular
Cancer
Therapeutics
Molecular Medicine in Practice
Molecular Profiling of Patients with Colorectal Cancer and
Matched Targeted Therapy in Phase I Clinical Trials
Rodrigo Dienstmann1, Danila Serpico1, Jordi Rodon1, Cristina Saura1, Teresa Macarulla1, Elena Elez1,
nchez-Olle
2, Claudia Aura3,
Maria Alsina1, Jaume Capdevila1, Jose Perez-Garcia1, Gessamí Sa
3
3
3
ndez-Losa , Ana Vivancos4, and Josep Tabernero1
Ludmila Prudkin , Stefania Landolfi , Javier Herna
Abstract
Clinical experience increasingly suggests that molecular prescreening and biomarker enrichment strategies
in phase I trials with targeted therapies will improve the outcomes of patients with cancer. In keeping with the
exigencies of a personalized oncology program, tumors from patients with advanced chemorefractory
colorectal cancer were analyzed for specific aberrations (KRAS/BRAF/PIK3CA mutations, PTEN and pMET
expression). Patients were subsequently offered phase I trials with matched targeted agents (MTA) directed at
the identified anomalies. During 2010 and 2011, tumor molecular analysis was conducted in 254 patients: KRAS
mutations (80 of 254, 31.5%), BRAF mutations (24 of 196, 12.2%), PIK3CA mutations (15 of 114, 13.2%), KRAS and
PIK3CA mutations (9 of 114, 7.9%), low PTEN expression (97 of 183, 53.0%), and high pMET expression (38 of
64, 59.4%). In total, 68 patients received 82 different MTAs: phosphoinositide 3-kinase (PI3K) pathway inhibitor
(if PIK3CA mutation, n ¼ 10; or low PTEN, n ¼ 32), PI3K pathway inhibitor plus MEK inhibitor (if KRAS
mutation, n ¼ 10; or BRAF mutation, n ¼ 1), second-generation anti-EGF receptor monoclonal antibodies (if
wild-type KRAS, n ¼ 11), anti-hepatocyte growth factor monoclonal antibody (if high pMET, n ¼ 10), mTOR
inhibitor plus anti-insulin-like growth factor-1 receptor monoclonal antibody (if low PTEN, n ¼ 5), and BRAF
inhibitor (if BRAF mutation, n ¼ 3). Median time-to-treatment failure on MTA was 7.9 versus 16.3 weeks for
their prior systemic antitumor therapy (P < 0.001). Partial response was seen in 1 patient [1.2%, PI3K inhibitor
with PIK3CA mutation] and stable disease >16 weeks in 10 cases (12.2%). These results suggest that matching
chemorefractory patients with colorectal cancer with targeted agents in phase I trials based on the current
molecular profile does not confer a significant clinical benefit. Mol Cancer Ther; 11(9); 2062–71. 2012 AACR.
Introduction
Colorectal cancer is the third most common cancer
worldwide, accounting for approximately 10% of total
cancer incidence and mortality (1). In the last decade, a
multidisciplinary approach to the treatment of metastatic
colorectal cancer along with the introduction of new
cytotoxic drugs and the addition of targeted therapies
directed against angiogenesis (bevacizumab, aflibercept,
and regorafenib) and the EGF receptor (EGFR) pathway
(cetuximab and panitumumab) have improved survival
endpoints considerably. Nevertheless, the prognosis in
metastatic colorectal cancer remains poor, and other than
the negative predictive effect of KRAS mutations for
Authors' Affiliations: 1Molecular Therapeutics Research Unit, Medical
Oncology Department, 2Database Management Office, 3Molecular Pathology Lab, and 4Cancer Genomics Lab, Vall d'Hebron Institute of Oncology,
noma de Barcelona,
Vall d'Hebron University Hospital, Universitat Auto
Barcelona, Spain
Corresponding Author: Josep Tabernero, Vall d'Hebron University Hos noma de Barcelona, P. Vall d'Hebron 119-129,
pital, Universitat Auto
Barcelona 08035, Spain. Phone: 3493-489-4301; Fax: 3493-274-6059;
E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-12-0290
2012 American Association for Cancer Research.
2062
response with EGFR-targeted agents, efficacious personalized treatment of metastatic colorectal cancer remains
an unmet need in clinical oncology.
Aberrant activation of the RAS/RAF/MEK and PI3K/
mTOR/AKT pathways, either by upstream EGFR stimulation or downstream mutations or deregulations, occurs
in virtually all colorectal tumors. Mutations in KRAS,
frequently found in codons 12 and 13, occur in as many
as 40% of patients with colorectal cancers and are strongly
associated with resistance to anti-EGFR monoclonal antibodies (mAb) as a result of constitutive pathway activation. Because cetuximab and panitumumab are not effective in all patients with wild-type (wt) KRAS metastatic
colorectal cancers, molecular markers downstream of
EGFR, namely, mutations in NRAS, BRAF, PIK3CA and
PTEN loss-of-function, may also predict lack of response
to anti-EGFR therapies in colorectal cancers (2). BRAF
mutations in colorectal cancers are mainly located in a
hotspot region in exon 15 (V600E) and occur in 5% to 15%
of the tumors. NRAS mutations are frequently found in
codon 61 and have been identified in as many as 5% of
colorectal cancer samples. KRAS, NRAS, and BRAF mutations are, however, mutually exclusive. Mutations in
PIK3CA, which encodes the catalytic p110a kinase subunit, have been reported in 15% of patients with colorectal
Mol Cancer Ther; 11(9) September 2012
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Colorectal Cancer Patients in Phase I Trials of Targeted Agents
cancers. As many as 70% involve the exon 9 helical
domain (such as E542K or E545K) or the exon 20 kinase
domain (such as H1047R; ref. 3). Loss-of-function mutations, deletions, or epigenetic silencing by promoter methylation of PTEN, a negative regulator of phosphoinositide
3-kinase (PI3K), have also been linked to resistance to antiEGFR mAbs in colorectal cancers (4–6). Loss of PTEN
expression, assessed by immunohistochemistry, is found
in as many as 40% of colorectal cancer tumors (4–6).
Importantly, the RAS/RAF/MEK and PI3K/AKT/
mTOR pathways also interact extensively: (i) each shows
activation of upstream receptor tyrosine kinases; (ii)
mutations in KRAS or BRAF and PIK3CA coexist in a
significant percentage of colorectal tumors; and (iii) complex networks allow compensatory parallel signaling
when one or the other pathway is inhibited (7).
Complex cross-talk involving EGFR and other pathways has also been identified in the past few years.
Mesenchymal–epithelial transition (MET) activation, for
example, occurs in 50% to 80% of colorectal tumors with
both mutant and wt KRAS (8). Preclinical cellular models
show cooperation between MET and KRAS to enhance
colorectal cancer tumorigenicity (9, 10). In addition, MET
hyperactivation, secondary to increased expression of
the MET receptor or its ligand [hepatocyte growth factor
(HGF)], may have a role in resistance to anti-EGFR therapies (11). The same is true for the insulin-like growth
factor-1 receptor (IGF1R) as well as HER2 and HER3
upregulation (12–14).
Multiple novel targeted agents have entered clinical
development. These include innovative mAbs targeting
EGFR, EGFR/HER3, MET (or HGF), and IGF1R, as well as
kinase inhibitors downstream of these pathways, mainly
BRAF, MEK, PI3K, mTORC1/2, and AKT. These agents
are being investigated in phase I trials using biomarker
enrichment strategies. Their study designs are based on
preclinical and early clinical response data showing the
potential predictive value of specific molecular aberrations. Examples include PIK3CA-activating mutations
and loss of PTEN expression and benefit with PI3K pathway inhibitors (15, 16), synergistic antitumor activity with
combination blockade of mTOR and IGF1R signaling (17,
18), RAS/RAF mutations and response to PI3K pathway
inhibitors plus MEK inhibitors (19–21), BRAF mutations
linked to antitumor activity of selective BRAF inhibitors
(22, 23), and MET hyperactivation and benefit with MET/
HGF targeting agents (24, 25).
Molecular biomarker–based patient selection in early
clinical trials has many putative advantages over an
unselected population-based approach. These include
(i) early clinical qualification of potential predictive biomarkers for molecularly targeted agents; (ii) further delineating underlying cancer biology; (iii) providing clinically
relevant therapeutic opportunities for patients with
advanced-stage cancer; and (iv) impacting novel drug
development by assisting in go versus no go decisions
and changing drug approval registration strategies for
promising agents. A caveat is, however, that finding a
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potential target in a patient’s tumor does necessarily
correlate with response to a therapeutic agent directed
toward the target. Early clinical validation is, therefore, a
key component of the drug development process (26).
Recent studies have examined the feasibility of using a
real-time molecular profile of patient tumors and matching
the identified aberration(s) with treatments targeted to the
specific aberration(s). Von Hoff and colleagues and Tsimberidou and colleagues showed that patients who received
MTAs had better response rates and improved time to
treatment failure (TTF; ref. 27, 28). As part of our personalized oncology program, tumors from patients with
advanced chemorefractory metastatic colorectal cancer
were analyzed for specific molecular aberrations [KRAS/
BRAF/PIK3CA mutations, PTEN, and phosphorylated
MET (pMET) expression] in the Vall d’Hebron Molecular
Pathology and Cancer Genomics Labs (Barcelona, Spain).
Patients with advanced cancer were then offered enrollment
on a molecularly appropriate phase I trial with an MTA
based on the hypothesis that biomarker enrichment strategies might improve their outcomes. We retrospectively
evaluated the benefit of MTA only in patients with metastatic colorectal cancers, unlike studies enrolling patients
with any advanced tumor type (27, 28). Our primary objective was to compare time TTF using a therapy selected by
the molecular profile of a patient’s tumor with the TTF for
the most recent unmatched therapy on which the patient
had experienced progression. We also describe the clinical
benefit rate with an MTA in patients with metastatic colorectal cancers treated in phase I clinical trials.
Patients and Methods
Patients and matched targeted therapy
All patients with pathologically confirmed metastatic
colorectal cancers refractory to standard therapy referred
to phase I clinical trials at the Molecular Therapeutic
Research Unit of Vall d’ Hebron Institute of Oncology
during 2010 and 2011 had archived formalin-fixed, paraffin-embedded (FFPE) tumor samples analyzed for specific
molecular aberrations. Informed consent was obtained at
baseline. In total, 254 patients/tumor samples were analyzed, and 68 patients were enrolled on specific first-inhuman phase I trials based on the results of tumor genetic
alterations/protein expression levels as well as logistic
factors, including study availability and eligibility criteria.
If more than one molecular deregulation was identified or
2 different matched targeted therapies were available for
one specific aberration, patients could receive more than
one MTA. These included second-generation anti-EGFR
mAbs [if (wt) KRAS], PI3K pathway inhibitors (if PIK3CA
mutation or low PTEN expression), mTORC1 inhibitor plus
anti-IGF1R mAb (if low PTEN expression), PI3K pathway
inhibitors plus MEK inhibitors (if KRAS or BRAF mutation),
BRAF inhibitor (if BRAF mutation) and anti-HGF mAb
(if high pMET expression). All phase I studies were conducted in accordance with the guidelines of the Vall d’
Hebron Institute of Oncology Institutional Review Board.
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Tissue samples and molecular analyses
PTEN and pMET expression levels were assessed by
immunohistochemistry using a histopathologic scoring
(H-score) system, which is based on a sum of the proportion of stained cells multiplied by the staining reactivity
[H-score ¼ (0 % staining cells negative) þ (1 % staining
cells weakly positive) þ (2 % staining cells moderately
positive) þ (3 % staining cells strongly positive); ref. 29].
An anti-PTEN antibody (Cell Signaling Technologies,
clone 138G6, cat# 9559) was used in a 1:100 dilution and
incubated at room temperature for 60 minutes. We considered low PTEN expression when there was an absence
of immunostaining (PTEN-null) or PTEN H-score 50
(range, 0–300) compared with the internal control. The
subgroup with PTEN-null expression (H-score ¼ 0) is
described separately. Paraffin-embedded cell pellets were
used as controls in each run (MDA231 and PC3 as positive
and negative controls, respectively) and also considered
as internal positive controls were nerve sheaths and vessels. An antibody against pMET (pMET Tyr1349; Epitomics, EP2367Y, cat# 2319-1) was used in a 1:100 dilution
and incubated at room temperature for 60 minutes. Positive expression was defined as H-score >30 (range, 0–300;
ref. 30).
For mutation detection, KRAS and PIK3CA were initially assessed by real-time PCR using a DxS Scorpions
technology (TheraScreen KRAS Mutation Kit and DxS
PI3K Mutation Test Kit; DxS). In this technique, allele- or
mutation-specific amplification is achieved by ARMS
(amplification refractory mutation system) technology
and its detection is conducted using Scorpions (bifunctional molecules containing a PCR primer covalently
linked to a probe; ref. 31). The following mutations were
evaluated: KRAS G12A, G12D, G12R, G12C, G12S, G12V,
G13D and PIK3CA H1047R, E542K, E545D, E545K. Standard Sanger sequencing of exons 9 or 20 of the gene
confirmed each PIK3CA mutation. Sanger sequencing was
also used to assess BRAF mutations in exon 15. Later on,
tumor genetic analyses by PCR-based and deep sequencing techniques were substituted by the OncoCarta Panel v
1.0, a multigene panel with 19 key oncogenes and 238
mutations, via the MassARRAY System (Sequenom, Inc.).
The MassARRAY System involves the following steps:
multiplex PCR of gene exons of interest, primer extension
reactions using IPLEX chemistry, and analysis of primer
extension products by matrix-assisted laser desorption/
ionization—time-of-flight (MALDI-TOF) mass spectrometry. A Vall d’Hebron pathologist evaluated total tumor
area in the tissue block, and a minimum 30% tumor
content was required for further processing. DNA was
extracted from five 10-mm slices of FFPE tumor samples
using the RecoverAll Total Nucleic Acid Isolation Kit
(Ambion). DNA was quantified with nanodrop (ND1000), 260:280 and 260:230 ratios were used for quality
control (QC) of sample purity. Additional QC was
obtained in low concentration samples by quantitative
PCR of 100-bp long amplicons in AKT1 (exon 1), KRAS
(exon 1), and PIK3CA (exon 20), dsDNA quantification
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with Quant-iT DNA BR (Invitrogen) and lab-on-a-chip
using High Sensitivity DNA Chips (Agilent).
The full mutation panel is not available for all patients
included in this study. This is because the prescreening
program was initially constructed on the basis of the
clinical trials available at the time of screening and their
inclusion criteria (i.e., there were no trials focused on
patients with PIK3CA- or BRAF-mutated tumors actively recruiting in early 2010). In addition, we gradually
added new technologies (OncoCarta Panel v 1.0 via the
MassARRAY System) to simultaneously assess multiple
mutations.
Treatment evaluation
We reviewed the electronic medical records of patients
enrolled in clinical trials according to molecular prescreening results, sex, age, Eastern Cooperative Oncology
Group performance status at the beginning of treatment
and detailed information on prior treatments. The formulation of the patient database and collection of data for the
purpose of this study were conducted retrospectively.
Previous therapies were classified into different
groups according to National Comprehensive Cancer
Network (NCCN) guidelines: (i) standard regimens,
including capecitabine/5-fluorouracil, oxaliplatin, irinotecan, bevacizumab, and cetuximab (monotherapies or
combinations) or (ii) nonstandard, if not an approved
chemotherapy regimen (such as single-agent gemcitabine
or pemetrexed used off-label, or mitomycin or capecitabine after progression to regimens containing infusional
5-fluorouracil) or an unmatched agent in another earlyphase clinical trial. Treatment on phase I studies continued until disease progression or death or unacceptable
toxicity occurred and was carried out according to the
specific requirements of each protocol. Efficacy was
assessed routinely by computed tomographic scans
and/or MRI at baseline before treatment initiation and
then every 2 cycles (6–8 weeks) as per each phase I study
protocol. In accordance with Response Evaluation Criteria in Solid Tumors (RECIST) v. 1.0 or 1.1 (depending on
study protocol), tumor responses were classified as complete response, partial response, stable disease, or progressive disease. TTF was defined as the time interval
from the start of a therapy to its discontinuation for any
reason, including disease progression, treatment toxicity,
or death, whichever occurred first. Patients with TTF on
MTA longer than 16 weeks (>2 radiologic RECIST assessments) were described separately. In addition, if the TTF
on MTA/TTF on prior therapy ratio was 1.3, then the
matched targeted therapy selected was defined as having
benefit for the patient (27).
Statistical analysis
Survival analyses were conducted using the Kaplan–
Meier method and compared with a log-rank test (TTF
with MTA vs. previous unmatched therapy). Subgroup
analysis was conducted considering standard versus nonstandard previous therapy. Exact 2-sided 95% confidence
Molecular Cancer Therapeutics
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Colorectal Cancer Patients in Phase I Trials of Targeted Agents
Table 1. Molecular profile of patients with colorectal cancer referred to phase I clinical trials
No. (%)
Tested
175/79 (68.9/31.1)
173/81 (68.1/31.9)
254
254
0
0
Analysis of single aberrations
KRAS mutation
Unknowna
G12D
G12V
G13D
G12C
G13V
G12F/G12S/K61H/K61L
BRAF mutation
V600E
K601E
PIK3CA mutation
E542K/E545K
Q546K
E110K/H1047L/R88Q
PTEN-low
PTEN-null
pMET high
80 (31.5)
17
20
15
9
6
2
1
24 (12.2)
23
1
15 (13.2)
5
2
1
97 (53)
45 (24.6)
38 (59.4)
254
0
196
58
114
140
183
71
64
190
Analysis of double aberrations
KRAS wild-type and PIK3CA mutation
KRAS mutation and PIK3CA mutation
KRAS mutation and PTEN-low
KRAS mutation and PTEN-null
BRAF mutation and PIK3CA mutation
BRAF mutation and PTEN-low
BRAF mutation and PTEN-null
PIK3CA mutation and PTEN-low
PIK3CA mutation and PTEN-null
pMET high and KRAS mutation
pMET high and BRAF mutation
pMET high and PIK3CA mutation
pMET high and PTEN low
6 (5.3)
9 (7.9)
38 (20.8)
14 (7.6)
0 (0)
4 (3.1)
2 (1.5)
4 (3.6)
1 (0.9)
16 (25)
2 (6.3)
2 (5.6)
4 (6.6)
114
114
183
183
92
130
130
110
110
64
32
36
61
140
140
71
71
172
124
124
144
144
190
222
218
193
Analysis of 3 mutations
No mutation (KRAS, BRAF, and PIK3CA wild-type)
Mutation in 1 gene (KRAS, BRAF, or PIK3CA)
Mutation in 2 genes (KRAS or BRAF and PIK3CA)
36 (39.1)
47 (51.1)
9 (9.8)
92
92
92
172
172
172
Biopsy site (primary/metastasis)
Mutation detection technique (DxS Scorpions
or sequencing/MassARRAY system)
a
Not tested
Total
254
254
KRAS mutation detected outside Vall d'Hebron Labs and tumor tissue left was not sufficient for confirmation on site.
intervals (CI) were calculated. All tests were 2-sided, and
P < 0.05 was considered statistically significant. All
statistical analyses were conducted using SPSS v.15.0
software (SPSS).
Results
Molecular prescreening
During 2010 and 2011, we analyzed tumor samples
from 254 patients with metastatic colorectal cancers
referred to phase I trials at our institution. Archived
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biopsy specimens were the only source of tissue used for
analysis. The median age of patients was 61.9 years (range,
32.8–85.1) and 65% were men. Detailed molecular profiles
of patients’ tumors are presented in Table 1. KRAS, BRAF,
and PIK3CA mutation rates are similar to previous reports
(3). PTEN expression was evaluated as being low in 53%
(97 of 183) of tumor samples. Of these, 46.4% (45 of 97) had
PTEN-null expression. The coexistence of KRAS mutations and PIK3CA mutations (7.9%) or KRAS mutations
and PTEN-low/null expression (20.8%/7.6%) confirms
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254 patients/ tumor samples analyzed
68 patients selected for phase I trials
based on molecular profile
82 matched targeted therapies
Figure 1. Study diagram and
illustration of the primary analysis
(TTF comparisons). In example 1,
patient received an MTA after
progression to one nonstandard
chemotherapy (CT) regimen. In
example 2, patient received 2
consecutive MTAs after
progression to standard CT
regimens. MTA, matched targeted
agent; TTF, time to treatment
failure.
TTF comparisons
Example 1:
Standard CT
Standard CT
Nonstandard CT
MTA
Example 2:
Standard CT
Standard CT
MTA
the frequent parallel activation of RAS/RAF/MEK and
PI3K/AKT/mTOR pathways in colorectal cancers. Compared with (wt) KRAS patients, those harboring a KRAS
mutation did not have a significantly higher prevalence of
PIK3CA mutations (7.9% vs. 5.3%, P ¼ 0.5). In addition,
approximately 60% of the tumors analyzed had high
pMET expression according to the cutoff value for inclusion on the anti-HGF trial. This aberration was not mutually exclusive with KRAS, BRAF, or PIK3CA mutations.
Matched targeted therapy
Sixty-eight patients were enrolled in 15 different
phase I clinical trials. There were a total of 82 matched
targeted therapies, with 8 patients participating in 2
clinical trials and 3 who were enrolled in 3 clinical
trials, as shown in Fig. 1. Table 2 presents patients’ demographics and clinical characteristics. Phase I trials included PI3K pathway inhibitors (National Clinical Trial
numbers: NCT01115751, NCT01219699, NCT01090960,
NCT00940498, NCT01058707, NCT00620594, NCT01068483,
NCT01449370), PI3K pathway inhibitors plus MEK
inhibitors (NCT01363232, NCT01155453), second-generation anti-EGFR mAbs (NCT01207323, NCT01117428),
anti-HGF mAb (NCT00969410), mTORC1 inhibitor plus
anti-IGF1R mAb (NCT00730379), and BRAF inhibitor
(NCT01143753). Detailed descriptions of matched targeted therapies, dose levels, and patient outcomes are
presented in Table 3.
Time to treatment failure on MTA
Median TTF on MTA was 7.9 weeks (95% CI, 7.6–8.1)
compared with 16.3 weeks (95% CI, 13.9–18.7) with the
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Mol Cancer Ther; 11(9) September 2012
MTA
prior systemic antitumor therapy (P < 0.001). As shown
in Fig. 2, analysis according to type of previous therapy
showed that if it was considered nonstandard (n ¼ 39), TTF
with MTA was 7.0 weeks (95% CI, 5.7–8.3) and TTF with
prior therapy was 8.7 weeks (95% CI, 7.3–10.1). Nonstandard therapy was another phase I trial with unmatched
agent in 21 patients and off-label chemotherapy regimens
Table 2. Patient demographics and clinical
characteristics
No. (N ¼ 68), %
Characteristic
Sex
Male
Female
Age, y
Median
Range
Number of prior treatment
regimens (metastatic)
Median
1–3
4–7
ECOG performance status
0
1
Tumor type
Colon
Rectum
43 (63.2)
25 (36.8)
62.8
37.2–82.6
3
52 (76.5)
16 (23.5)
23 (33.8)
45 (66.2)
41 (60.3)
27 (39.7)
Abbreviation: ECOG, Eastern Cooperative Oncology Group.
Molecular Cancer Therapeutics
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BRAF mutation
BRAF inhibitor
(n ¼ 3)
Abbreviations: PR, partial response; SD, stable disease.
PTEN low
mTORC1 inhibitor
plus anti-IGF1R
mAb (n ¼ 5)
3
2
PTEN-low and KRAS
mutation
PTEN-low and KRAS
wild-type
3
1
pMET high and BRAF
mutation
BRAF mutation
4
pMET high and KRAS
wild-type
pMET high
Anti-HGF mAb
(n ¼ 10)
1
5
5
5
KRAS wild-type
KRAS wild-type and
PTEN-low
KRAS wild-type þ
BRAF mutation
KRAS wild-type
Second-generation
anti-EGFR mAb
(n ¼ 11)
1
1
6
3
4
pMET high and KRAS
mutation
KRAS mutation
KRAS mutation and
PTEN-null
KRAS mutation and
PIK3CA mutation
BRAF mutation and
PTEN-null
KRAS or BRAF
mutation
PI3K pathway inhibitor
plus MEK inhibitor
(n ¼ 11)
10
PTEN-low and KRAS
mutation
PIK3CA mutation and
KRAS wild-type
PIK3CA mutation and
KRAS mutation
6
22
PTEN-low and KRAS
wild-type
PTEN low or
PIK3CA mutation
PI3K pathway inhibitor
(n ¼ 42)
No.
Molecular profile
Rationale
Targeted therapy
<1/3 MTD: 1
MTD: 2
MTD: 5
<1/3 MTD: 1
1/3 to <MTD: 4
MTD: 5
MTD: 11
1/3 to <MTD: 6
MTD: 5
<1/3 MTD: 5
1/3 to <MTD: 1;
MTD: 36
Dose level
PR: 0 SD >16 wks: 0
PR: 0 SD >16 wks: 1
(20%)
PR: 0 SD >16 wks: 1
(10%)
PR: 0 SD >16 wks: 3
(27.3%)
PR: 0 SD >16 wks: 0
PR: 1 (2.4%)
SD >16 wks: 5
(11.9%)
Response rate
Need for combination
therapy.
Wrong biomarker?
Target or pathway
redundancy?
Wrong biomarker?
Need for combination
therapy. Treatment at
biologically inactive
doses.
Presence of
downstream pathway
aberrations (PTENlow and BRAF
mutation). Acquired
resistance to antiEGFR mAbs
Treatment at
biologically inactive
doses.
Wrong biomarker?
Need for combination
therapy. Resistance
to monotherapy in the
presence of KRAS
mutations.
Reasons for lack of
response
Table 3. Matched targeted therapies in phase I clinical trials based on molecular profile, dose level, and patient outcome
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Colorectal Cancer Patients in Phase I Trials of Targeted Agents
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7.9 (95%CI, 7.6–8.1)
Overall
(n = 82)
16.3 (95%CI, 13.9–18.7)
7.0 (95%CI, 5.7–8.3)
Prior therapy nonstandard
regimen as per NCCN (n = 39)
Figure 2. Comparisons of TTF on
MTA (light gray bars) and prior
unmatched therapy (dark gray
bars). Subgroup analysis
according to type of prior therapy.
NCCN, National Comprehensive
Cancer Network; MTA, matched
targeted agent; TTF, time to
treatment failure.
MTA
8.7 (95%CI, 7.1–10.1)
Prior unmatched therapy
8.1 (95%CI, 7.2–9.1)
Prior therapy standard
regimen as per NCCN (n = 43)
21.9 (95%CI, 15.0–28.7)
0
5
10
15
20
25
TTF (wk)
in 18 patients. On the other hand, if previous therapy was
an approved standard regimen (n ¼ 43), TTF with MTA
was 8.1weeks (95% CI, 7.2–9.1) and TTF with prior therapy
was 21.9 weeks (95% CI, 15.0–28.7). Overall, 64 patients
(78%) received maximum tolerated doses (MTD) or recommended phase 2 doses (RP2D), 9 patients (11%) had doses
lower than one third of the MTD and 9 patients (11%)
received intermediate doses (that ranged from one third to
less than the MTD). In total, 10 matched therapies had to be
discontinued because of adverse events (12.2%).
Tumor response and clinical benefit with MTA
One patient whose tumor harbored PIK3CA R88Q and
KRAS G12C mutations (detected using the MassARRAY
system) had a confirmed partial response to a specific
PI3K-a inhibitor as monotherapy. This is the only patient
who has continued on treatment for longer than 24 weeks.
Stable disease longer than 16 weeks was seen in 10 cases
(12.2%): 2 patients on specific PI3K-a inhibitor (both with
PIK3CA mutations), 2 patients on PI3K/mTOR inhibitors,
and one patient on mTORC1/2 inhibitor [all with low
PTEN and a (wt) KRAS/BRAF/PIK3CA profile], one
patient on mTORC1 inhibitor plus anti-IGF1R mAb
(PTEN-null and KRAS mutant), 3 patients on secondgeneration anti-EGFR mAb [all with (wt) KRAS/BRAF
profiles and refractory to cetuximab or panitumumab],
and one patient on anti-HGF mAb [the one with the
highest pMET H-score (200) plus (wt) KRAS status; only
2 patients had an H-score < 100]. None of the 11 patients
who received a PI3K pathway inhibitor plus an MEK
inhibitor had disease stabilization for longer than 16
weeks (3 had a simultaneous KRAS mutation and
PTEN-null expression, one had a KRAS mutation plus a
PIK3CA mutation, and one had a BRAF mutation and
PTEN-null expression). A TTF ratio 1.3 was achieved in
13 of the 82 matched therapies (15.9%). There was no
difference in terms of TTF on MTA according to site of
mutation analysis [primary (n ¼ 53) vs. metastasis (n ¼
29); 7.9 weeks in both subgroups], number of previous
lines of chemotherapy [1–3 (n ¼ 52) vs. 4 (n ¼ 30); 7.9 and
8.0 weeks, respectively], number of molecular aberrations
2068
Mol Cancer Ther; 11(9) September 2012
[0 or 1 (n ¼ 44) vs. 2 (n ¼ 38); 8.4 and 6.1 weeks,
respectively], and dose level [MTD (n ¼ 64) vs. lower
than MTD (n ¼ 18); 7.9 weeks in both subgroups].
Discussion
Our results suggest that matching patients with chemorefractory metastatic colorectal cancers with targeted agents
in early clinical trials based on their current molecular
profiles does not result in longer TTF compared with their
prior systemic therapy. In addition, only a small percentage of study patients derived benefit in tumor response or
disease stabilization, comparable with statistics reported
in the literature of patients treated on phase I trials without
molecular selection (32). These findings are disparate from
those in a recently published series of patients with PIK3CA
mutant breast and gynecologic malignancies enrolled in
phase I trials with PI3K/mTOR inhibitors (33). However,
all responses in this study were observed in patients
treated with combination therapies, mainly mTORC1 inhibitors plus cytotoxic agents or bevacizumab.
Some caveats deserve consideration when looking at
the results of this study. First, it is a retrospective study in a
single institution. Despite the homogeneous patient population, the mechanisms of action of the targeted agents
are heterogeneous. Notably, the biomarkers selected for
enrichment of the phase I trials were primarily exploratory. Different techniques (DxS Scorpions, sequencing,
MassARRAY system) with different sensitivities were
used to identify the most frequent mutations in KRAS,
BRAF, and PIK3CA (of note, all PIK3CA mutations
detected by DxS Scorpions or the MassARRAY system
were confirmed by Sanger sequencing). In addition, analyzing archival tumor samples from the time of diagnosis
may not reflect the molecular aberrations that drive metastatic disease as a result of clonal evolution and selection
pressure from previous treatments (34). This is particularly true in regard to protein expression levels, such as
PTEN (5). Furthermore, a limitation inherent to all phase I
clinical trials is that patients may have been treated at
nonbiologically active doses, even though 78% received
MTD/RP2D doses and were treated in the expansion
Molecular Cancer Therapeutics
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Published OnlineFirst June 21, 2012; DOI: 10.1158/1535-7163.MCT-12-0290
Colorectal Cancer Patients in Phase I Trials of Targeted Agents
cohorts of each protocol. Finally, the timing of tumor
assessment during a phase I trial (every 6–8 weeks) is
usually shorter than restaging intervals for patients
receiving standard therapies (every 8–12 weeks), which
is another possible bias.
Many patients in our series received PI3K pathway
inhibitors based on PTEN expression levels, irrespective
of coexisting oncogenic aberrations (e.g., KRAS mutations). The predictive value of PTEN expression for determining response to PI3K pathways inhibitors in colorectal
cancer is, however, still pending. Moreover, the optimal
method for assessing PTEN loss of function has not yet
been established, that is, a cutoff point to define positive
and negative expression levels according to immunohistochemical assay results. Preclinical experiments showed
that PIK3CA mutations and PTEN aberrations render
tumors sensitive to PI3K pathway inhibitors, whereas
simultaneous mutations in the mitogen-activated protein
kinase (MAPK) pathway (KRAS, BRAF) can mediate
resistance to therapy (35, 36). Preliminary clinical data
suggested a negative predictive role for tumors harboring
KRAS mutations in response to mTORC1 inhibitors (37).
Nevertheless, efficacy of mTORC1/2 inhibitors was
observed in KRAS/BRAF-mutant colorectal cancer xenograft models, and PI3K/mTOR inhibitors induced tumor
regressions in genetically engineered (wt) PIK3CA mouse
models (38, 39). Interestingly, the only patient with colorectal cancer achieving a partial response in our series had
a molecular profile with a simultaneous PIK3CA and
KRAS mutation. PI3K pathway inhibitors have also
shown activity in KRAS-mutant breast and ovarian cancer
(33, 40). Despite this positive result, most of our patients
with PI3K pathway activation treated with single-agent
kinase inhibitors did not derive benefit from therapy,
especially those with low PTEN expression and coexisting
KRAS mutations.
It is possible that a treatment approach with a combination of agents could overcome resistance to PI3K pathway inhibitors in colorectal cancers. In preclinical models,
inhibiting the PI3K pathway showed synergy with cytotoxic agents, enhancing its efficacy by potentiating apoptosis (41). In addition, dual inhibition of the RAF/MEK
and PI3K/AKT/mTOR pathways seems to be required
for complete abrogation of downstream effectors in RASmutant tumors (19–21). PIK3CA and PTEN mutations
strongly reduce the sensitivity of RAS-mutant cells to
MEK inhibitors (42). Preclinical studies provide a clear
rationale for the coinhibition of these frequently co-activated pathways, and many early clinical trials testing this
hypothesis are underway. The clinical impact of such a
parallel pathway blockade remains unclear, but none of
the 11 patients treated with PI3K pathway inhibitors plus
MEK inhibitors in our series had benefit in terms of
response or disease stabilization. A recently published
large data set reported similarly disappointing results,
with no partial responses or disease stabilizations for
more than 16 weeks in 16 patients with metastatic colorectal cancers treated using the same approach (43).
www.aacrjournals.org
Remarkably, as in our series, some patients who received
PI3K pathway inhibitors plus MEK inhibitors had simultaneous RAS/RAF and PI3K/AKT/mTOR activation. It is
also important to highlight the fact that the use of full
doses of both targeted agents was rarely achieved because
of overlapping toxicities. Interestingly, in patient-derived
metastatic colorectal cancer xenografts exhibiting KRAS
and BRAF mutations, concomitant inhibition of PI3K/
mTOR and MEK pathway activity proved to be more
effective than single pathway inhibition, although the best
response was limited to tumor growth arrest rather than
overt tumor regression (21). This observation is potentially important for further drug development in RAS/RAFmutant colorectal cancers.
Regarding BRAF inhibitors in BRAF-mutant colorectal
cancers, none of the 4 patients treated in our series with
selective BRAF inhibitors or PI3K pathway inhibitors
plus MEK inhibitors had signs of antitumor activity. In
an expansion cohort of the phase I study with vemurafenib at the RP2D, only 1 of 19 patients had a confirmed
partial response (44). These findings support preclinical
data suggesting that BRAF-mutant colorectal cancers
might respond to combined targeted therapy with BRAF
and EGFR inhibitors (45). A combination strategy also
appears hopeful when agents targeting the MET/HGF
pathway are used. Preliminary clinical data suggest that
adding an MET tyrosine kinase inhibitor or an anti-HGF
mAb to an approved anti-EGFR mAb might increase the
response rate in (wt) KRAS metastatic colorectal cancer
(46, 47). Interestingly, the patients who had the greatest
benefit with a MTA in our data set had a molecular
profile with (wt) KRAS/BRAF and received second-generation anti-EGFR mAbs (3 of 11 patients refractory to
cetuximab or panitumumab had stable disease for more
than 16 weeks).
These retrospective data, while not definitive, are
certainly hypothesis-generating. With the switch from
histology- to molecular-driven therapy, phase I trials in
clinical oncology are providing an arena for early
hypothesis testing. The one-size-fits-all approach for
drug development of molecular targeted agents in solid
tumors has yielded disappointing results, and patients
with metastatic colorectal cancers enrolled in biomarker-driven early clinical trials do not appear to derive
significant benefit. A strong biologic hypothesis, solid
preclinical data on the mechanisms of primary/
acquired resistance and tumor models that better mimic
the clinical disease are needed to further drug development. Our data confirm that prescreening strategies
for early clinical trials and molecular profiling in
patients with metastatic colorectal cancers are feasible.
For personalized therapy to become a reality for these
patients, a more comprehensive analysis of molecular
aberrations appears to be critically important.
Disclosure of Potential Conflicts of Interest
J. Tabernero has had a role as consultant/advisor for Amgen, Genentech, Merck-Serono, Novartis, Roche, and Sanofi-Aventis. No potential
conflicts of interest were disclosed by the other authors.
Mol Cancer Ther; 11(9) September 2012
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2069
Published OnlineFirst June 21, 2012; DOI: 10.1158/1535-7163.MCT-12-0290
Dienstmann et al.
Authors' Contributions
Conception and design: R. Dienstmann, D. Serpico, J. Rodon, C. Saura, E.
Elez, J. Tabernero
Development of methodology: R. Dienstmann, D. Serpico,
J. Rodon, M. Alsina, G. Sanchez-Olle, J. Hernandez-Losa, A.
Vivancos, J. Tabernero
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): R. Dienstmann, J. Rodon, C. Saura, T. Macarulla,
E. Elez, M. Alsina, J. Capdevila, G. Sanchez-Olle, C. Aura, L. Prudkin,
S. Landolfi, J. Hernandez-Losa, A. Vivancos, J. Tabernero
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Dienstmann, J. Rodon, M. Alsina,
G. S
anchez-Olle, A. Vivancos, J. Tabernero
Writing, review, and/or revision of the manuscript: R. Dienstmann,
D. Serpico, J. Rodon, C. Saura, E. Elez, J. Capdevila, J. Perez-Garcia,
J. Hern
andez-Losa, A. Vivancos, J. Tabernero
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Dienstmann, D. Serpico, G.
Sanchez-Olle
Study supervision: R. Dienstmann, J. Rodon, M. Alsina, J. Tabernero
Acknowledgments
The authors thank Debora Moreno and Nuria Murtra from Database
Management Office, Jose Jimenez from Molecular Pathology Lab of Vall
d’Hebron Institute of Oncology, and Joann Aaron, MA, for editorial support.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received March 21, 2012; revised May 14, 2012; accepted May 23, 2012;
published OnlineFirst June 21, 2012.
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