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Genomic Medicine Frontier in Human Solid Tumors:

Prospects and Challenges


Rodrigo Dienstmann, Jordi Rodon, Jordi Barretina, and Josep Tabernero
Rodrigo Dienstmann, Jordi Rodon, and
Josep Tabernero, Vall dHebron Univer-
sity Hospital, Barcelona, Spain; and
Jordi Barretina, Novartis Institutes for
Biomedical Research, Cambridge, MA.
Published online ahead of print at
www.jco.org on April 15, 2013.
Authors disclosures of potential con-
icts of interest and author contribu-
tions are found at the end of this
article.
Corresponding author: Josep
Tabernero, PhD, Medical Oncology
Department, Vall dHebron University
Hospital, P. Vall dHebron 119-122,
Barcelona, Spain 08035; e-mail:
jtabernero@vhebron.net.
2013 by American Society of Clinical
Oncology
0732-183X/13/3115w-1874w/$20.00
DOI: 10.1200/JCO.2012.45.2268
A B S T R A C T
Recent discoveries of genomic alterations that underlie and promote the malignant phenotype,
together with an expanded repertoire of targeted agents, have provided many opportunities to
conduct hypothesis-driven clinical trials. The ability to prole each unique cancer for actionable
aberrations by using high-throughput technologies in a cost-effective way provides unprecedented
opportunities for using matched therapies in a selected patient population. The major challenges
are to integrate and make biologic sense of the substantial genomic data derived from multiple
platforms. We dene two different approaches for the analysis, interpretation, and clinical
applicability of genomic data: (1) the genomically stratied model originates from the one test-one
drug paradigm and is currently being expanded with an upfront multicategorical approach
following recent advances in multiplexed genotyping platforms; and (2) the comprehensive
assessment model is based on whole-genome, -exome, and -transcriptome data and allows
identication of novel drivers and subsequent therapies in the experimental setting. Tumor
heterogeneity and evolution of the diverse populations of cancer cells during cancer progression,
inuenced by the effects of systemic treatments, will need to be addressed in the new scenario
of early drug development. Logistical issues related to prescreening strategies and trial allocation,
in addition to concerns in the economic and ethical domains, must be taken into consideration.
Here we present a historical view of how increased understanding of cancer genomics has been
translated to the clinic and discuss the prospects and challenges for further implementation of a
personalized treatment strategy for human solid tumors.
J Clin Oncol 31:1874-1884. 2013 by American Society of Clinical Oncology
INTRODUCTION
A wide range of genomic alterations can lead to the
development of cancer. Recent advances in mas-
sively parallel sequencing technologies allow faster,
more sensitive, and more precise analyses of cancer
genomes.
1
This has revolutionized the study of tu-
mor biology and has begun to challenge the para-
digm that has guided optimal cancer treatment to
date.
2
Cancer genomics refers to the study of tumor
genomes at different levels, including changes in the
DNA sequence (copy number alterations, muta-
tions, and rearrangements), the epigenome (DNA
methylation and histone modication patterns),
and the transcriptome (gene or microRNA expres-
sionchanges), as depictedinFigure 1. The spectrum
of genomicdysregulations that promotetumorigen-
esis includes gene inactivation (eg, promoter silenc-
ing, gene deletion, mutations), changes in gene
expression(eg, copy number amplication, methyl-
ation), and mutations or gene rearrangements that
result in dominant protein activation.
3,4
Emerging
technologies provide the ability to identify aberra-
tions that play crucial roles in tumorigenesis and
progression, denedas driver lesions. Suchevents
canconfer critical tumor dependencies, withaddic-
tion of the cancer cell to a particular molecular
pathway, despite various other concomitant pas-
senger genomic alterations. Identication of bio-
logically important genes and pathways frequently
disrupted across different cancer types can generate
clinically relevant diagnostic, prognostic, and thera-
peutic information, and these aberrations are often
referred to as actionable. A subset of oncogene
mutations may also be druggable, ie, targets for
therapeutic development.
2,5
By compiling -omics data from several hun-
dred tumors per cancer type, ongoing global re-
search initiatives such as The Cancer Genome Atlas
(TCGA)
6,7
and the International Cancer Genome
Consortium (ICGC)
8
will uncover many types of
driver genomic alterations that might play an im-
portant role in tumorigenesis. In addition, the gen-
erationof genetic predictions of drugresponseinthe
preclinical setting with efforts such as the Genomics
of Drug Sensitivity in Cancer
9
and the Cancer Cell
Line Encyclopedia
10
will further help translate
genomic data into matched therapeutic regimens.
JOURNAL OF CLINICAL ONCOLOGY
R E V I E W A R T I C L E
VOLUME 31 NUMBER 15 MAY 20 2013
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
Over the last decade, many disease-specic alterations in cancer
genomics have been translated to the clinic. Given the unprecedented
opportunities for improvements in the outcome of patients with can-
cer, we discuss the prospects and challenges for further implementa-
tion of a personalized treatment strategy in human solid tumors.
UNDERSTANDING THE CANCER GENOME LANDSCAPE AND
TRANSLATING ADVANCES INTO THERAPEUTICS
Successful Application of Cancer Genomics
Recent examples of therapies that target driver dependencies
have proved that major clinical responses can be seen in genetically
denedtumor subtypes, evenwiththe genomic complexity or aggres-
siveness typical of most malignancies.
11
With the current treatment
armamentarium and the large number of experimental drugs under
development, virtually all kinases with aberrant activity can be effec-
tively inhibited.
12
This enables clinical researchers to investigate ther-
apies that target selected kinase aberrations for individual patients
with cancer in proof-of-concept studies. Pioneering work in the eld
of targeted interference of the oncogenes to which cancer cells are
addicted began with the discovery of ERBB2 (HER2) amplication in
breast cancer.
13
Proling for a genetic alterationeffectively targetedby
a monoclonal antibody (mAb) therapy, trastuzumab, resulted in im-
provedsurvival inanaggressive subtype of anepithelial cancer.
14
This
was followed by the development of the rst tyrosine kinase inhibitor
(TKI), imatinib, which targets the BCR-ABL fusion gene of patients
with chronic myeloid leukemia.
15
The dramatic responses seen in
hematologic malignancies and the discovery that imatinib inhibits
additional tyrosine kinases, including KIT, which is constitutively
activated by mutations in gastrointestinal stromal tumors, paved the
way for its application in solid tumors.
16
The imatinib story taught us
that targeted small-molecule drugs could be used to treat cancers
driven by specic molecular mechanisms when protein kinases and
downstream pathways are activated. It was then found that the fre-
quency and duration of response to the TKI imatinib markedly varies
withmutations indiverse domains of KIT
17
andthat tumors mayhave
several distinct clones bearing different KITmutations.
18
The impres-
sive responses with TKIs getinib and erlotinib in a subset of non
small-cell lung cancer (NSCLC), later dened as epidermal growth
factor receptor (EGFR)-mutated, have shown that even the most
frequent solid tumors could benet from a targeted approach, pro-
vided that the sensitive population of patients whose tumors bear the
specic driver genomic dysregulationcanbe identied.
19
Later on, the
characterization of actionable mutations, such as KRAS mutations
and the absence of response to anti-EGFR mAbs in colorectal
cancer,
20-22
allowed restriction of labeled indications for targeted
agents to avoid treating patients who do not benet. Another impor-
tant lesson was that targeted therapies can be active in the setting of
loss of tumor suppressor genes and not only by gain-of-function
mutations in tyrosine kinase genes. The efcacy of poly(ADP-ribose)
High-throughput genotyping platforms (multiplexed screens): detection of selected somatic mutations. Allows
identification of rare mutations (such as BRAF
V600E
in lung cancer) but are limited to the known variants that have
been chosen for analysis. Favor oncogenes over tumor suppressor genes, for which only frequently mutated sites
are evaluated. Not suited for the analysis of gene copy number variations and does not allow the identification of
gene rearrangements. Difficulties in detecting small insertions or deletions. Decreased sensitivity in tumor samples
with high stromal admixture.
Targeted massively parallel sequencing: detection of somatic mutations with increased sensitivity. Permits increased
sequencing coverage of predefined regions of interest, such as coding exons of known oncogenes and tumor
suppressor genes, in addition to pharmacogenomic polymorphisms. Also suited for detection of gene copy number
variations and predefined gene rearrangements. Identification of small insertions/deletions remains difficult with
current algorithms.
Genome
Epigenome
Transcriptome
Whole-exome sequencing: detection of point mutations, small insertions/deletions, pharmacogenomic
polymorphisms, and gene copy number variations with increased sensitivity. Not suited for discovery of
gene rearrangements.
Whole-genome sequencing: discovery of novel gene rearrangements, complex insertions/deletions, and microbial
infections as well as identification of copy number alterations. Accurate point mutation detection remains a limitation,
requiring significantly more coverage.
RNA sequencing: assessment of gene expression profiles, alternative splicing, chromosome rearrangements/
gene fusion transcripts as well as mutations in coding sequences. Accurate point mutation detection remains a
limitation because the wide dynamic range of gene expression levels makes it difficult to achieve the necessary
depth of coverage required to call genotypes with high confidence in low-abundance messenger RNAs.
Bisulfite sequencing and chromatin immunoprecipitation sequencing: provide high-resolution
maps of DNA methylation and histone modifications that have a role in the composition and regulation of the
cancer transcriptome.
Fig 1. Emerging genomic platforms.
Genomic Medicine in Solid Tumors: Prospects and Challenges
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
polymerase (PARP) inhibitors, suchas olaparib, inBRCA1/2-mutated
malignancies
23
clinically conrmed the concept of synthetic lethal-
ity, which states that if an alteration affecting one or the other gene
product individually (BRAC1/2 or PARP) is compatible withsurvival,
alterations in both genes simultaneously cause cell death.
24
The next
instructive advance was the discovery that driver mutations at various
points within cell signaling modules may be effectively targeted by
using novel potent inhibitors, suchas the mutatedcytoplasmic serine/
threonine kinase RAF in melanoma. The failure of the multitargeted
RAF inhibitor sorafenib in BRAF
V600E
-mutated melanomas was fol-
lowed by dramatic responses with potent BRAF inhibitors specically
selective for the BRAF
V600E
mutation, such as vemurafenib.
25
Finally,
recurrent druggable translocations were identied in epithelial can-
cers, such as the EML4 anaplastic lymphoma kinase (ALK) rearrange-
ment and c-ros oncogene 1 (ROS1) fusions in small subsets of
NSCLC.
26,27
The existence of ALK/ROS1 inhibitors in clinical devel-
opment, namely crizotinib, signicantly decreased the elapsed time
between target discovery and successful use of targeted therapies.
28,29
Whether common genomic events transversing different cancer
lineages will translate into response to similar therapies remains to be
fully tested. Some examples showing that tumors should likely be
treated with targeted agents on the basis of their specic molecular
alterations rather than on an organ of origin include the clinical
activity of olaparib in breast, ovarian, and prostate cancers harboring
BRCA1/2 mutations,
24
vemurafenib in BRAF
V600E
-mutated mela-
noma and thyroid cancer,
25
crizotinib in lung cancer and anaplastic
large-cell lymphomas harboring ALK translocations,
30
and vismo-
degib (hedgehog pathway inhibitor) in medulloblastoma and basal
cell carcinomas with loss of function mutations in PTCH1.
31,32
Con-
versely, a specic genetic abnormality may not confer the same level of
sensitivity to an agent across all cancers, as exemplied by trastu-
zumab, which has been shown to benet patients with HER2-
amplied breast and gastric cancer
14,33
but not those with ovarian
or endometrial cancer.
34,35
The same appears to be true in the case
of BRAF
V600E
-mutated colorectal cancer and the poor response
to vemurafenib.
36
Together withadvances ingenome sequencing technologies, im-
portant discoveries in the eld of epigenomics were made in the last
decade. The most paradigmatic example is hypermethylation of CpG
islands located in the promoter region of O
6
-methylguanineDNA
methyltransferase as a predictor of good therapeutic response to alky-
lating agents in gliomas.
37
In addition, by using microarray technolo-
gies for transcriptional proling, expression signatures based on
multigene sets were extensively studied in solid tumors. By means of
sophisticated bioinformatics tools, gene proles of coordinately up-
regulatedanddownregulatedtranscripts that correlate withparticular
tumor subtypes, stage, andprognosis weredeveloped.
38
Awell-known
example is the molecular classicationof breast cancer intove major
categories with different clinical measures, including treatment re-
sponse,
39
which was recently expanded to include integrated analysis
of copy number variations.
40
Insights gained from these studies help
explicate the biologic underpinnings of each tumor subtype and the
respective molecular drivers. Indeed, cross-study comparisons have
shown that different tumor lineages may have signicant overlap in
genomic aberrations, such as basal-like breast cancers, high-grade
serous ovarian cancers, and serous endometrial cancers.
12
Interest-
ingly, different genetic aberrations leading to the same nal event
(impaired homologous recombination, for example) can potentially
prognosticate benet fromspecic targetedagents (PARPinhibitors).
The ovarianTCGAstudy, for example, identiedgermline or somatic
defects in BRCA1/2 in approximately 50% of high-grade serous tu-
mors,
7
whichmayexplainthe clinical activityof olaparibinthis cancer
subtype irrespective of mutation analysis.
41
Tumors are clearly undergoing reclassication on the basis of
genetic criteria. Commonmalignancies are being fragmented accord-
ing to genomic analysis, such as colorectal cancer (KRAS/BRAF/
NRAS/PIK3CA wild-type and KRAS-, NRAS-, BRAF- and PIK3CA-
mutated tumors or combinations), melanoma (BRAF-mutated,
NRAS-mutated, or KIT-mutated tumors), and NSCLC (EGFR-
mutated, KRAS-mutated, BRAF-mutated, ALK- or ROS1-rearranged
carcinomas). Modern sequencing technologies have recently identi-
ed novel driver mutations segregated from more frequent aberra-
tions in many tumor types,
42-48
some of which have clear therapeutic
relevance. Using whole-transcriptome sequencing, in-frame fusion
transcripts of KIF5B (the kinesin family 5B gene) and the RET onco-
gene were unraveled in 1% to 2% of lung adenocarcinomas. In
preclinical models, TKIs with activity against RET suppress fusion-
induced cell growth, introducing potential newavenues for the treat-
ment of patients with this specic aberration.
42
Other examples
include a recurrent MAGI3-AKT3 fusion leading to constitutive acti-
vationof the AKTkinase foundintriple-negative breast cancer, which
is abolished by ATP-competitive AKTsmall-molecule inhibitors,
43
as
well as genetic rearrangements leading to PTEN inactivation or gene
fusions activating BRAF in subsets of prostate cancers, providing a
rationale for clinical trials exploring the activity of PI3K pathway and
BRAF/MEKinhibitors, respectively, inthese subsets of patients.
44,45
In
rare tumors such as malignant myxoid/round-cell sarcomas, PIK3CA
mutations were detected in 18% of patients, also highlighting the
potential benet of genomic analysis of individual tumor samples.
48
The identication of recurrent mutations in isocitrate dehydrogenase
(IDH1/2) genes in gliomas and the resulting hypermethylating phe-
notype has stimulated renewed attention to altered metabolism in
cancer biology and the development of drugs that selectively target
aberrant metabolicpathways inadditiontohypomethylatingagents.
49
Furthermore, systematic genomic proling incancer cell lines has also
provided a powerful biomarker discovery platform to guide rational
cancer therapeutic strategies, as exemplied by the marked sensitivity
of Ewing sarcoma cells harboring the EWS-FLI1 gene translocationto
PARP inhibitors.
50
Dealing With Tumor Heterogeneity and Resistance to
Targeted Therapies
Initial reports fromtheaforementionedgenomecharacterization
consortia have revealed the immense complexity of the cancer ge-
nome as well as a striking inter- and intratumor heterogeneity at the
whole-genome level in solid tumors. Topographic differences in mu-
tations, chromosome copy number variations, and gene expression
signatures conrmthat multiple clonal subpopulations of tumor cells
exist within a single neoplasm. In fact, conventional histopathology
has repeatedly shownextensive heterogeneity of commonneoplasms,
as exempliedby regional separationof subclones harboring different
patterns of HER2 amplication in breast cancer.
51
Interestingly, the
unique ndingwithnext-generationsequencingtechniques is that the
history of a tumor is encoded by its heterogeneity.
52
Analyses of
matched primary and metastatic tumors have characterized distinct
patterns of branching or Darwinian-like tumor clonal evolution,
Dienstmann et al
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with acquisition of necessary driver mutations that underlie tumor
progression.
53-55
As was recently reported in renal cell carcinoma,
common alterations in driver genes may be distinct across different
regions of the tumor.
55
In addition, comparative studies suggest that
the mutational signatures found in metastatic samples reect the
patterns of subclones that already exist inthe primary tumor, withlow
frequency subpopulations in the primary site being enriched in the
metastatic site. Some of these subclones may acquire additional mu-
tations that may be correlated to organ-specic metastasis.
54,55
More-
over, it is anticipated that the coexistence of large numbers of
passenger alterations canleadtonovel tumor phenotypes as a result of
genetic interactions among these mutations.
56
This remarkable tumor heterogeneity will likely represent a ma-
jor challenge topersonalizedmedicine andbiomarker development.
57
In the clinical scenario, it could explain the mixed responses to tar-
geted therapies.
58
Indeed, heterogeneity between primary and meta-
static tumors for only a limited number of markers (hormonal
receptors and HER2 status in breast cancer) has been associated with
markedly worsened outcomes.
59,60
Whether these outcomes reect
inappropriate use of targeted therapies or greater aggressiveness of
tumors with increased genomic instability that results in the genera-
tion of multiple subclones remains to be ascertained. Conversely,
molecular discordances betweenprimary andmetastatic disease differ
among cancer types. In colorectal cancer and NSCLC, for example,
there is high concordance for KRAS and EGFR mutations, respec-
tively.
61,62
In fact, the effectiveness of the currently available targeted
treatments such as cetuximab and panitumumab for patients with
advanced KRAS wild-type colorectal cancer and getinib or erlotinib
in EGFR-mutated NSCLC has largely been demonstrated from trials
that have identied genetic mutations in archived diagnostic samples
rather than in new biopsies from metastatic lesions. However, for
advanced cancers that have progressed through multiple treatments,
analysis of archived tumor tissue from a primary site probably does
not accurately reect the actual disease biology. Biopsying one single
metastatic site likely underestimates the real tumor genomics land-
scape and only partially reveals the problem of intratumor heteroge-
neity. Multiple sampling for systematic genomic analysis may not be
feasible, and it is also unclear whether such an aggressive approach
wouldactually change the paradigmof a personalizedtreatment strat-
egy, given the knowledge we have today.
Nevertheless, it is clear that even for tumors with thousands of
mutations, a limited number of pathways are frequently altered and
drive cancer biology.
63
In glioblastomasone of the rst cancer
types to be subjected to comprehensive -omics prolingmapping
genomic changes ontobothknownandinferredcellular networks has
identied three frequently disrupted pathways with key roles in tu-
morigenesis, which are independently altered in most tumors: p53,
retinoblastoma [RB], and receptor tyrosine kinase.
6
Pathway conver-
gence was alsodemonstratedinheadandneck carcinomas, because at
least 30% of the tumors analyzed by whole-exome sequencing har-
bored mutations in genes that regulate squamous differentiation (for
example, NOTCH1, IRF6, and TP63), implicating their dysregulation
as a major driver of carcinogenesis in this tumor type.
64
In estrogen
receptorpositive breast cancer, distinct tumor phenotypes have
been linked to specic patterns of somatic mutations that map into
known activating pathways (p53/RB, PI3K/AKT/mammalian tar-
get of rapamycin, and mitogen-activated protein kinase).
65
Eluci-
dation of dysregulated pathways in cancer by using functional
protein proling platforms can further identify distinct molecular
networks that drive cancer phenotypes.
66
Moreover, the aforemen-
tioned subclonal mutations in focal tumor regions are unlikely to
provide growth advantage to the tumor at large and, therefore, are
not expected to be clinically useful targets of rst-line therapy.
57,67
High-throughput RNA sequencing studies have also revealed that
only a limited number of mutations are signicantly expressed.
68
These ndings may have a positive impact on targeted therapeutics
by enabling a customized approach, even for genomically complex
and heterogeneous neoplasms.
Conversely, clinical responses to targeted agents are consistently
abrogated by the development of drug resistance. High-sensitivity
genomic analysis in relapsed tumor samples has revealed emer-
gence of secondary mutations, such as KIT
T670I
in imatinib-
resistant gastrointestinal stromal tumors,
69
EGFR
T790M
in getinib- and
erlotinib-resistant lung cancer,
70
ALK
L1196M
in crizotinib-resistant
ALK-rearranged lung cancer,
71
and Smoothened (SMO)
D473H
in me-
dulloblastoma progressing to hedgehog pathway inhibitors.
72
These
mutations impede durable effects of kinase inhibitors either through
alterations in the drug target (gatekeeper mutations) or by changes in
the conformational state of the kinase that led to increased ATP afn-
ity. They presumably represent positive selectionof rare cell subpopu-
lations that are already present in the primary tumor.
73
It remains to
be determined whether massively parallel sequencing of pre- and
postrelapse tumors can identify such latent mutations and potentially
predict likely forms of adaptive resistance. In addition, it is still un-
known whether passenger mutations might convert into alternative
driver mutations on functional blockade of the original driver muta-
tion with targeted agents. So far, relatively few whole-genome,
-exome, or -transcriptome studies have been performed in the setting
of progressiontodruggabletumor dependencies. Theidenticationof
a somatic MEK1
C121S
mutation in a patient with melanoma progress-
ing to vemurafenib and the in vitro conrmation that it increases
kinase activity and confers robust resistance to both RAF and MEK
inhibitions indicates that such studies will probably direct the devel-
opment of alternative therapeutic options.
74
Other recently published
studies detected the emergence of KRAS mutations in both tumor
biopsies and circulating tumor DNA (ctDNA) from the serum of
patients with metastatic colorectal cancer who initially had KRAS
wild-type tumors and responded to cetuximab- or panitumumab-
based therapies, with later development of secondary resistance.
75,76
These data emphasize a clonal selection process achieved under treat-
ment pressure as the major determinant of the nal clinical out-
come.
77
Another frequent resistance mechanismrevealedby genomic
and proteomic analyses with potential for guiding second-line treat-
ment decisions is the activation of parallel signaling pathways, such as
MET amplication in the setting of EGFR-mutated NSCLC cancer
progressing to EGFR TKIs.
78
In large academic cancer centers, specic protocols for rebiopsy-
ing tumors at the time of progression to molecular targeted therapy
are becoming standard of care for patients eligible for further treat-
ment lines.
79
In EGFR-mutated NSCLC with acquired resistance to
erlotinib, for example, secondary mutations have been identied in
more thanhalf the patients,
80
providing anindicationfor treatment in
clinical trials with promising combination strategies.
81
Tissue that is
collected as part of a clinical trial (at baseline, during treatment, or at
the time of tumor progression) has great potential to advance scien-
tic knowledge by revealing how well a drug is affecting the target of
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interest, by uncovering new oncogenic signaling pathways, by estab-
lishing predictors of favorable or unfavorable outcomes, and by iden-
tifying mechanisms of drug resistance. Mandatory biopsies are
appropriate wheninvestigators weigh the risks against the necessity of
the correlative question.
82
However, various hurdles, includingethical
concerns and procedural risks, must be considered when protocols
mandate biopsies. Importantly, the experience of large centers shows
that biopsies inearly-phase clinical trials are safe, withless thana 1.5%
riskof serious complications.
83
Figure 2summarizes the scheduling of
tumor biopsies andthe opportunities for genomic analysis (withfocus
on turnaround time) during the curative or palliative settings, while
patients are receiving standard or experimental therapies.
CONCEPTUAL APPROACHES TO GENOMIC MEDICINE
IN ONCOLOGY
Given the evolving nature of genomic discoveries in cancer that result
from novel technologies and the expanded repertoire of targeted
agents, two major conceptual approaches for the analysis, interpreta-
tion, and clinical applicability of data have been delineated in the last
fewyears. As showninFigure 3 andsummarizedinTable 1, a targeted
or genomically stratied model originates fromthe one test-one drug
paradigm with the requirement of companion diagnostics for tar-
geted therapies. Advances in multiplexed genotyping platforms are
Biopsy
(diagnosis)
Surgery
Relapse
* ) y s p o i b ( * ) y s p o i b (
Neoadjuvant
Curative setting
Palliative setting
Standard treatment
Palliative setting
Experimental treatment
4 T 3 T 2 T 1 T
(biopsy)*
T5
(biopsy)*
(pharmacodynamics)
e n i l n e n i l n e n i l d n o c e S e n i l t s r i F t n a v u j d A
Fig 2. Scheduling of tumor biopsies and the opportunities for genomic analysis. Neoadjuvant setting: small sample from biopsy. Treatment decision in days (Time 1
[T1]). Predictive or prognostic role in a curative setting (eg, human epidermal growth factor receptor 2 status in breast cancer). Low tolerance for false-positive or
false-negative results. Adjuvant setting: usually larger sample from surgery. Four to 6 weeks before treatment decisions (T2) allows more extensive genomic analysis
in central laboratories. Predictive or prognostic role in a curative setting (eg, Oncotype DX/MammaPrint in breast cancer, KIT status in GI stromal tumors). Palliative
setting (standard treatment): small sample from biopsy, if any. Treatment decision in days (T3). Tests oriented to guide therapy (eg, EGFR mutation in lung cancer, BRAF
mutation in melanoma, KRAS mutation in colorectal cancer). Performing comprehensive genomic analysis of an archival (formalin xed, parafn embedded or fresh
frozen) sample (preferably from a metastatic site) while the patient is receiving standard treatment may allow treatment selection in the experimental setting based
on central laboratory results. Palliative setting (experimental treatment): small sample from biopsy, if any (threshold for indicating biopsy varies according to academic
institution and availability of clinical trials). Treatment decision needs to be made in a few weeks (T4) because of the patients anxiety or symptomatic deterioration.
Tests oriented to suggest therapy (a higher degree of indetermination is acceptable since it is an experimental environment and the test or the drug may not be
sufciently developed or suitable for the purpose). Examples include multiplexed genomic platforms and targeted, massively parallel sequencing. Better if performed
locally at the site, but central analysis might be an option in some circumstances (investigational gene expression proles). Allows inclusion in early proof-of-concept
clinical trials and identication of tumor vulnerabilities (and experimental treatment options) not originally expected for a specic histologic subtype. At the time of
progressive disease after response to a targeted agent, rebiopsying the metastatic site for research purposes (identication of resistance mechanisms) and to
potentially guide alternative treatment options in clinical trials is recommended. Treatment decision needs to be made in few weeks (T5) if the patient is expected to
tolerate additional therapies. Examples include analysis of secondary mutations and alternative pathway activation. (*) Optional.
Examples:
1. Nonsmall-cell lung cancer,
ALK rearrangement by FISH = crizotinib
2. Breast cancer, low-risk gene
expression profile by OncotypeDX =
tamoxifen
3. Melanoma, BRAF
V600
mutation by
real-time PCRbased test = vemurafenib
Examples:
1. Melanoma, BRAF, KIT, or NRAS
mutation = vemurafenib, imatinib,
MEK inhibitor
2. Colorectal, KRAS, BRAF, NRAS,
PIK3CA = not cetuximab or
panitumumab-based therapy
Genomically stratied/multicategorical model
Omniscientreductionist model
One test - one drug (companion diagnostics)
Aberration A One assay Drug A
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,

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i
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a
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y
t
e
s

o
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d
a
t
a
Mutation A Drug A
Mutation A Drug A
Mutation B Drug B
Mutation C Drug C
Mutation N Drug N
Mutation B Drug B
Mutation C Drug C
Mutation N Drug N
Fig 3. Conceptual approaches to genomic medicine in oncology. FISH, uorescent in situ hybridization; PCR, polymerase chain reaction.
Dienstmann et al
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currently engendering an upfront multicategorical approach that can
be used to decide among different potential treatments. Genes that
predict sensitivity or resistance to cancer therapies are targeted for
sequencing using small amounts of tumor DNA.
84
The prospect is to
use massively parallel sequencing technologies, which allow evalua-
tion of a more comprehensive spectrum of alterations, including not
only point mutations but also copy number variations and fusion
events, by using a targeted capture-sequencing approach. At the pres-
ent time, clinical investigators are facing the alternative conceptual
model that pursues a personalized oncology paradigm through
comprehensive assessment of genomic data. Different from the
genomically stratied approach, it is hypothesis-generating by allow-
ing identication of novel drivers and matched targeted therapies in
the experimental setting. This omniscient model is grounded on
whole-genome, -exome, and -transcriptome sequencing platforms
but is ultimately reductionist and focused on actionable aberrations.
Bioinformatic algorithms based on available databases (eg, Cancer
Gene Census, Catalogue of Somatic Mutations inCancer, andMolec-
ular Signatures Database) can be used to selectively identify somatic
aberrations in high-priority cancer genes that might have immediate
implications for patient care (including the variants that have been
predicted to confer tumor sensitivity or resistance to approved or
experimental therapies) as well as those involving known biologically
relevant pathways intumor cells. The remaining variants of unknown
clinical signicance are also catalogued because they may ultimately
represent actionable aberrations after additional research in the eld.
Computational tools can help predict the effect of an amino acid
switch on protein structure and function,
85
although experimental
validation with transformation assays is the most powerful method.
International databases that include mutation type, patient demo-
graphics, and treatment and outcome data are needed to ensure ro-
bust statistical validity for these rare events.
2
GENOMIC-BASED CLINICAL TRIALS: MULTIPLE STEPS OF A
COMPLEX WORKFLOW
Using genomic proling to select patients for clinical trials with
matchedtreatments that are expectedtoprovide benet is timely. The
ultimate goal of such trials is to determine which mutation proles
correlate with sensitivity/lack of resistance to specic targeted thera-
pies and whether treatment outcomes are consistent among different
cancer histologies.
2
Inaddition, genomic-basedtrials canalsogenerate
valuable information regarding cancer biology, clinically qualify po-
tential predictive biomarkers, accelerate patient benet, and assist in
the decisiononwhether a novel targetedagent warrants further devel-
opment.
86
Some studies have shown that real-time molecular prol-
ing of tumors from actual patients and treatment with matched
Table 1. Conceptual Approaches to Genomic Medicine in Oncology
Genomically Stratied/Multicategorical Model Omniscient-Reductionist Model
Derivation from one test-one drug (companion diagnostics) to a
multiplexed, multicategorical approach.
Derivation from the Human Genome Project and the evolution of technology.
Comprehensive throughput.
Focused on unicausality whereby one aberration is effectively
targeted by one drug (or combinations of drugs).
Opened to multicausality of drivers but is reductionist because of current knowledge
and the available druggable options (typically used as monotherapies).
Diseases are categorized into different subsets by genomic analysis.
Aberration-drug pairs are predened. Occasional overlap among
categories may confound treatment options (colon cancer KRAS
plus PIK3CA mutation, Oncotype DX intermediate risk).
Diseases are dened by patterns of recurrent alterations in pathways (convergent
evolution). Each patient has a unique disease with a combination of alterations
that are unexpected before analysis: personalized medicine. Need for a
functional understanding of each alteration (systems biology) may complicate
decisions.
Hypothetical-deductive reasoning (preconceived theory). The
aberrations selected for analysis take into consideration
preclinical/clinical data.
Inductive reasoning can be applied (when data generate a new theory). Deductive
reasoning is also used to simplify and prioritize data analysis and to identify
actionable aberrations.
Attitude: Ask questions you want to know the answers to. Attitude: Ask all questions, but listen only to answers that please you. Describe
reality as it is but reduce it later to correspond with your theory.
The paradigms are embedded in the question and established prior to
analysis (ie, PI3K inhibitors work well in PIK3CA-mutated tumors).
Decision after the analysis is straightforward (if BRAF
V600
-
mutated gene is present, treat with a BRAF inhibitor).
No preconceived notion before analysis. A comprehensive analysis follows (quasi-
omniscient) with subsequent reductionism to what we can understand.
Decision after the analysis is not straightforward because one could identify 10
alterations in known oncogenes/tumor suppressor genes, but the patient is
treated with only one drug (ie, a PI3K inhibitor based on current knowledge or
best guess).
If patient fails to respond, genomic aberrations that may represent
primary resistance mechanisms cannot be dened. Therefore, it
is not hypothesis-generating. Targeted assays may evade
opportunities for discoveries of new drivers and predictive
biomarkers.
If patient fails to respond, potential genomic aberrations that may represent primary
resistance mechanisms can be identied after re-analysis of original assay results
(considering repetition of the same event in other patients). Therefore, it is
hypothesis-generating but not easily reproducible. By allowing identication of
novel drivers and hypothesis-driven therapies, a single patient can provide many
lessons for investigators when an identiable, druggable aberration has a good
response to treatment.
Analysis can be performed locally at the patients facility or in a
centralized laboratory.
Analysis has to be done centrally since few sites can afford the cost of analysis or
interpretation of data.
Quick, relatively low performance but applicable solutions, including
enrollment in clinical trials with enrichment strategies.
Slow, high performance, hard to interpret and apply to the individual. Applicable to
clinical trials for enrichment strategies but has limitations such as turnaround
time. Fits well with the research/experimental setting and relies on bioinformatics
expertise.
If multiple platforms are used to identify different aberrations (such as
multiplexed genotyping for mutation detection plus uorescent in
situ hybridization for copy number variations and
rearrangements), the cost may become high.
Current platforms of comprehensive genomic analysis are becoming less expensive.
Genomic Medicine in Solid Tumors: Prospects and Challenges
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targeted agents can increase response rates and improve time to pro-
gression compared with unselected therapies.
87,88
Recently, academic
groups have demonstrated the feasibility of a targeted, massively par-
allel sequencing approach to assist in determining mutation-driven
inclusioncriteria for clinical trials.
89
By usinga combinationof whole-
genome and-exome sequencing plus sequencing of transcribedRNA,
others have proven that informative mutations in tumors of selected
patients can be identied within 3 to 4 weeks, a time that is short
enough to be clinically useful.
90
Although the potential benets of
high-throughput assays for individual patients with cancer have al-
ready been demonstrated,
91
the next logical step is to facilitate larger
clinical trials in oncology with biomarker-informed therapies.
As targeted therapeutics are developed to treat small subsets of
individual disease populations, changes will be required in current
clinical trial designs and research frameworks. Early-phase clinical
trials will best be served by taking advantage of enrichment strategies,
witha focus onpatients who are unlikely to benet fromstandard-of-
care chemotherapy lines and more likely to benet from a novel
therapy because of the presence or absence of specic tumor genetic
abnormalities.
92
Patients with excellent responses to targeted thera-
pies based on novel driver mutations should be reported to advance
theeldbeyondanecdotal observation. PhaseII trials will benet from
exploiting novel designs, including studies that subclassify a specic
disease into discrete molecular categories to test different agents in
separate cohorts (eg, the I-SPY 2 [Investigation of Serial Studies to
Predict Your Therapeutic Response With Imaging and Molecular
Analysis] trial in breast cancer
93
and the BATTLE [Biomarker-
Integrated Approaches of Targeted Therapy for Lung Cancer Elimi-
nation] trial in NSCLC
94
), as well as histology-independent trials (eg,
the MO28072trial withvemurafenibinmultiple solidtumors harbor-
ing BRAF
V600
mutations). In genomic trials of selected populations,
many patients need to be proled for sufcient accrual to provide
appropriate statistical power. If the magnitude of treatment effect is
greater than expected with an unselected approach, the number of
patients required per arm to demonstrate improved outcomes is sig-
nicantly reduced. Conversely, when the predictive value of a specic
molecular aberration is unknown, large randomized trials with strat-
ication strategies are still required, with biomarker-positive and
-negative patients being allocated to the standard-of-care or targeted
therapy arm.
95
Many unsolved biologic questions, scientic concerns regarding
platformselection, and logistical issues related to prescreening strate-
gies andtrial allocation, inadditionto challenges inthe economic and
ethical domains, will need to be overcome before genomic-driven
trials and personalized cancer medicine can become a reality and
supplant currently accepted modalities, as summarized in Table
2.
2,11,92,96
Importantly, academic institutions, pharmaceutical compa-
nies (that control which new agents are available for clinical testing),
and third-party payers (for provision of marketed drugs) will need to
work in a collaborative environment to allowrational drug combina-
tions tobe readilytestedandavoidduplicationof efforts. Clinical trials
that test matched targeted agents in the setting of rare molecular
aberrations or that integrate biomarker development can only be
accomplished with multi-institutional cooperative networks that fa-
cilitate data sharing and technology exchange. Finally, global efforts
are needed to prospectively validate genomic tools. Successful initia-
tives include adjuvant clinical trials in early breast and colon cancer
that evaluate gene expression proling platforms, such as Oncotype
DX (Genomic Health, Redwood City, CA) and MammaPrint/Colo-
Print (Agendia, Amsterdam, the Netherlands), which are expected to
assist in clinical decision making by enhancing the prediction of out-
come compared with traditional pathology.
FUTURE DIRECTIONS AND CONCLUSIONS
The ability to prole patients with each cancer for actionable aberra-
tions in a cost-effective way provides unprecedented possibilities for
matched therapies in a selected patient population. The major chal-
lenge will be to integrate and make biologic sense of the massive
amount of genomic data derived from multiple platforms. The low
frequency of many signicantly mutated genes represents another
important limitation of correlative analyses. Therefore, to obtain a
complete picture of the biology underlying eachcancer subtype, it will
be mandatory to map specic patterns of somatic mutations into
cellular pathways linkedtotumor biology. Inaddition, because cancer
genomes are not exclusively disrupted at the DNA sequence level but
are also driven by various permutations in genetic regulation, it is
imperative to investigate other genomic dimensions, such as DNA
methylation status, to understand the phenotypic heterogeneity dis-
played by most solid tumors.
97
Studies that use massively parallel
sequencing of primary tumor samples have identied recurrent mu-
tations in genes related to histone modication, proteins involved in
chromatin remodeling, and transcription factors, known targets of
epigenetic modiers.
65,98-102
Several international projects are in the
process of assessing epigenomic events in human cancers and how
genetic mechanisms affect epigenetic effectors.
103,104
To date, it re-
mains tobe shownwhether data obtainedfromepigenetic prolingby
using deep sequencing can predict drug response. Furthermore, to
implement true personalizedmedicine, one shouldconsider the char-
acteristics of the tumor and also the host-tumor relationship (tumor
microenvironment and immune response) and host-drug relation-
ship (metabolism genes and pharmacogenomics). The signicant
clinical activity of immune-checkpoint-pathway inhibitors such as
antiCTLA-4 (ipilimumab)
105
and antiprogrammed death 1 (PD-
1)
106
mAbs in a variety of solid tumors is a practical example that
shows that not all actionable/druggable cancer targets are products of
genetic aberrations. Germline genetic aberrations inproteins involved
in drug metabolism and apoptosis, which may modify toxicity and
response to targeted agents, should also be assessed.
We envisiona future inwhichgenomics-drivencancer medicine
will address treatment of many solid tumor subsets. Novel potential
targets in selected malignancies are increasingly being described. Ulti-
mately, however, solid tumors will not be universally impacted with
genomic proling alone. More likely, the management of rare subsets
of commoncancers andsome relatively rare tumor types withspecic
aberrations will change dramatically with the widespread availability
of genomic tools. Examples include NRAS-mutated melanomas and
benet with MEKinhibitors
107
or CDK4-amplied liposarcomas and
antitumor activity of CDK4 inhibitors.
108
In addition, the develop-
ment and validation of predictive markers will certainly guide the
reappropriation of existing therapies, such as histone deacetylase,
DNAmethyltransferases, and proteasome inhibitors.
Key issues related to clonal evolution and tumor heterogeneity
must be identiedandaddressed. Systematic analyses of the evolution
of cancer clonal architecture during therapy will identify ubiquitous
Dienstmann et al
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driver events present in all regions of the tumor that could be more
efciently targeted.
57
Because therapy-induced selection of drug-
resistant mutations limits the efcacy of targeted therapies, methods
that allow massively parallel sequencing from small samples, such as
circulating tumor cells or free ctDNA from serum (also known as
liquid biopsies), are likely to be the mainstay technologies for clinical
laboratory testing in the future and may also provide a cost-effective
alternative to biopsy. Other advantages include lack of invasiveness
andlackof spatial sampling errors. Indeed, ultrasensitive ctDNAanal-
ysis platforms have demonstrated sufcient accuracy in reecting
aberrations present in primary and metastatic breast tumors
109,110
as
well as detecting mutant KRAS clones in colorectal cancer.
75,76
These
advances may greatly enhance successful application of genomic test-
ing in cancer management by more efciently monitoring disease
status over time and allowing real-time resequencing and molecular
proling at multiple points during the disease course, particularly of
patients progressing to targeted treatment approaches.
In conclusion, the genomic information derived from modern
sequencing technologies provides a newparadigmfor clinical investi-
gation. Treatment decisions for each individual patient are dened by
Table 2. Challenges for Implementing Genomic-Driven Trials and Personalized Cancer Therapy
Prescreening strategies
Population: The focus is on chemorefractory patients with common malignancies who are considering clinical trials versus those with rare diseases with no
standard treatment options.
Timing: Timing choice is one of the following: immediately before considering clinical trials versus a broader prescreening program for patients with
metastatic disease and high possibility of actionable aberrations.
Location: Local analysis is done in academic centers (need for secondary verication?) versus centralized analysis in reference institutions.
Tissue for analysis
Quantity and quality of DNA: Recommendation for tissue is 70% tumor cellularity with 10% necrotic tumor tissue.
Fresh frozen versus FFPE: Snap freezing tumor tissues in liquid nitrogen is the optimal method for nucleic acid preservation. There is good correlation with
mutational output between matched fresh-frozen and FFPE tumor samples with next-generation sequencing results. RNA extracted from FFPE tissue is
often of poor quality.
Primary tumor versus real-time biopsies: Clonal evolution, selection pressure from prior therapies, and tumor heterogeneity may affect the results.
Need for standardization: Collection, handling, and storage of specimens needs to be standardized.
Platform selection
Sensitivity: Deep sequencing technology increases the sensitivity of mutation detection, particularly in the setting in which there is a high stromal admixture.
Reliability, precision, accuracy, and interlaboratory reproducibility of data: Bioinformatic analysis and stringent quality control can reduce errors.
Turnaround time: Sequencing analysis is completed in a clinically relevant time frame.
Laboratory CLIA/GCLP certication: Results that affect clinical decision-making must be validated.
Type of analysis: The genome is comprehensively assessed for structural rearrangements, copy number alterations, point mutations, insertions, deletions,
and gene expression proles versus focusing on actionable aberrations with immediate clinical implications (such as druggable kinases or selected tumor
suppressor genes). There is balance between analytical coverage (ie, genome coverage), statistical power, and cost-effectiveness when high-throughput
platforms are used.
Multidisciplinary treatment decision
Results can be grouped into three categories: (1) those that may have a direct impact on care of the patient with the cancer of interest, (2) those that may
be biologically important but are not currently actionable, and (3) those of unknown importance.
1. How is a driver distinguished from a passenger genetic alteration?
2. How does one dene which mutations engender sensitivity to specic targeted therapeutics?
3. How are nondruggable aberrations managed?
4. How are aberrations of unknown biologic/clinical signicance dealt with?
5. How is therapy selected in the case of multiple aberrations?
Data integration among multiple platforms: Bioinformatics pipelines for data analysis are required.
Trial allocation
There is a need for multi-institutional trial networks to assess novel agents that target specic aberrations; there are potential geographical limitations.
What should be done when eligibility criteria are too stringent or slots are not available in genomic-driven early clinical trials? And when patients present
with clinical deterioration at the time of recruitment?
Is it possible to offer treatment outside clinical trials with matched targeted agents approved for another indication (such as off-label vemurafenib in
BRAF
V600
-mutated lung cancer) or to prescribe marketed drugs not previously tested in combination regimens?
Reimbursement and nancial issues
Cost of prescreening and diagnostic approaches (eg, expenditures for training personnel with appropriate expertise and setting up certied laboratories) and
the resulting therapeutic implications (when analysis indicates off-label treatments) must be addressed.
From a societal point of view, the benets (in terms of cost savings) of avoiding empiric prescription of expensive drugs when a personalized cancer
approach is adopted must be taken into consideration.
From an economic point of view, patients receiving targeted therapies are expected to remain on treatment for longer periods, with benets for
pharmaceutical companies.
Ethical issues
Privacy and condentiality should be addressed at all times.
Informed consent form should discuss how to deal with incidental ndings, such as germline mutations associated with risk for other diseases (eg, long QT
syndrome) and those that provide risk information relevant to family members (such as mutations in BRCA1/2 and cystic brosis genes).
Results should be disclosed to patients but only those associated with sufcient risk and established clinical utility should be communicated, ensuring
proper understanding of health and social implications.
Clinicians have an undened obligation to nd suitable on-trial or off-trial options for patients whose tumors have undergone molecular proling.
Abbreviations: CLIA, Clinical Laboratory Improvement Amendments; FFPE, formalin xed, parafn embedded; GCLP, Good Clinical Laboratory Practice.
Genomic Medicine in Solid Tumors: Prospects and Challenges
www.jco.org 2013 by American Society of Clinical Oncology 1881
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
the evolving knowledge of specic molecular aberrations and their
possible consequences. Nevertheless, the extreme genomic complex-
ity and mutability of cancer mandates the use of comprehensive se-
quencing and gene expression platforms as well as analysis of
functional protein pathway activation patterns so that a personalized
treatment strategy in human solid tumors can best be implemented.
AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
Although all authors completed the disclosure declaration, the following
author(s) and/or an authors immediate family member(s) indicated a
nancial or other interest that is relevant to the subject matter under
consideration in this article. Certain relationships marked with a U are
those for which no compensation was received; those relationships marked
with a C were compensated. For a detailed description of the disclosure
categories, or for more information about ASCOs conict of interest policy,
please refer to the Author Disclosure Declaration and the Disclosures of
Potential Conicts of Interest section in Information for Contributors.
Employment or Leadership Position: Jordi Barretina, Novartis (C)
Consultant or Advisory Role: Josep Tabernero, Amgen (C), Genentech
(C), Merck Serono (C), Novartis (C), Roche (C), sano-aventis (C),
Bayer Pharmaceuticals (C) Stock Ownership: None Honoraria: None
Research Funding: None Expert Testimony: None Other
Remuneration: None
AUTHOR CONTRIBUTIONS
Conception and design: Rodrigo Dienstmann, Jordi Rodon, Jordi
Barretina, Josep Tabernero
Provision of study materials or patients: All authors
Manuscript writing: All authors
Final approval of manuscript: All authors
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Dienstmann et al
1884 2013 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY
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Copyright 2013 American Society of Clinical Oncology. All rights reserved.
Acknowledgment
We thank Josep Corco (Universitat Internacional de Catalunya) for his invaluable help in conceptual denitions of genomic approaches and
Joann Aaron for English editing.
Genomic Medicine in Solid Tumors: Prospects and Challenges
www.jco.org 2013 by American Society of Clinical Oncology
Information downloaded from jco.ascopubs.org and provided by at ASCO on March 6, 2014 from 158.232.241.130
Copyright 2013 American Society of Clinical Oncology. All rights reserved.