Clinical Chemistry / Predicting Response in Colorectal Cancer
Biomarker-Based Prediction of Response to Therapy
for Colorectal Cancer
Current Perspective
Jeffrey S. Ross, MD,1,2 Jorge Torres-Mora, MD,1 Nikhil Wagle, MD,3 Timothy A. Jennings, MD,1
and David M. Jones, MD1
Key Words: Colorectal cancer; CRC; Biomarkers; Molecular classification; Pharmacogenomics; Pharmacogenetics; Review
DOI: 10.1309/AJCP2Y8KTDPOAORH
Abstract
The diagnosis and management of colorectal
cancer (CRC) has been impacted by the discovery and
validation of a wide variety of biomarkers designed to
facilitate a personalized approach for the treatment
of the disease. Recently, CRC has been reclassified
based on molecular analyses of various genes and
proteins capable of separating morphologic types of
tumors into molecular categories. At the same time,
a number of new prognostic and predictive single
genes and proteins have been discovered that are
designed to reflect sensitivity and/or resistance to
existing therapies. Multigene predictors have also
been developed to predict the risk of relapse for
intermediate-stage CRC after completion of surgical
extirpation. More recently, a number of biomarkers
tested by a variety of methods have been proposed as
specific predictors of chemotherapy and radiotherapy
response. Other markers have been successfully used
to predict toxic effects of standard therapies. In this
review, a series of novel biomarkers are considered
and compared with standard-of-care markers
for their potential use as pharmacogenomic and
pharmacogenetic predictors of disease outcome.
478 Am J Clin Pathol 2010;134:478-490
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Colorectal cancer (CRC) is the second leading cause of
cancer mortality in the United States.1 There are 160,000 new
cases of CRC diagnosed each year and 57,000 CRC-related
deaths in the United States.1 Despite a slow decline during the
last 20 years, for men, the age-adjusted incidence is 61.2 cases
per 100,000 and for women, it is 44.8 per 100,000.1
Pathologists have traditionally had a major role in the
care of patients with newly diagnosed CRC in the initial
establishment of the diagnosis of invasive disease and in
the morphologic classification and final staging of surgi-
cally resected specimens.2,3 In the last 30 years, there has
been significant advancement in the understanding of the
molecular origins of CRC and characteristics of tumor aggres-
siveness.4-7 This major expansion of genomic and proteomic
data has simultaneously contributed to the discovery of novel
targeted therapies for patients with CRC in whom recurrent
and metastatic disease have developed.8-10 Moreover, the use
of gene sequencing, expression profiling, and other molecular
techniques has introduced putative biomarkers that have been
reported to predict the clinical response and toxic effects for
the nontargeted traditional antineoplastic drugs that have been
used to the treat the disease.11 These new techniques have led
to the discovery of a number of biologically interesting genes
and pathways associated with CRC development and progres-
sion, as well as new biomarkers that are emerging as bedside
clinical tests for the prediction of response to therapy for the
disease. As a result, practicing pathologists have now become
responsible for overseeing the performance of these emerg-
ing tests designed to personalize the selection of postsurgical
treatment for CRC.
This review is focused primarily on the discovery and
development of predictive biomarkers designed to predict
© American Society for Clinical Pathology
 Clinical Chemistry / Review Article

therapy-specific disease outcome parameters such as overall

and recurrence-free survival. Prognostic markers are also
considered, and their interactions with predictive markers and
indirect impact on therapy selection and disease outcome are
also reviewed.

Molecular Classification of CRC

In the past several years, several groups have attempted
to produce a molecular classification of CRC based on a num-
ber of features, including chromosomal instability (tendency
for chromosome breakage [CIN]), microsatellite instability
(defective DNA repair capability [MSI]), and frequent CpG

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island hypermethylation (gene silencing due to methylation of
the promoter gene sequence [CIMP]).12-15 In contrast with the
breast cancer molecular “portraits” system that has achieved
wide acceptance by pathologists and oncologists,16 the CRC
❚Image 1❚ Sessile serrated adenoma with invasive
molecular classification continues to undergo refinement in
adenocarcinoma. Low-power magnification of a sessile
the number of classes and their clinical practice applicability.
serrated polyp/adenoma with an area of invasive carcinoma
The molecular classifications of CRC have uniformly
arising in the center of the polyp. Invasive colorectal cancer
attempted to link DNA stability and gene expression status
(CRC) associated with sessile serrated precursor lesions
with 7 factors: (1) tumor type, (2) tumor grade, (3) extent and
appears to be a distinct molecular subclass of sporadic CRC.
distribution of associated inflammatory changes, (4) presence
Approximately 10% to 15% of sporadic CRCs arising in the
of “serrated” architecture, (5) hereditary basis, (6) pathologic
proximal colon may feature a serrated precursor lesion with
stage, and (7) general prognosis.12-15 However, it should be
diploid microsatellite instability, frequent DNA methylation,
noted that the molecular profiles that characterize the discrete
KRAS mutation, loss of chromosome 1p, and overexpression
pathogenic pathways (CIN, serrated, and CIMP) differ sig-
of genes such as Ephrin receptor B2 and hypoxia-inducible
nificantly from the gene expression patterns that reflect the
factor 1-α12-15 (H&E, ×20).
predictive and prognostic gene expression profiles that are
based primarily on clinical outcome associations.
A proposed molecular classification of CRC is summa-
rized in ❚Table 1❚, and examples of the morphologic features of sequencing, and routine and methylation-specific messenger
specific molecular classes are shown in ❚Image 1❚, ❚Image 2❚, RNA (mRNA) expression profiling. To date, although of sig-
and ❚Image 3❚. This classification has been developed by using nificant interest, the molecular classification of CRC has not
a variety of molecular techniques, including fluorescence in achieved widespread clinical use, nor has it been validated or
situ hybridization, comparative genomic hybridization, DNA reproduced by multiple institutions on a large scale.

❚Table 1❚
The Molecular Classification of CRC*

CIN MSI CIMP

Features Aneusomy + LOH Replication error Gene silencing

Genes BUB1, AURKA, APC, p53 MSH2, MLH1, MSH6 BRAF, p53, p21
Status Present/absent L/H 0/L/H
Pathology — HNPCC, mucinous, signet-ring cell, Crohn Serrated histologic features
reaction, TILs, necrosis, high tumor grade

APC, adenomatous polyposis coli; AURKA, aurora kinase A; BRAF, raf murine sarcoma viral oncogene homolog B1; BUB1, budding uninhibited by benzimidazoles 1 homolog;
CIMP, CpG island hypermethylation; CIN, chromosomal instability; CRC, colorectal cancer; H, high; HNPCC, hereditary nonpolyposis colorectal cancer; KRAS, Kirsten
rat sarcoma; L, low; LOH, loss of heterozygosity; MGMT, O-guanine methyltransferase; MLH1, mutL homolog 1; MSH2, mutS homolog 2; MSH6, mutS homolog 6; MSI,
microsatellite instability; TILs, tumor infiltrating lymphocytes; WT, wild type.
* Modified from Ogino and Goel.13 The 6 molecular classes of CRC are as follows: 1 (10%) MSI-H/CIMP-H: sporadic MSH-H, BRAF mutation, good prognosis, proximal,

female, high grade; 2 (5%) MSI-H/CIMP-L: HNPCC, mucinous, KRAS mutation, p53 WT; 3 (5%-10%) MSI-L/CIMP-H: BRAF mutation, CIN–, p53 WT, female, elderly,
signet ring, poor prognosis; 4 (5%) MSI-L/CIMP-L: MGMT methylation, KRAS mutation; 5 (30%-35%) MSI-L/CIMP-L: KRAS mutation, CIN–, male; and 6 (40%) MSI-L/
CIMP 0: CIN, KRAS WT, BRAF WT, dista.

© American Society for Clinical Pathology Am J Clin Pathol 2010;134:478-490 479

479 DOI: 10.1309/AJCP2Y8KTDPOAORH 479
Ross et al / Predicting Response in Colorectal Cancer
❚Image 2❚ Molecular class 1 colorectal cancer (CRC) with
high microsatellite instability (MSI-H) and high CpG island
hypermethylation. This image is taken from a poorly
differentiated CRC arising in the proximal colon in an elderly
woman. Note the high nuclear grade and intratumoral
lymphocytic infiltrates. In other regions, signet-ring cell
histologic features and mucinous differentiation were noted.
This case of sporadic MSI-H CRC has been termed medullary
carcinoma in some studies and typically features a favorable
prognosis (H&E, ×200).
Prediction of Response to Primary Treatment
for CRC
Pathologic Staging
The staging of resected CRC by pathologists remains
the cornerstone for the prediction of future disease relapse
and progression.2 After many decades of use of the staging
method of Dukes et al, the current TNM staging system has
now achieved near universal use.2,17 Recent attention has
focused on understaging of disease caused by the evaluation
of an insufficient number of retrieved lymph nodes from the
resected pericolorectal mesentery or perirectal adventitia.18
The minimum number of lymph nodes recovered for accurate
CRC TNM staging has varied from 10 to 18.18,19
Molecular Staging
Although it has achieved standard of practice for breast
cancer and significant use in selected cases of malignant mela-
noma, the sentinel lymph node biopsy has not, to date, been
widely applied to CRC surgery.20 This generally reflects the
widely held opinion that, unlike breast cancer, there appears
to be no clinical benefit to the sparing of regional draining
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❚Image 3❚ Molecular class 2 colorectal cancer (CRC)
with high microsatellite instability and low/0 CpG island
hypermethylation. Low-power magnification of a well-
differentiated CRC with mucinous features and an intense
peritumoral lymphocytic reaction arising in the proximal
colon of an elderly woman. This group includes hereditary
nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome
cases, but approximately 50% of tumors in this group are
sporadic and unrelated to HNPCC/Lynch syndrome.12-15
Although these lesions also arise in the proximal colon,
often in elderly women, and may feature peritumoral
lymphocytic reactions and mucinous features, they do not
display intratumoral lymphocytes, high tumor grade, or
signet-ring cell features (H&E, ×20).
lymph nodes in sentinel lymph node–negative CRC resec-
tions. In addition, the introduction of molecular detection of
micrometastatic deposits of CRC using real time–polymerase
chain reaction (RT-PCR) in sentinel lymph nodes has also
not achieved widespread clinical use.21,22 Finally, a variety of
molecular assays have been developed to assess the status of
CRC surgical resection margins, but this approach has been
limited to research studies.23
Oncotype Dx Colon Cancer Test
The Oncotype Dx test for breast cancer (Genomic Health,
Redwood City, CA) is now widely used to plan therapy for
early-stage disease.24 A similar Oncotype Dx mRNA expres-
sion profile of 12 target genes using RT-PCR on formalin-
fixed, paraffin-embedded tissue samples was developed to
create a recurrence score and test its ability to predict CRC
recurrence in stage II tumors treated by surgery. The Oncotype
Dx Colon Cancer Test (Genomic Health) has been validated
in a large clinical trial (QUASAR) of primary CRC cases
© American Society for Clinical Pathology

treated with adjuvant chemotherapy.25 The validation study
showed the test to independently predict risk of recurrence
and disease-free and overall survival.26 In that study, multi-
variate analysis indicated that the recurrence score retained
prognostic significance (P = .008) independent of mismatch
repair, T stage, nodes examined, grade, and lymphovascular
invasion.26 The current format of this test is not designed to
predict the response of a specific therapy.
ColoPrint
By using a set of 38 prognosis-related gene probes on a
microarray platform, the ColoPrint (Agendia BV, Amsterdam,
the Netherlands) gene signature was validated on a cohort of
178 samples from patients with stage II and III colon cancer.
There were 61% low-risk and 39% high-risk cases. There
was a significant difference in distant metastasis-free survival
between low risk (89% disease free) and high risk (62% dis-
ease free) with a hazard ratio (HR) of 3.19 (P = 8.5e–4). The
performance of the profile was significant for untreated (P =
.0082) and treated patients (P = .016).27
Circulating Tumor Cells
A number of recently introduced technologies have
enabled the detection of circulating tumor cells (CTCs) in
patients with early-stage and advanced CRC.28 In a recent
meta-analysis of 36 published studies, the identification of
CTCs in peripheral blood was an independent predictor of
reduced recurrence-free (HR, 3.24) and overall (HR, 2.28)
survival.29 However, to date, the ability of CTC determina-
tions to guide therapy selection and the changing of therapy in
CRC has not been confirmed in prospective clinical trials.30
Other Prognostic Factors
Compiled from a series of reviews,31-36 ❚Table 2❚ lists a
series of prognostic biomarkers studied in patients with CRC.
Although a significant number of prognostic factors have
achieved independent prognostic significance for disease-free
and overall survival, no single routine or molecular test has
achieved widespread use in daily practice.37,38 To date, the
pathologic stage of disease based on primary resection speci-
men evaluation remains the cornerstone of prognosis assess-
ment and likely success of the primary therapy for CRC.39,40
Cytotoxic Chemotherapy for CRC: Biomarkers
for the Prediction of Efficacy and Toxicity
5-Fluorouracil and Capecitabine
5-Fluorouracil (5-FU) and its oral prodrug, capecitabine
(Xeloda) are the cornerstones of multiagent chemotherapy for
relapsed and metastatic CRC.10,11 These drugs are part of the
2 major regimens for CRC treatment: FOLFIRI (5-FU, folinic
© American Society for Clinical Pathology
Clinical Chemistry / Review Article
acid [Leucovorin], and irinotecan [Campostar]) and FOLFOX
(5-FU, folinic acid [Leucovorin], and oxaliplatin [Eloxatin]).
Three biomarkers have been studied widely as potential pre-
dictors of 5-FU-based chemotherapy response and potential
serious drug toxic effects.
Thymidylate Synthase
Treatment of CRC with 5-FU has long been associated
with wide variations in tumor response and drug toxicity.41,42
Direct inhibition of thymidylate synthase (TS, also abbrevi-
ated TYMS) is considered the major mechanism of 5-FU anti-
cancer activity. Although a series of early reports indicated
that increased TS expression was associated with resistance
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to 5-FU treatment,43 numerous recent studies designed to
measure TS protein expression by slide-based immuno-
histochemical analysis and mRNA expression by RT-PCR
as a predictor of 5-FU response in CRC have failed to
achieve consensus or widespread clinical adoption.40-42 Thus,
although the majority of studies have found that TS overex-
pression is an unfavorable prognostic and predictive factor for
5-FU–treated CRC, the test is not routinely used before the
selection of 5-FU–based therapies. Germline polymorphisms
in the TS gene seem to have a major role in the regulation of
TS expression, but this approach has focused more on predict-
ing 5-FU toxic effects than efficacy.42,43 TS polymorphisms
associated with low TS expression are consistently associated
with increased 5-FU drug toxic effects, including bone mar-
row suppression, mucositis, and diarrhea.
Dihydropyrimidine Dehydrogenase
Dihydropyrimidine dehydrogenase (DPD, also abbrevi-
ated DPYD) is an enzyme associated with the breakdown
of administered 5-FU. Compared with TS, studies of DPD
expression as a predictor of 5-FU efficacy are generally less
convincing.11,44,45 In certain patients with germline polymor-
phisms of the DPD gene, significant reduction in enzymatic
activity is present, resulting in severe toxic effects when 5-FU
is administered.11,44,45
Methylenetetrahydrofolate Reductase
Although a number of studies have linked a reduction in
folic acid pools associated with the presence of methylenetet-
rahydrofolate reductase (MTHFR) germline polymorphisms,
large studies have not confirmed the prognostic or predictive
value of MTHFR genotyping.46-48 MTHFR polymorphisms
have also been associated with capecitabine toxic effects.11
DNA Mismatch Repair Status
CRCs deficient in DNA mismatch repair (molecular class-
es 1 and 2 with high MSI [MSI-H] tumors) typically feature a
proximal anatomic location, mucinous features, lymphocytic
infiltration, and pushing margins. There is preclinical evidence
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❚Table 2❚
Prognostic Biomarkers for CRC
Biomarker Test Substrate Slide Based Test Type Prognostic Significance

Tumor type Surgical pathology Yes Morphology Limited; mucinous and signet-ring carcinomas may have
report worse prognosis
Tumor grade Surgical pathology Yes Morphology Very limited; vast majority of tumors are well to moderately
report differentiated
Tumor TNM stage Surgical pathology Yes Morphology High; pathologic stage at diagnosis the single most
report powerful prognostic factor for CRC
Tumor border Surgical pathology Yes Morphology Moderate; infiltrating vs round tumor border associated
report with worse prognosis
Presence of tumor Surgical pathology Yes Morphology Moderate; single-cell tumor budding at the tumor border
budding report has independent adverse impact in some studies
Microsatellite Tumor DNA No (MLH1, Genotyping, PCR, High MSI associated with favorable prognosis and
instability MSH2/6 IHC HPLC, capillary favorable response to chemotherapy
may predict electrophoresis
for MSI status)
CIN and Tumor DNA Yes Feulgen spectroscopy; Confirmed unfavorable prognostic factors for patients
aneuploidy cytogenetics with CRC

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18q deletion/LOH Tumor DNA Yes/yes Cytogenetics/SMAD4 Deletion of 18q and loss of SMAD4 expression linked
(DCC/Smad4) IHC to adverse prognosis in CRC in most but not all studies
p53 mutation Tumor DNA Yes/no IHC/gene sequencing Controversial (Failure to universally confirm p53
mutation as an unfavorable prognostic factor for CRC
may reflect on the suboptimal accuracy of using IHC
as a predictor of gene mutation status.)
Oncotype Dx Tumor mRNA No RT-PCR (FFPE Geneset in validation stage for predicting risk of relapse
Colon Cancer samples) for resected specimens
ColoPrint Tumor mRNA No Genomic microarray Geneset in validation phase for predicting risk of relapse
(fresh/frozen samples) for resected specimens
TILs Surgical pathology Yes IHC High TILs associated with high MSI and favorable
report prognosis
Ki-67 Surgical pathology Yes IHC High Ki-67 labeling index associated with high
report proliferation, poor differentiation, and adverse prognosis
p21 Tumor protein Yes IHC Loss of expression associated with cell cycle dysregulation
and adverse prognosis
p27 Tumor protein Yes IHC Loss of expression associated with cell cycle dysregulation
and adverse prognosis
PTEN deletion Tumor protein Yes IHC PTEN expression loss associated with activation of PIK3CA
pathway, resistance to apoptosis, and adverse disease outcome
KRAS mutation Tumor DNA No Genotyping Meta-analyses indicate KRAS mutation status not an
independent prognostic factor for CRC
BRAF mutation Tumor DNA No Genotyping BRAF V600E mutation linked to overall survival in low and
stable MSI tumors36
PIK3CA mutation Tumor DNA No Genotyping PIK3CA mutation associated with adverse prognosis37
EGFR Tumor DNA Yes FISH/CISH EGFR gene copy number, mutation status, or protein
expression not been universally confirmed as independent
prognostic factors for CRC
Tumor DNA No Genotyping
Tumor protein Yes IHC
HER2 Tumor protein Yes IHC HER2 not established as a prognostic factor or therapy
target for CRC
Tumor DNA Yes FISH/CISH
BCL2 Tumor protein Yes IHC Altered expression associated with clinical outcome in some
but not all studies
β-Catenin Tumor protein Yes IHC Altered expression associated with clinical outcome in some
but not all studies
E-cadherin Tumor protein Yes IHC Altered expression associated with clinical outcome in some
but not all studies
TGF-β Tumor protein Yes IHC Altered expression associated with clinical outcome in some
but not all studies
VEGF Tumor protein Yes IHC Altered expression associated with clinical outcome in some
but not all studies; VEGF ligand the target of bevacizumab-
based treatment for metastatic CRC
uPA Tumor protein No Tissue ELISA Independent predictor of survival in several major studies;
no established slide-based IHC assay available
MMPs/TIMPS Tumor protein Yes IHC Altered expression associated with clinical outcome in some
but not all studies
Telomerase Tumor DNA No/yes TRAP assay/hTERT Telomerase activation/expression an independent prognostic
assay factor for CRC in most studies
TS Germline DNA No Genotyping Overexpression appears to be controlled by genetic poly-
morphisms; overexpression associated with unfavorable
prognosis in most but not all CRC studies
Tumor mRNA No RT-PCR
Tumor protein Yes IHC
CTCs Whole cells Yes Immunomagnetic bead Presence of CTCs in CRC an independent predictor of
capture adverse outcome

CIN, chromosomal instability; CISH, chromogenic in situ hybridization; CRC, colorectal carcinoma; CTCs, circulating tumor cells; EGFR, epidermal growth factor receptor; ELISA,
enzyme linked immunosorbent assay; FFPE, formalin-fixed, paraffin-embedded; FISH, fluorescence in situ hybridization; HPLC, high-performance liquid chromatography;
hTERT, human telomere reverse transcriptase; IHC, immunohistochemical analysis; LOH, loss of heterozygosity; MMP, matrix metalloproteinase; mRNA, messenger RNA;
MSI, microsatellite instability; RT-PCR, real-time polymerase chain reaction; TGF, transforming growth factor; TILs, tumor infiltrating lymphocytes; TIMP, tissue inhibitor of
metalloproteinase; TRAP, telomere repeat amplification protocol; TS, thymidylate synthase; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor.

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that these tumors are resistant to 5-FU but are sensitive to
irinotecan and mitomycin C.49
In summary, the aforementioned genetic and molecular
tests designed to predict efficacy and toxic effects for 5-FU
and capecitabine have shown a high percentage of false-
negative results, possibly due to incomplete sequencing and
pathway analyses.50 In the future, a combination of more
thorough (“deep”) sequencing of germline and tumor cells
combined with gene expression profiling and analysis of
epigenetic events may ultimately lead to routine evaluation of
these biomarkers in the personalized selection of antineoplas-
tic therapy for CRC.
Irinotecan (CPT-11; Campostar)
In the FOLFIRI regimen, the topoisomerase I inhibi-
tor irinotecan is combined with the 5-FU/folinic acid regi-
men.51-53 Irinotecan is metabolized to its active form, SN-38,
which itself is conjugated and eliminated by the liver-
derived enzyme UDP-glucuronosyltransferase, also known as
UGT1A1. Several biomarkers have been proposed to predict
efficacy and toxic effects for irinotecan-based regimens.
Topoisomerase I
Measuring tumor cell topoisomerase I expression has
correlated with irinotecan response in several large stud-
ies,54,55 but this procedure is not currently performed as part
of the selection of therapy for CRC.
UGT1A1
Polymorphisms in the UGT1A1 gene have been strong-
ly linked to toxicity of irinotecan-based treatment for
CRC.11,51-53,56,57 One particular polymorphism, homozygosity
for the 7-repeat allele (also known as UGT1A1*28) is associat-
ed with severe diarrhea when irinotecan is administered.11,56,57
Although UGT1A1 genotyping is being performed on a regu-
lar basis in some facilities, widespread use has not occurred
owing to the presence of conflicting negative data58 and the
lack of current endorsement for testing by specialty societies
(eg, American Society of Clinical Oncology [ASCO] and
College of American Pathologists) or regulatory agencies.
Other Predictive Biomarkers
A variety of additional biomarkers have been proposed
to predict irinotecan efficacy,59,60 but these have predomi-
nantly featured retrospective analysis of patient cohorts, and
prospective, randomized studies of biomarker-driven selec-
tion or rejection of irinotecan for the treatment of recurrent or
metastatic CRC are lacking.
Oxaliplatin
Although long popular in Europe, oxaliplatin-based mul-
tiagent chemotherapy (FOLFOX) for CRC was introduced
© American Society for Clinical Pathology
Clinical Chemistry / Review Article
and became more widely adopted in the United States dur-
ing the last 10 years.61-63 Given that platin-based drugs are
DNA-damaging agents, the focus on discovery of predictive
biomarkers has been on genes and pathways associated with
DNA.64
Excision Repair Cross-Complementing C1
The DNA excision repair protein, excision repair cross-
complementing C1 (ERCC1), has been the focus of study
for the prediction of non–small cell lung cancer response
to the standard first-line platin-based drugs plus paclitaxel
chemotherapy regimen.65 Recent studies have also shown
that ERCC1 expression may also predict for resistance to
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FOLFOX treatment of CRC.66-68 However, there is no cur-
rent consensus as to the best way to test CRC specimens
for ERCC1 expression, with advocates for protein detection
by immunohistochemical analysis and mRNA detection by
RT-PCR. In addition, polymorphisms of the ERCC1 gene
have been associated with decreased expression of ERCC1
protein and improved survival after FOLFOX chemothera-
py.11,66 Given the established success in predicting resistance
to platin-based drugs in lung cancer and the initial results of
similar studies in CRC, it is possible the ERCC1 testing will,
at some point in the future, become a routine procedure for
patients with high-risk primary and metastatic CRC.
Excision Repair Cross-Complementing C2
Excision repair cross-complementing C2 (also known
as ERCC2 and XPD) is another nucleotide excision repair
enzyme that may be a predictive biomarker for CRC treatment
efficacy.69-71
Ribonucleotide Reductase M1
Ribonucleotide reductase M1 (RRM1) expression has
been linked to response of lung cancer to DNA-damaging
agents such as platin-based drugs and gemcitabine. In CRC,
only limited studies have been performed, and it is not cur-
rently known whether RRM1 expression can guide selection
of oxaliplatin-based therapy for the disease.72
X-Ray Repair Complementing Defective Repair in Chinese
Hamster Cells 1
X-ray repair complementing defective repair in Chinese
hamster cells 1 (XRCC1) is a DNA base repair enzyme
that has been linked to prognosis in CRC.66,69,73,74 XRCC1
polymorphisms have been associated with reduced XRCC1
expression and enhanced response to FOLFOX.74-76
Glutathione S-Transferase π 1
Glutathione S-transferase π 1 (GSTP1) is an enzyme
that facilitates glutathione conjugation and detoxification of a
number of drugs, including oxaliplatin.11 GSTP1 expression
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❚Table 3❚
Biomarkers Proposed to Predict Clinical Resistance to Cetuximab/Panitumumab Therapy

Incidence CAP Level of

Biomarker Detection Method Positive Result (%) Evidence Category*

EGFR IHC Membranous and cytoplasmic > 85 IV

staining
KRAS mutation Direct sequencing and pyrosequencing; Mutation in codons 12, 13, or 61 27-53 I (codons 12 and 13); IIA
PCR for mutant DNA in exons 2 and 3 (codons 61 and 146)
BRAF mutation Direct sequencing; PCR for mutant DNA V600E point mutation 8-13 IIA

PIK3CA mutation Direct sequencing and pyrosequencing; Point, missense, and frameshift 3-15 IIB
PCR for mutant DNA mutations
p53 mutation Direct sequencing and pyrosequencing; Point, missense, and frameshift 1-5 IIB
PCR for mutant DNA; Roche AmpliChip mutations.
IHC Overexpression of “mutant” protein 1-5 IIB

PTEN deletion IHC Loss of immunoreactivity in tissue 4-5 IIB

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sections
Direct sequencing for chromosomal Segmental gene deletion 4-5 IIB
deletion
Methylation-specific PCR Promoter gene silencing 4-5 IIB

Fcγ receptor Germline genotyping RII and RIII polymorphisms Unknown III
polymorphism

ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; EGFR, epidermal growth factor receptor; FDA, US Food and Drug Administration;
IHC, immunohistochemical analysis; NCCN, National Comprehensive Cancer Network; PCR, polymerase chain reaction.
* “Predicted” level of evidence as defined by the CAP.82 Categories are as follows: I, factors definitively proven based on evidence from multiple, statistically robust published

trials and generally used in patient management; IIA, factors extensively studied biologically and/or clinically and repeatedly shown to have prognostic value for outcome
and/or predictive value for therapy that is of sufficient import to be included in the pathology report but remains to be validated in statistically robust studies; IIB, factors
shown to be promising in multiple studies but lacking sufficient data for inclusion in category I or IIA; III, factors not yet sufficiently studied to determine their prognostic
value; IV, factors well studied and shown to have no prognostic significance.

has been linked to resistance to FOLFOX chemotherapy in Therapeutic Antibodies for CRC: Biomarkers
CRC in some studies,66,77 but not in others.78 Loss of GSTP1 for the Prediction of Efficacy and Toxicity
expression associated with germline polymorphisms has
frequently been associated with oxaliplatin-related adverse Anti–Epidermal Growth Factor Receptor Antibody
events, especially neurotoxicity.79 Drugs (Cetuximab and Panitumumab)

DNA Polymerase β In the last decade, regulatory agencies have approved

DNA polymerase β (POLB) is another gene associated
with the excision repair pathway. In a recent study, overex-
pression of POLB was an unfavorable prognostic factor for
CRC, but the authors concluded that the marker was suited to
predicting response only to cisplatin.80
In summary, a number of biomarkers have been estab-
lished as predictive factors for response of recurrent and meta-
static CRC to both of the competing FOLFIRI and FOLFOX
regimens. Although these tests are not currently recommended
for daily clinical use by regulatory authorities or specialty soci-
eties, there is great potential for a personalized approach to che-
motherapy selection for patients with CRC to ultimately reach
mainstream clinical status. In the adjuvant setting, current opin-
ion based on clinical trial results now holds that the FOLFOX
regimen is superior to the FOLFIRI regimen.81 However, for
an individual patient with high-risk primary CRC, there may
be germline and tumoral genomic information whose eventual
clinical validation can lead to a specific selection of agents,
rather than using the “one-size-fits-all” approach.
the use of 2 monoclonal antibodies, cetuximab (Erbitux) and
panitumumab (Vectibix) that target the human epidermal
growth factor receptor 1 (EGFR) for the treatment of EGFR-
overexpressing CRC.82,83 Both antibody therapeutics were
developed with an EGFR expression test (immunohistochem-
ical analysis) to confirm patient eligibility for treatment. More
recently, a panel of biomarkers has been proposed to direct
treatment with these agents ❚Table 3❚. These biomarkers are
evaluated in Table 3 for their level of evidence using College
of American Pathologists evaluation criteria.84
EGFR Expression, Amplification, and Mutation
Although a positive EGFR tumor cell immunostain is
required for use of cetuximab and panitumumab, EGFR
expression at the protein or mRNA levels has not correlated
with drug response.82,83 EGFR gene amplification mea-
sured by fluorescence in situ hybridization, in contrast, has
correlated with antibody treatment efficacy. EGFR somatic
gene mutation, a major predictor of efficacy of anti-EGFR

484 Am J Clin Pathol 2010;134:478-490 © American Society for Clinical Pathology

484 DOI: 10.1309/AJCP2Y8KTDPOAORH
 Clinical Chemistry / Review Article

patients with newly diagnosed CRC considered for anti-EGFR

Impact
No impact on drug response
Validated in numerous studies
and meta-analyses
Validated in several but not all
retrospective studies
Validated in several but not all
retrospective studies
Validated in several but not all
retrospective studies
Validated in several but not all
retrospective studies
Validated in several but not all
Regulatory Status
Required by FDA for patient
eligibility
Adopted by FDA, NCCN, and
ASCO
Nearing formal adoption
Under evaluation
Under evaluation
Under evaluation
Under evaluation
antibody therapeutics are first tested for KRAS mutation, and
only patients with wild-type KRAS gene tumors are considered
eligible to receive these drugs.
A number of issues remain concerning the widespread
use of KRAS mutation testing in CRC. Currently, the actual
mutation test has not been standardized, and fully validated
tests appear to be lacking in clinical practice. A variety of
approaches for the detection of KRAS mutation have been
used, including direct sequencing, high-resolution pyro-
sequencing, and amplification-refractory mutation detection
using PCR with a bifunctional self-probing primer.94,95 The
latter method has recently achieved the highest sensitivity

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retrospective studies
Validated in several but not all Under evaluation
retrospective studies
Validated in several but not all Under evaluation
retrospective studies
Not validated Under evaluation
tyrosine kinase small molecule drugs in non–small cell lung
cancer (gefitinib and erlotinib), has not successfully predicted
response to anti-EGFR antibodies.84
KRAS Mutation
During clinical development, it was noted that cetuximab
showed evidence of benefit in only 10% to 20% of patients with
metastatic CRC.85 Thus, to save cost and improve potential
patient outcomes, investigators sought novel biomarkers that
could predict resistance to anti-EGFR therapy for CRC. The
KRAS oncogene is a major component of the ras/raf pathway
known to regulate the cell cycle in normal cells and CRC cells.
Studies have indicated that KRAS mutation is found in between
27% and 53% of CRC cases.85-90 The vast majority of KRAS
mutations are located in the second and third exons in codons
12, 13, and 61. Mutated KRAS CRC, with few exceptions,
appears to be completely resistant to benefit from treatment by
cetuximab and panitumumab in the metastatic setting. Data in
support of this association appear fully confirmed for mutations
in codons 12 and 13.91-93 For codons 61 and 146, the evidence
for conferring resistance to cetuximab and panitumumab is not
as robust as for codons 12 and 13.93,94 In mid 2009, the ASCO
recommended that all patients with metastatic CRC who are
candidates for anti-EGFR antibody therapy should have their
tumors tested for KRAS mutations in a Clinical Laboratory
Improvement Amendments–accredited laboratory. If KRAS
mutations are identified in codons 12 or 13, ASCO recom-
mends that patients with metastatic CRC should not receive
anti-EGFR antibody therapy as part of their treatment. More
recently, the US Food and Drug Administration has incor-
porated this ASCO provisional opinion, and essentially all
and specificity in a direct comparison study.96 Currently,
there is no US Food and Drug Administration clearance or
approval for a KRAS mutation test. Depending on the nature
of the tissue block on which the KRAS mutation testing is
going to be performed and the inherent sensitivity of the
mutation detection assay to be used, it may be necessary to
perform some type of microdissection to eliminate nonma-
lignant areas so that the dilution of the KRAS mutation in
the cancer cells will not be missed. Other current issues con-
cerning KRAS mutation testing include whether frameshift
and missense mutations should also be detected and reported
and whether codon 61 should be sequenced to search for
mutations in addition to codons 12 and 13.
BRAF Mutation
The BRAF gene is located distal to the KRAS gene in
the ras/raf pathways. The V600E mutation in the BRAF gene
has been identified in up to 13% of CRCs.97-100 There is an
inverse relationship with KRAS mutation results, with the
V600E BRAF mutations seen only in KRAS wild-type tumors.
Patients with BRAF-mutated CRCs may be similarly resistant
to the therapeutic benefits of anti-EGFR antibody drugs, as
are patients with KRAS-mutated tumors.96,98,99 However, 1
recent study could only demonstrate BRAF mutation as a
general adverse prognostic factor and not a predictor of anti-
EGFR antibody efficacy.97 Thus, it is estimated that BRAF
testing, when widely incorporated into the workup of a newly
diagnosed CRC specimen, would identify an additional 8%
to 10% of cases that, although KRAS wild-type, are BRAF
mutated and, thus, similarly resistant to cetuximab/panitu-
mumab. Although BRAF inhibitors are not currently under
evaluation for the treatment of CRC, it is possible that BRAF
mutation testing will be needed to identify patients for this
type of targeted therapy approach.
PIK3CA Mutation
The PIK3CA gene is an important oncogene and occa-
sionally mutated in CRC. Some but not all preliminary stud-
ies suggest that CRCs with PIK3CA mutations are resistant

© American Society for Clinical Pathology Am J Clin Pathol 2010;134:478-490 485

485 DOI: 10.1309/AJCP2Y8KTDPOAORH 485
Ross et al / Predicting Response in Colorectal Cancer
to the anti-EGFR antibody drugs.99-104 Unlike BRAF muta-
tions, PIK3CA mutations are identified in KRAS-mutated and
KRAS wild-type CRC.99-104 It is also estimated that only 3%
to 10% of patients whose tumors are in the KRAS wild-type
group will have PIK3CA mutations, so the potential contri-
bution of PIK3CA+ mutation status to the group of CRCs
resistant to the cetuximab and panitumumab antibodies will
be quite small. Given the current conflicting data for this
predictive biomarker, it is not anticipated that in the short-
term future PIK3CA mutation testing will be performed in
routine clinical practice for determining eligibility for anti-
EGFR antibody therapy. However, PIK3CA mutation testing
might be performed for the purpose of identifying patients
as candidates to enroll in clinical trials testing the efficacy
of agents targeting the PIK3CA/molecular targets of the
rapamycin (mTOR) biologic pathway.
p53 Mutation
Although immunohistochemical analysis has been used
to predict p53 gene mutation status in colorectal and other
malignancies, significant concern about accuracy has led to
a reduction in the use of this approach. For the prediction of
resistance to the cetuximab and panitumumab drugs, immu-
nohistochemical analysis is not thought by most investigators
to be sensitive and specific enough to be used clinically.
Thus, a mutation sequencing–based test must be applied to
attempt to identify accurately the status of the p53 gene. It
is estimated that an additional 1% to 5% of KRAS wild-type
CRCs will have a p53 gene mutation. It is interesting that,
in contrast with some studies, KRAS wild-type p53-mutated
CRCs have been linked to positive response to cetuximab
therapy.105 However, data are limited for this biomarker,
and widespread adoption of p53 mutation testing in CRC is
not anticipated for the foreseeable future.
PTEN Expression
The PTEN tumor suppressor gene is an important cell
cycle regulator in CRCs. The loss of PTEN expression is seen
in approximately 40% of patients with CRC, mostly in the
KRAS-mutated group. However, approximately 5% of tumors
that are KRAS wild-type will have loss of PTEN expression
measured by immunohistochemical analysis. Recent studies
have shown that PTEN expression loss in KRAS wild-type
tumors was associated with cetuximab resistance.102,104,106
Further study of this biomarker will be needed to confirm its
role as a therapy selector for CRC.
Fcγ Receptor Status and Antibody-Dependent Cellular
Cytotoxicity Response
Antibody-dependent cellular cytotoxicity (ADCC) is
considered a major aspect of the mechanism of action of
monoclonal antibody therapeutics.106-108 Interactions with the
486 Am J Clin Pathol 2010;134:478-490
486 DOI: 10.1309/AJCP2Y8KTDPOAORH
Fc receptor may be a critical step in the activation of natural
killer lymphocytes and ADCC response. Preliminary studies
have linked germline polymorphisms and posttranslational
modifications (glycosylation and fucosylation) of the Fcγ
receptor with impaired ADCC response associated with
monoclonal antibody therapeutics such as cetuximab and
panitumumab.107-109 The clinical development of Fc recep-
tor assays to predict cetuximab and panitumumab resistance
will require validation of these retrospective observations in
prospective trials.
Bevacizumab (Avastin)
A variety of antiangiogenesis approaches have been
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evaluated for the treatment of metastatic CRC. The anti–
vascular endothelial growth factor (VEGF) ligand antibody
therapeutic, bevacizumab, is approved for first-line treatment
of metastatic CRC.110-112 Bevacizumab is routinely combined
with cytotoxic agents to take advantage of its antiangiogenesis
and vascular stabilization mechanisms of action. To date, no
biomarkers have emerged that are capable of predicting effi-
cacy for this agent.110-113
Novel Cytotoxic Agent and Antibody
Therapeutics in Clinical Trials
A wide variety of novel therapies in various stages of
clinical trial evaluation for the treatment of relapsed and
metastatic CRC have targeted mitogen-activated extracellular
kinase (MEK inhibitors), polyadenosine diphosphate–ribose
polymerase (PARP inhibitors), VEGF (VEGF inhibitors),
insulin-like growth factor 1 receptor (anti-IGF antibod-
ies), proteasome (bortezomib and other novel proteasome
inhibitors), mTOR (mTOR inhibitors), histone deacetylase
(HDAC inhibitors), and cyclooxygenase (COX 1 and 2 inhibi-
tors).113-115 As these studies progress, there will be keen inter-
est as to the role of biomarkers in the clinical development of
these agents.
Summary
The molecular classification of CRC is in its early
stages of development and refinement. Although interest-
ing associations have been discovered that have important
clinical ramifications such as the association of MSI-H
with Lynch syndrome (hereditary nonpolyposis colorectal
cancer), the day-to-day clinical usefulness for the molecular
classification has not, to date, been realized. In contrast, the
integration of new molecular diagnostic tests into the clini-
cal management of CRC has achieved significant recent
success associated with the use of KRAS oncogene mutation
© American Society for Clinical Pathology

testing to predict resistance to anti-EGFR antibody therapy.
Recent evidence strongly suggests that BRAF mutation test-
ing will soon achieve similar standard-of-practice useful-
ness. For the PIK3CA, p53, PTEN and Fcγ receptor tests,
more evidence will be required before they can also be
used as markers of cetuximab and panitumumab resistance.
Similarly, the markers of efficacy for 5-FU, irinotecan, and
oxaliplatin have not, to date, achieved standard-of-practice
usefulness. Clinical trials that use the various predictive
biomarkers for these drugs must still be conducted in a
randomized, prospective manner before they can become
universally accepted. The markers of drug toxic effects,
such as the UGT1A1 genotyping for irinotecan toxic effects,
also require further validation on a large scale before they
can achieve everyday use. However, it is entirely possible
that a number of these markers will become accepted and
widely used along with still yet to be developed biomarkers
all designed to personalize the clinical management of this
common life-threatening malignancy.
From the 1Department of Pathology and Laboratory Medicine,
Albany Medical College, Albany, NY; 2Foundation Medicine,
Cambridge, MA; and 3Dana Farber Cancer Institute and Harvard
Medical School, Boston, MA.
Address reprint requests to Dr Ross: Dept of Pathology, Mail
Code 81, Albany Medical College, 47 New Scotland Ave, Albany,
NY 12208.
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