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

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DOI: 10.1309/AJCP2Y8KTDPOAORH

Colorectal cancer (CRC) is the second leading cause of

Abstract cancer mortality in the United States.1 There are 160,000 new
The diagnosis and management of colorectal cases of CRC diagnosed each year and 57,000 CRC-related
cancer (CRC) has been impacted by the discovery and deaths in the United States.1 Despite a slow decline during the
validation of a wide variety of biomarkers designed to last 20 years, for men, the age-adjusted incidence is 61.2 cases
facilitate a personalized approach for the treatment per 100,000 and for women, it is 44.8 per 100,000.1
of the disease. Recently, CRC has been reclassified Pathologists have traditionally had a major role in the
based on molecular analyses of various genes and care of patients with newly diagnosed CRC in the initial
proteins capable of separating morphologic types of establishment of the diagnosis of invasive disease and in
tumors into molecular categories. At the same time, the morphologic classification and final staging of surgi-
a number of new prognostic and predictive single cally resected specimens.2,3 In the last 30 years, there has
genes and proteins have been discovered that are been significant advancement in the understanding of the
designed to reflect sensitivity and/or resistance to molecular origins of CRC and characteristics of tumor aggres-
existing therapies. Multigene predictors have also siveness.4-7 This major expansion of genomic and proteomic
been developed to predict the risk of relapse for data has simultaneously contributed to the discovery of novel
intermediate-stage CRC after completion of surgical targeted therapies for patients with CRC in whom recurrent
extirpation. More recently, a number of biomarkers and metastatic disease have developed.8-10 Moreover, the use
tested by a variety of methods have been proposed as of gene sequencing, expression profiling, and other molecular
specific predictors of chemotherapy and radiotherapy techniques has introduced putative biomarkers that have been
response. Other markers have been successfully used reported to predict the clinical response and toxic effects for
to predict toxic effects of standard therapies. In this the nontargeted traditional antineoplastic drugs that have been
review, a series of novel biomarkers are considered used to the treat the disease.11 These new techniques have led
and compared with standard-of-care markers to the discovery of a number of biologically interesting genes
for their potential use as pharmacogenomic and and pathways associated with CRC development and progres-
pharmacogenetic predictors of disease outcome. 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

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

478 DOI: 10.1309/AJCP2Y8KTDPOAORH
 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.

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479 DOI: 10.1309/AJCP2Y8KTDPOAORH 479
Ross et al / Predicting Response in Colorectal Cancer

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❚Image 2❚ Molecular class 1 colorectal cancer (CRC) with ❚Image 3❚ Molecular class 2 colorectal cancer (CRC)
high microsatellite instability (MSI-H) and high CpG island with high microsatellite instability and low/0 CpG island
hypermethylation. This image is taken from a poorly hypermethylation. Low-power magnification of a well-
differentiated CRC arising in the proximal colon in an elderly differentiated CRC with mucinous features and an intense
woman. Note the high nuclear grade and intratumoral peritumoral lymphocytic reaction arising in the proximal
colon of an elderly woman. This group includes hereditary
lymphocytic infiltrates. In other regions, signet-ring cell
nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome
histologic features and mucinous differentiation were noted.
cases, but approximately 50% of tumors in this group are
This case of sporadic MSI-H CRC has been termed medullary
sporadic and unrelated to HNPCC/Lynch syndrome.12-15
carcinoma in some studies and typically features a favorable Although these lesions also arise in the proximal colon,
prognosis (H&E, ×200). 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).
Prediction of Response to Primary Treatment
for CRC

Pathologic Staging lymph nodes in sentinel lymph node–negative CRC resec-

The staging of resected CRC by pathologists remains tions. In addition, the introduction of molecular detection of
the cornerstone for the prediction of future disease relapse micrometastatic deposits of CRC using real time–polymerase
and progression.2 After many decades of use of the staging chain reaction (RT-PCR) in sentinel lymph nodes has also
method of Dukes et al, the current TNM staging system has not achieved widespread clinical use.21,22 Finally, a variety of
now achieved near universal use.2,17 Recent attention has molecular assays have been developed to assess the status of
focused on understaging of disease caused by the evaluation CRC surgical resection margins, but this approach has been
of an insufficient number of retrieved lymph nodes from the limited to research studies.23
resected pericolorectal mesentery or perirectal adventitia.18
The minimum number of lymph nodes recovered for accurate Oncotype Dx Colon Cancer Test
CRC TNM staging has varied from 10 to 18.18,19 The Oncotype Dx test for breast cancer (Genomic Health,
Redwood City, CA) is now widely used to plan therapy for
Molecular Staging early-stage disease.24 A similar Oncotype Dx mRNA expres-
Although it has achieved standard of practice for breast sion profile of 12 target genes using RT-PCR on formalin-
cancer and significant use in selected cases of malignant mela- fixed, paraffin-embedded tissue samples was developed to
noma, the sentinel lymph node biopsy has not, to date, been create a recurrence score and test its ability to predict CRC
widely applied to CRC surgery.20 This generally reflects the recurrence in stage II tumors treated by surgery. The Oncotype
widely held opinion that, unlike breast cancer, there appears Dx Colon Cancer Test (Genomic Health) has been validated
to be no clinical benefit to the sparing of regional draining in a large clinical trial (QUASAR) of primary CRC cases

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

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 Clinical Chemistry / Review Article

treated with adjuvant chemotherapy.25 The validation study acid [Leucovorin], and irinotecan [Campostar]) and FOLFOX
showed the test to independently predict risk of recurrence (5-FU, folinic acid [Leucovorin], and oxaliplatin [Eloxatin]).
and disease-free and overall survival.26 In that study, multi- Three biomarkers have been studied widely as potential pre-
variate analysis indicated that the recurrence score retained dictors of 5-FU-based chemotherapy response and potential
prognostic significance (P = .008) independent of mismatch serious drug toxic effects.
repair, T stage, nodes examined, grade, and lymphovascular
invasion.26 The current format of this test is not designed to Thymidylate Synthase
predict the response of a specific therapy. Treatment of CRC with 5-FU has long been associated
with wide variations in tumor response and drug toxicity.41,42
ColoPrint Direct inhibition of thymidylate synthase (TS, also abbrevi-
By using a set of 38 prognosis-related gene probes on a ated TYMS) is considered the major mechanism of 5-FU anti-
microarray platform, the ColoPrint (Agendia BV, Amsterdam, cancer activity. Although a series of early reports indicated
the Netherlands) gene signature was validated on a cohort of that increased TS expression was associated with resistance

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178 samples from patients with stage II and III colon cancer. to 5-FU treatment,43 numerous recent studies designed to
There were 61% low-risk and 39% high-risk cases. There measure TS protein expression by slide-based immuno-
was a significant difference in distant metastasis-free survival histochemical analysis and mRNA expression by RT-PCR
between low risk (89% disease free) and high risk (62% dis- as a predictor of 5-FU response in CRC have failed to
ease free) with a hazard ratio (HR) of 3.19 (P = 8.5e–4). The achieve consensus or widespread clinical adoption.40-42 Thus,
performance of the profile was significant for untreated (P = although the majority of studies have found that TS overex-
.0082) and treated patients (P = .016).27 pression is an unfavorable prognostic and predictive factor for
5-FU–treated CRC, the test is not routinely used before the
Circulating Tumor Cells selection of 5-FU–based therapies. Germline polymorphisms
A number of recently introduced technologies have in the TS gene seem to have a major role in the regulation of
enabled the detection of circulating tumor cells (CTCs) in TS expression, but this approach has focused more on predict-
patients with early-stage and advanced CRC.28 In a recent ing 5-FU toxic effects than efficacy.42,43 TS polymorphisms
meta-analysis of 36 published studies, the identification of associated with low TS expression are consistently associated
CTCs in peripheral blood was an independent predictor of with increased 5-FU drug toxic effects, including bone mar-
reduced recurrence-free (HR, 3.24) and overall (HR, 2.28) row suppression, mucositis, and diarrhea.
survival.29 However, to date, the ability of CTC determina-
tions to guide therapy selection and the changing of therapy in Dihydropyrimidine Dehydrogenase
CRC has not been confirmed in prospective clinical trials.30 Dihydropyrimidine dehydrogenase (DPD, also abbrevi-
ated DPYD) is an enzyme associated with the breakdown
Other Prognostic Factors of administered 5-FU. Compared with TS, studies of DPD
Compiled from a series of reviews,31-36 ❚Table 2❚ lists a expression as a predictor of 5-FU efficacy are generally less
series of prognostic biomarkers studied in patients with CRC. convincing.11,44,45 In certain patients with germline polymor-
Although a significant number of prognostic factors have phisms of the DPD gene, significant reduction in enzymatic
achieved independent prognostic significance for disease-free activity is present, resulting in severe toxic effects when 5-FU
and overall survival, no single routine or molecular test has is administered.11,44,45
achieved widespread use in daily practice.37,38 To date, the
pathologic stage of disease based on primary resection speci- Methylenetetrahydrofolate Reductase
men evaluation remains the cornerstone of prognosis assess- Although a number of studies have linked a reduction in
ment and likely success of the primary therapy for CRC.39,40 folic acid pools associated with the presence of methylenetet-
rahydrofolate reductase (MTHFR) germline polymorphisms,
large studies have not confirmed the prognostic or predictive
Cytotoxic Chemotherapy for CRC: Biomarkers value of MTHFR genotyping.46-48 MTHFR polymorphisms
for the Prediction of Efficacy and Toxicity have also been associated with capecitabine toxic effects.11
5-Fluorouracil and Capecitabine
DNA Mismatch Repair Status
5-Fluorouracil (5-FU) and its oral prodrug, capecitabine CRCs deficient in DNA mismatch repair (molecular class-
(Xeloda) are the cornerstones of multiagent chemotherapy for es 1 and 2 with high MSI [MSI-H] tumors) typically feature a
relapsed and metastatic CRC.10,11 These drugs are part of the proximal anatomic location, mucinous features, lymphocytic
2 major regimens for CRC treatment: FOLFIRI (5-FU, folinic infiltration, and pushing margins. There is preclinical evidence

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Ross et al / Predicting Response in Colorectal Cancer

❚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.

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

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that these tumors are resistant to 5-FU but are sensitive to and became more widely adopted in the United States dur-
irinotecan and mitomycin C.49 ing the last 10 years.61-63 Given that platin-based drugs are
In summary, the aforementioned genetic and molecular DNA-damaging agents, the focus on discovery of predictive
tests designed to predict efficacy and toxic effects for 5-FU biomarkers has been on genes and pathways associated with
and capecitabine have shown a high percentage of false- DNA.64
negative results, possibly due to incomplete sequencing and
pathway analyses.50 In the future, a combination of more Excision Repair Cross-Complementing C1
thorough (“deep”) sequencing of germline and tumor cells The DNA excision repair protein, excision repair cross-
combined with gene expression profiling and analysis of complementing C1 (ERCC1), has been the focus of study
epigenetic events may ultimately lead to routine evaluation of for the prediction of non–small cell lung cancer response
these biomarkers in the personalized selection of antineoplas- to the standard first-line platin-based drugs plus paclitaxel
tic therapy for CRC. chemotherapy regimen.65 Recent studies have also shown
that ERCC1 expression may also predict for resistance to

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Irinotecan (CPT-11; Campostar) FOLFOX treatment of CRC.66-68 However, there is no cur-
In the FOLFIRI regimen, the topoisomerase I inhibi- rent consensus as to the best way to test CRC specimens
tor irinotecan is combined with the 5-FU/folinic acid regi- for ERCC1 expression, with advocates for protein detection
men.51-53 Irinotecan is metabolized to its active form, SN-38, by immunohistochemical analysis and mRNA detection by
which itself is conjugated and eliminated by the liver- RT-PCR. In addition, polymorphisms of the ERCC1 gene
derived enzyme UDP-glucuronosyltransferase, also known as have been associated with decreased expression of ERCC1
UGT1A1. Several biomarkers have been proposed to predict protein and improved survival after FOLFOX chemothera-
efficacy and toxic effects for irinotecan-based regimens. py.11,66 Given the established success in predicting resistance
to platin-based drugs in lung cancer and the initial results of
Topoisomerase I similar studies in CRC, it is possible the ERCC1 testing will,
Measuring tumor cell topoisomerase I expression has at some point in the future, become a routine procedure for
correlated with irinotecan response in several large stud- patients with high-risk primary and metastatic CRC.
ies,54,55 but this procedure is not currently performed as part
of the selection of therapy for CRC. Excision Repair Cross-Complementing C2
Excision repair cross-complementing C2 (also known
UGT1A1 as ERCC2 and XPD) is another nucleotide excision repair
Polymorphisms in the UGT1A1 gene have been strong- enzyme that may be a predictive biomarker for CRC treatment
ly linked to toxicity of irinotecan-based treatment for efficacy.69-71
CRC.11,51-53,56,57 One particular polymorphism, homozygosity
for the 7-repeat allele (also known as UGT1A1*28) is associat- Ribonucleotide Reductase M1
ed with severe diarrhea when irinotecan is administered.11,56,57 Ribonucleotide reductase M1 (RRM1) expression has
Although UGT1A1 genotyping is being performed on a regu- been linked to response of lung cancer to DNA-damaging
lar basis in some facilities, widespread use has not occurred agents such as platin-based drugs and gemcitabine. In CRC,
owing to the presence of conflicting negative data58 and the only limited studies have been performed, and it is not cur-
lack of current endorsement for testing by specialty societies rently known whether RRM1 expression can guide selection
(eg, American Society of Clinical Oncology [ASCO] and of oxaliplatin-based therapy for the disease.72
College of American Pathologists) or regulatory agencies.
X-Ray Repair Complementing Defective Repair in Chinese
Other Predictive Biomarkers Hamster Cells 1
A variety of additional biomarkers have been proposed X-ray repair complementing defective repair in Chinese
to predict irinotecan efficacy,59,60 but these have predomi- hamster cells 1 (XRCC1) is a DNA base repair enzyme
nantly featured retrospective analysis of patient cohorts, and that has been linked to prognosis in CRC.66,69,73,74 XRCC1
prospective, randomized studies of biomarker-driven selec- polymorphisms have been associated with reduced XRCC1
tion or rejection of irinotecan for the treatment of recurrent or expression and enhanced response to FOLFOX.74-76
metastatic CRC are lacking.
Glutathione S-Transferase π 1
Oxaliplatin Glutathione S-transferase π 1 (GSTP1) is an enzyme
Although long popular in Europe, oxaliplatin-based mul- that facilitates glutathione conjugation and detoxification of a
tiagent chemotherapy (FOLFOX) for CRC was introduced number of drugs, including oxaliplatin.11 GSTP1 expression

© American Society for Clinical Pathology Am J Clin Pathol 2010;134:478-490 483

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

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

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retrospective studies and specificity in a direct comparison study.96 Currently,
Validated in several but not all Under evaluation there is no US Food and Drug Administration clearance or
retrospective studies
Validated in several but not all Under evaluation approval for a KRAS mutation test. Depending on the nature
retrospective studies of the tissue block on which the KRAS mutation testing is
Not validated Under evaluation
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
tyrosine kinase small molecule drugs in non–small cell lung the cancer cells will not be missed. Other current issues con-
cancer (gefitinib and erlotinib), has not successfully predicted cerning KRAS mutation testing include whether frameshift
response to anti-EGFR antibodies.84 and missense mutations should also be detected and reported
and whether codon 61 should be sequenced to search for
KRAS Mutation mutations in addition to codons 12 and 13.
During clinical development, it was noted that cetuximab
showed evidence of benefit in only 10% to 20% of patients with BRAF Mutation
metastatic CRC.85 Thus, to save cost and improve potential The BRAF gene is located distal to the KRAS gene in
patient outcomes, investigators sought novel biomarkers that the ras/raf pathways. The V600E mutation in the BRAF gene
could predict resistance to anti-EGFR therapy for CRC. The has been identified in up to 13% of CRCs.97-100 There is an
KRAS oncogene is a major component of the ras/raf pathway inverse relationship with KRAS mutation results, with the
known to regulate the cell cycle in normal cells and CRC cells. V600E BRAF mutations seen only in KRAS wild-type tumors.
Studies have indicated that KRAS mutation is found in between Patients with BRAF-mutated CRCs may be similarly resistant
27% and 53% of CRC cases.85-90 The vast majority of KRAS to the therapeutic benefits of anti-EGFR antibody drugs, as
mutations are located in the second and third exons in codons are patients with KRAS-mutated tumors.96,98,99 However, 1
12, 13, and 61. Mutated KRAS CRC, with few exceptions, recent study could only demonstrate BRAF mutation as a
appears to be completely resistant to benefit from treatment by general adverse prognostic factor and not a predictor of anti-
cetuximab and panitumumab in the metastatic setting. Data in EGFR antibody efficacy.97 Thus, it is estimated that BRAF
support of this association appear fully confirmed for mutations testing, when widely incorporated into the workup of a newly
in codons 12 and 13.91-93 For codons 61 and 146, the evidence diagnosed CRC specimen, would identify an additional 8%
for conferring resistance to cetuximab and panitumumab is not to 10% of cases that, although KRAS wild-type, are BRAF
as robust as for codons 12 and 13.93,94 In mid 2009, the ASCO mutated and, thus, similarly resistant to cetuximab/panitu-
recommended that all patients with metastatic CRC who are mumab. Although BRAF inhibitors are not currently under
candidates for anti-EGFR antibody therapy should have their evaluation for the treatment of CRC, it is possible that BRAF
tumors tested for KRAS mutations in a Clinical Laboratory mutation testing will be needed to identify patients for this
Improvement Amendments–accredited laboratory. If KRAS type of targeted therapy approach.
mutations are identified in codons 12 or 13, ASCO recom-
mends that patients with metastatic CRC should not receive PIK3CA Mutation
anti-EGFR antibody therapy as part of their treatment. More The PIK3CA gene is an important oncogene and occa-
recently, the US Food and Drug Administration has incor- sionally mutated in CRC. Some but not all preliminary stud-
porated this ASCO provisional opinion, and essentially all 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- Fc receptor may be a critical step in the activation of natural
tions, PIK3CA mutations are identified in KRAS-mutated and killer lymphocytes and ADCC response. Preliminary studies
KRAS wild-type CRC.99-104 It is also estimated that only 3% have linked germline polymorphisms and posttranslational
to 10% of patients whose tumors are in the KRAS wild-type modifications (glycosylation and fucosylation) of the Fcγ
group will have PIK3CA mutations, so the potential contri- receptor with impaired ADCC response associated with
bution of PIK3CA+ mutation status to the group of CRCs monoclonal antibody therapeutics such as cetuximab and
resistant to the cetuximab and panitumumab antibodies will panitumumab.107-109 The clinical development of Fc recep-
be quite small. Given the current conflicting data for this tor assays to predict cetuximab and panitumumab resistance
predictive biomarker, it is not anticipated that in the short- will require validation of these retrospective observations in
term future PIK3CA mutation testing will be performed in prospective trials.
routine clinical practice for determining eligibility for anti-
EGFR antibody therapy. However, PIK3CA mutation testing Bevacizumab (Avastin)
might be performed for the purpose of identifying patients A variety of antiangiogenesis approaches have been

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as candidates to enroll in clinical trials testing the efficacy evaluated for the treatment of metastatic CRC. The anti–
of agents targeting the PIK3CA/molecular targets of the vascular endothelial growth factor (VEGF) ligand antibody
rapamycin (mTOR) biologic pathway. therapeutic, bevacizumab, is approved for first-line treatment
of metastatic CRC.110-112 Bevacizumab is routinely combined
p53 Mutation with cytotoxic agents to take advantage of its antiangiogenesis
Although immunohistochemical analysis has been used and vascular stabilization mechanisms of action. To date, no
to predict p53 gene mutation status in colorectal and other biomarkers have emerged that are capable of predicting effi-
malignancies, significant concern about accuracy has led to cacy for this agent.110-113
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
Novel Cytotoxic Agent and Antibody
to be sensitive and specific enough to be used clinically.
Therapeutics in Clinical Trials
Thus, a mutation sequencing–based test must be applied to
attempt to identify accurately the status of the p53 gene. It A wide variety of novel therapies in various stages of
is estimated that an additional 1% to 5% of KRAS wild-type clinical trial evaluation for the treatment of relapsed and
CRCs will have a p53 gene mutation. It is interesting that, metastatic CRC have targeted mitogen-activated extracellular
in contrast with some studies, KRAS wild-type p53-mutated kinase (MEK inhibitors), polyadenosine diphosphate–ribose
CRCs have been linked to positive response to cetuximab polymerase (PARP inhibitors), VEGF (VEGF inhibitors),
therapy.105 However, data are limited for this biomarker, insulin-like growth factor 1 receptor (anti-IGF antibod-
and widespread adoption of p53 mutation testing in CRC is ies), proteasome (bortezomib and other novel proteasome
not anticipated for the foreseeable future. inhibitors), mTOR (mTOR inhibitors), histone deacetylase
(HDAC inhibitors), and cyclooxygenase (COX 1 and 2 inhibi-
PTEN Expression tors).113-115 As these studies progress, there will be keen inter-
The PTEN tumor suppressor gene is an important cell est as to the role of biomarkers in the clinical development of
cycle regulator in CRCs. The loss of PTEN expression is seen these agents.
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
Summary
measured by immunohistochemical analysis. Recent studies
have shown that PTEN expression loss in KRAS wild-type The molecular classification of CRC is in its early
tumors was associated with cetuximab resistance.102,104,106 stages of development and refinement. Although interest-
Further study of this biomarker will be needed to confirm its ing associations have been discovered that have important
role as a therapy selector for CRC. clinical ramifications such as the association of MSI-H
with Lynch syndrome (hereditary nonpolyposis colorectal
Fcγ Receptor Status and Antibody-Dependent Cellular cancer), the day-to-day clinical usefulness for the molecular
Cytotoxicity Response classification has not, to date, been realized. In contrast, the
Antibody-dependent cellular cytotoxicity (ADCC) is integration of new molecular diagnostic tests into the clini-
considered a major aspect of the mechanism of action of cal management of CRC has achieved significant recent
monoclonal antibody therapeutics.106-108 Interactions with the success associated with the use of KRAS oncogene mutation

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

486 DOI: 10.1309/AJCP2Y8KTDPOAORH
 Clinical Chemistry / Review Article

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more evidence will be required before they can also be Rev Cancer. 2009;9:489-499.
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490 DOI: 10.1309/AJCP2Y8KTDPOAORH