REVIEW ARTICLE
Molecular Pathology of Colorectal Cancer
Shuko Harada, MD and Diana Morlote, MD
Abstract: Colorectal cancer (CRC) is the third most commonly
diagnosed cancer. This review gives an overview of the current
knowledge of molecular mechanisms of colorectal carcinogenesis and
the role of molecular testing in the management of CRC. The
majority of CRCs arise from precursor lesions such as adenoma,
transforming to adenocarcinoma. Three molecular carcinogenesis
pathways have been identified; (1) chromosomal instability, (2)
microsatellite instability (MSI), and (3) CpG island methylator phe-
notype, each account for ~85%, 15%, and 17%, respectively. Evalu-
ation of MSI status, extended RAS mutation analysis, and BRAF
mutation analysis are recommended by the guideline published by
joint effort from professional societies. MSI testing is important for
identification of Lynch syndrome patients and prognostic and pre-
dictive markers. Extended RAS testing is an important predictive
marker for antiepidermal growth factor receptor therapy. BRAF p.
V600 mutation status can be used as prognostic marker, but not
predictive marker for antiepidermal growth factor receptor therapies.
Emerging technologies utilizing high throughput sequencing have
introduced novel biomarkers and testing strategies. Tumor mutation
burden predicts immunotherapy response in addition to MSI status.
Liquid biopsy can be utilized when adequate tissue sample is not
available or for monitoring therapy response. However, assay stand-
ardization and guidelines and recommendations for utilization of
these assay will be needed. The advancement in CRC research and
technologies will allow better prognostication and therapy strat-
ification for the management of patients with CRCs.
Key Words: colorectal carcinoma, microsatellite instability (MSI),
extended RAS, BRAF mutation, Lynch syndrome
(Adv Anat Pathol 2020;27:20–26)
C olorectal cancer (CRC) is the third most commonly
diagnosed cancer and the third leading cause among all
cancer deaths in the United States, although the incidence has
been decreasing.1 Globally, CRC is the second and third
leading cause of cancer death in men and women, respectively.2
The vast majority of CRC develop sporadically, whereas <10%
of cases result from a hereditary cancer syndrome.3 The
majority of CRCs arise from precursor lesions such as ade-
noma, transforming to adenocarcinoma. Three molecular car-
cinogenesis pathways have been identified; (1) chromosomal
instability (CIN),4 (2) microsatellite instability (MSI),5,6 and (3)
CpG island methylator phenotype (CIMP).7 Prognosis is
determined by multiple factors including TNM stage, location
of the tumor, and histologic subtypes. In addition, there are
molecular biomarkers that predict prognosis and therapy
response. This review gives an overview of the current knowl-
edge of molecular mechanisms of colorectal carcinogenesis and
the role of molecular testing in the management of CRC.
From The University of Alabama at Birmingham, Birmingham, AL.
The authors have no funding or conflicts of interest to disclose.
Reprints: Shuko Harada, MD, Department of Pathology, The University
of Alabama at Birmingham, 1802 6th Avenue South, WP P210E,
Birmingham, AL 35249 (e-mail: sharada@uabmc.edu).
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
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MOLECULAR MECHANISMS OF COLORECTAL
CARCINOGENESIS
Chromosomal Instability
The CIN pathway, which accounts for ~85% of CRCs, is
characterized by alteration of the number and structure of
chromosomes, such as loss of chromosome 17p and 18q,
leading to aneuploidy (an abnormal chromosome number),
subkaryotypic amplification, chromosomal rearrangement,
and loss of heterozygosity at tumor suppressor gene loci.4,8 In
addition, CIN tumors accumulate mutations in oncogenes and
tumor suppressor genes including APC, TP53, KRAS, and
BRAF.4 According to the well-accepted Vogelstein’s model,
inactivation of APC occurs first in the hyperproliferative epi-
thelium, followed by oncogenic mutations, most commonly
KRAS, and eventually chromosomal alteration and inactiva-
tion of the tumor suppressor gene TP53.9 Typically, these
changes lead to the classic adenoma-carcinoma sequence. The
Cancer Genome Atlas Network (TCGA) analysis found gains
of 1q, 7p, 7q, 8p, 8q, 12q, 13q, 19q, 20p, and 20q and loss of
1p, 4q, 5q, 8q, 14q, 20p, and 22q in CIN tumors.10 The study
also found 24 genes significantly mutated; in addition to the
genes expected APC, TP53, SMAD4, PIK3CA and KRAS,
frequent mutations were found in ARID1A, SOX9, and
FAM123B.10
The most clinically relevant pathways involved in CIN
tumors are the Wnt and MAPK (mitogen-activating protein
kinase) pathways.11 The Wnt pathway is activated when the
Wnt protein binds to the Frizzled family receptor, which
removes Axin from the “destruction complex.” This leads to
β-catenin to accumulate in the nucleus and activates tran-
scription. When the signal is not activated, β-catenin is
degraded by APC-dependent destruction complex in the
cytoplasm. The Wnt signaling pathway was altered in 93%
of all tumors including biallelic inactivation of APC or
activation of CTNNB1 (β-catenin).10 These changes lead to
uncontrolled nuclear accumulation of β-catenin and cell
proliferation. MAPK pathway is activated by a receptor
tyrosine kinase. Activating mutation in one of the signaling
molecules involved in this pathway, such as RAS, RAF, or
ERK, leads to the signaling molecule becoming con-
stitutively active, results in uncontrolled cell proliferation.
Microsatellite Instability
MSI, which accounts for about 15% of CRCs, is
characterized by generalized instability of short tandemly-
repeated DNA sequences known as microsatellites.12 MSI
may result from either mutation of one of mismatch repair
(MMR) genes, MLH1, MSH2, MSH6, or PMS2, or
silencing of the MLH1 promoter by hypermethylation.
Normally, when 2 strands of DNA replicate and nucleotide
mismatch occur, these errors are corrected by a MMR
enzyme. However, defects in this function result in a high
frequency of replication errors because of the slippage of the
DNA polymerase.3,8 Lynch syndrome is the most common
hereditary colon cancer syndrome, which is characterized by
Adv Anat Pathol  Volume 27, Number 1, January 2020
MSI.5 Lynch syndrome is associated with germline muta-
tions in one of the MMR genes and increases lifetime risk of
CRC to about 80% with early onset.5 Sporadic MSI tumors
can occur because of methylation of CpG-rich promoter
sequence of MLH1. These cancers tend to arise in proximal
colon, tend to exhibit poor differentiation, mucinous cell
type, and prominent lymphocytic infiltration.3 MSI tumors
with hypermethylation account for three-quarters of
hypermutated CRCs, whereas a quarter had somatic MMR
gene mutation and polymerase ε mutations.10
CpG Island Methylator Phenotype
The CIMP pathway is characterized by widespread
hypermethylation of numerous promoter CpG island loci and
consequent inactivation of tumor suppressor genes.7 CIMP
tumors were initially identified in 30% to 35% of CRCs, but
DNA methylation analysis of CIMP-specific promoters
revealed that the CIMP pathway accounts for 17% of CRC.13
Although CIN and MSI pathways are usually exclusive,10 the
CIMP pathway overlaps substantially with the MSI pathway.
In fact, sporadic MSI CRCs are almost exclusively associated
with CIMP-associated methylation of the MLH1 promoter
region.14 CIMP-positive tumors are shown to represent a
distinct subset with high BRAF mutation.13,14 CIMP has a
strong association with the serrated neoplasia.15 CIMP
tumors tend to arise in the proximal colon, at an older age,
and are more common in female individuals.16
MICROSATELLITE INSTABILITY TESTING
MMR Deficiency by Immunohistochemistry (IHC)
Testing for MMR deficiency (dMMR) can be performed
by IHC for the 4 major MMR proteins (MLH1, MSH2, PMS2,
and MSH6) or by MSI polymerase chain reaction (PCR)-based
assay.17,18 Initial screening can be performed by either method-
ology depending on the availability. In general, IHC does not
require a special laboratory to perform and can be done at low
cost. In addition, the assay can help screen for a possible
defective gene. Therefore, MMR IHC is generally preferred.
However, limitations include the fact that not all pathogenic
mutations result in loss of expression and interpretation is
somewhat subjective.15 For the IHC assay, if all proteins are
expressed in the nucleus, the tumor is considered microsatellite
stable (MSS). Loss of 1 or 2 protein expression indicates MMR
deficiency, which highly correlates with MSI. As MLH1/PMS2
and MSH2/MSH6 form functional pairs and MLH1 and MSH2
are needed to stabilize the complex, when MLH1 or MSH2 are
lost, PMS2 or MSH6 are also lost (Table 1). The most common
MMR-deficient pattern is the loss of MLH1/PMS2 because of
TABLE 1. Interpretation of Mismatch Repair Protein Immunohistochemistry Test Results and Further Action
Nuclear Expression of Protein
MLH1 PMS2 MSH2 MSH6
+ + + +
− − + +
+ − + +
+ + − −
+ + + −
MSI indicates microsatellite instability; MSS, microsatellite stable.
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Molecular Pathology of Colorectal Cancer
MLH1 defect (Fig. 1). Only a small percentage of MLH1 loss
tumors are because of Lynch syndrome, that is, because of
germline MLH1 mutation, whereas the majority of them result
from silencing of MLH1 expression because of promoter
hypermethylation.19 Hypermethylation of the MLH1 promoter
is often a characteristic of CIMP tumors. These tumors often
harbor a BRAF p.V600E mutation. BRAF p.V600 mutational
analysis can be performed ahead of MLH1 promoter methyl-
ation analysis in tumors with MLH1 loss. The presence of a
BRAF mutation strongly favors sporadic pathogenesis.17 In
addition, BRAF mutation is associated with poor prognosis,
especially in CIMP-low tumors.13 The absence of a BRAF
mutation does not exclude sporadic etiology and requires pro-
moter methylation analysis to exclude a diagnosis of Lynch
syndrome. Loss of other proteins (MSH2, MSH6, or PMS2)
increases a probability for Lynch syndrome and genetic coun-
seling and germline gene sequencing is recommended (Table 1).
Germline mutations are typically nonsense or frameshift muta-
tions, which result in loss of function. A hit to the second allele
(second hit), as a result of either additional loss-of-function
mutation or loss of heterozygosity, silences the function of the
MMR gene and results in tumor formation.
Microsatellite Instability Testing by Polymerized
Chain Reaction
MSI can be determined by a PCR-based assay. MSI is
defined as alterations in the lengths of microsatellites, also
referred as short tandem repeats, because of deletion or inser-
tion of repeating units to produce novel-length alleles in tumor
DNA when compared with the normal/germline DNA from
the same individual.20 There are > 1 million microsatellite loci
throughout the genome.21 To standardize MSI analysis, the
reference panel, referred to as the Bethesda panel, for the
detection of MSI was proposed by a 1997 National Cancer
Institute (NCI) workshop and consists of 5 microsatellite
markers, 2 mononucleotide loci [big adenine tract (BAT)-25
and BAT-26) and 3 dinucleotide loci (D2S123, D5S346, and
D17S250).22 Commercial assay kits have become available
since then, including the one from Promega Corp. (Madison,
WI), which uses 5 nearly monomorphic mononucleotide
microsatellite loci (BAT-25, BAT-26, NR-21, NR-24, and
MONO-27).20 The performance of these assays is similar, but
mononucleotide loci are shown to be more sensitive and specific
than dinucleotide loci.23 An MSI-H (high) tumor is defined as
the one that shows shift of the size in ≥ 2 out of 5 microsatellite
loci, whereas a shift only at 1 locus is considered MSI-L (low).21
Emerging Technologies
Recently, cartridge-based automated PCR assay was
developed by Biocartis (Mechelen, BE) (Idylla MSI). The
Interpretation Action
MSS No further action
MSI, MLH1 loss MLH1 promoter heypermethylation analysis,
if no methylation, MLH1 mutation analysis,
and genetic counseling
MSI, PMS2 loss PMS2 mutation analysis and genetic counseling
MSI, MSH2 loss MSH2 mutation analysis and genetic counseling
MSI, MSH6 loss MSH6 mutation analysis and genetic counseling
www.anatomicpathology.com | 21
Harada and Morlote Adv Anat Pathol  Volume 27, Number 1, January 2020

FIGURE 1. Mismatch repair immunohistochemistry showing loss of MLH1 (B). PMS2 is also lost when MLH1 is lost (E). MSH2 and MSH6
are intact (C and D, respectively). Hematoxylin and eosin staining image is shown in (A). All 200× magnification. Note the presence of
internal control staining in lymphocytes and stromal cells (B and E).

performance has been shown to be comparable with the
conventional PCR-based assay with 93.6% agreement and
lower failure rate (4% vs. 11.9%).24 Although further vali-
dation by large number of the cases is warranted, these
automated tools may give small laboratories the ability to
perform MSI assays on cases with equivocal results by IHC.
Next-generation sequencing (NGS) can also be used for
determination of MSI status.25,26 Zhu et al25 showed MSI by
NGS achieved 98% sensitivity and 100% specificity for the
detection of MSI status as compared with the PCR assay.
Waalkes et al26 also demonstrated high sensitivity and specificity
for CRC (both 100%), whereas endometrial and prostate cancer
had false-negative cases. An advantage of using NGS is the
ability to analyze over 100 loci for instability, whereas the assay
requires special instrumentation and a bioinformatics pipeline.
Comparison of different techniques is summarized in Table 2.

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Adv Anat Pathol  Volume 27, Number 1, January 2020
TABLE 2. Comparison of Assay Technologies to Determine Mismatch Repair Gene Status23–25
MMR IHC MSI PCR
Description MLH1, MSH2, MSH6, 5 mononucleotide
PMS2 protein expression microsatellite loci, PCR,
determined by IHC and fragment analysis
Sensitivity 94% 83%-98%
Specificity 100% 100%
Pros No need of molecular Require small amount
laboratory; work on low of tumor; scalable;
tumor cellularity samples; objective interpretation
identify a defective gene
Cons Some mutations do not Need molecular lab; need Further validation study
result in expression loss; normal tissue; labor
subjective interpretation intensive
IHC indicates immunohistochemistry; MMR, mismatch repair; MSI, microsatellite instability; NGS, next-generation sequencing; PCR, polymerase chain
reaction.
Testing Algorithm
MSI/MMR testing was introduced initially to identify
Lynch syndrome patients. Bethesda guidelines were devel-
oped by the NCI to identify individuals who should be
tested for MSI.27,28 Those included age under 50 years,
strong family history, presence of synchronous or meta-
chronous colorectal or Lynch syndrome-associated tumors,
and CRC with MSI-H histology.28 However, 10 of 23 pro-
bands with Lynch syndrome were over 50 years of age and 5
did not meet the Amsterdam criteria or the Bethesda
guidelines.29 Therefore, universal screening of CRC for
Lynch syndrome has been recommended by many pro-
fessional organizations including the National Compre-
hensive Cancer Network (2014).30
In addition to identifying Lynch syndrome patients,
MSI testing can be used as a prognostic and predictive
marker. MSI tumors tend to be earlier stage and have lower
recurrence rates than MSS tumors.31 The evidence suggests
that MSI tumors do not benefit from fluorouracil-based
adjuvant chemotherapy in stage II or stage III colon
cancer.32 Recently, MSI status was recognized as a bio-
marker for immune checkpoint inhibitor therapy.33 This will
be discussed in the separate section.
PREDICTIVE MARKERS FOR TARGETED
THERAPIES
Extended RAS
Signaling pathway through the epidermal growth fac-
tor receptor (EGFR) plays an important role in CRC
(Fig. 2). Anti-EGFR monoclonal antibodies such as cetux-
imab and panitumumab have been shown to bind to the
extracellular domain of EGFR and block downstream sig-
naling, that is, RAS/MRAF/MEK/ERK pathway, and have
a significant clinical benefit in patients with metastatic
CRC.34 However, if a downstream signaling molecule is
mutated, the anti-EGFR antibody cannot block the path-
way, resulting in resistance. Activation mutations in the
KRAS gene are seen in 30% to 50% CRC. Five randomized
trials of cetuximab or panitumumab, when used as either
monotherapy or combination with chemotherapy, have
shown that patients with metastatic CRC may benefit from
these therapies. Both agents are approved by the US Food
and Drug Administration (FDA) for treatment of metastatic
CRC. Stratified analyses of data from these trials by KRAS
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Molecular Pathology of Colorectal Cancer
Automated MSI NGS
7 microsatellite loci, PCR, Over 100 microsatellite
analyzed by high-resolution loci, NGS analysis
melting detection
No data 98%
No data 100%
Short hands-on time; Ability to analyze many
fast turnaround time more loci, reduce
equivocal results
Expensive; need a special
is needed; difficult to instrument and
troubleshoot when failed bioinformatics
mutational status, not detected (“wild type”) or abnormal
(mutated), indicated that patients with KRAS mutation in
codon 12 or 13 did not derive benefit from treatment with
cetuximab or panitumumab.35 Therefore, the American
Society of Clinical Oncology (ASCO) published its clinical
opinion stating all patients with metastatic CRC who are
candidates for anti-EGFR antibody therapy should have
their tumor tested for KRAS mutations in a CLIA-certified
laboratory. If KRAS mutation in codon 12 or 13 is detected,
then patients should not receive anti-EGFR antibody ther-
apy as part of their treatment.35 In addition to codons 12
and 13 of KRAS, mutations in other codons of KRAS and in
NRAS are found to render tumors resistant to anti-EGFR
antibody therapy.36,37
Therefore, a recent guideline from the American Society
for Clinical Pathology, the College of American Pathologists,
the Association for Molecular Pathology, and the ASCO
recommends mutational testing for EGFR signaling pathway
genes as they provide clinically actionable information as
negative predictors of benefit to anti-EGFR monoclonal
antibody therapies for targeted therapy of CRC.17 The
guideline states that patients with colorectal carcinoma being
FIGURE 2. EGFR pathway. EGFR indicates epidermal growth
factor receptor; mTOR, mammalian target of rapamycin; P,
phosphorylation; PI3K, phosphatidylinositol 3 kinase.
www.anatomicpathology.com | 23
Harada and Morlote
considered for anti-EGFR therapy must receive RAS muta-
tional testing. Mutational analysis should include KRAS and
NRAS codons 12 and 13 of exon 2, 59, and 61 of exon 3, and
117 and 146 of exon 4 (“expanded” or “extended” RAS). In
addition, BRAF p.V600 [BRAF c.1799 (p.V600)] mutational
analysis should be performed in CRC tissue in patients with
colorectal carcinoma for prognostic stratification. However,
they state there is insufficient evidence to recommend BRAF
p.V600 mutational status as a predictive biomarker for
response to anti-EGFR inhibitors.17 Additional biomarkers
have been investigated for possible targeted therapies. How-
ever, currently there is insufficient evidence to recommend
PIK3CA or PTEN mutational analysis of colorectal carci-
noma tissue for therapy selection outside of a clinical trial.17
Testing Algorithm
Extended RAS and BRAF mutation analysis can be
performed by any technologies that can detect sequence
variations, that is, Sanger sequence, allele-specific real-time
PCR, pyrosequencing, or NGS. When the guideline rec-
ommended testing exons 2, 3, and 4 of KRAS and NRAS,
then single-gene assays became cumbersome and inefficient.
NGS-based targeted gene panels have become more efficient
and cost-effective. In fact, there is a significant increase in
laboratories using NGS for RAS testing from ∼10% in 2014
to 2015 to 23.1% in 2015 to 2016.38 In the same study, they
found that only a third to a quarter of participating labo-
ratories met the guideline recommendations for extended
RAS testing, suggesting a delay in implementation at the
time of survey38 although much improvement is expected by
now. NGS-based assays, however, require a specific instru-
ment, a complex workflow, a bioinformatics pipeline, higher
quality and quantity of DNA, and longer turnaround time.
Recently, a rapid and fully automated system has been
evaluated, and concordances between the Idylla NRAS-
BRAF or KRAS Mutation Test and reference test results
were found in almost perfect agreement.39–41 A study by
Franczak et al42 showed that the Idylla platform retrieved
49/67 (73.1%) samples that did not reach DNA quality
requirements for NGS. A caveat is an assay does not cover
some rare variants (eg, KRAS p.G12F, p.G13C) compared
with sequence-based assays. Initial quick screening for
extended RAS and BRAF status can be performed by an
automated system and large NGS-targeted panel can be per-
formed for patients with recurrent or resistant advanced-stage
FIGURE 3. Biomarker testing algorithm for colon cancer. IHC indicates immunohistochemistry; MSI, microsatellite instability; NGS, next-
generation sequencing; PCR, polymerase chain reaction.
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Adv Anat Pathol  Volume 27, Number 1, January 2020
cancer looking for additional targeted therapy or clinical trial
options. Overall, a molecular testing algorithm on the basis of
current NCCN guidelines (version 2.2019; www.nccn.org/
professionals/physician_gls/pdf/colon.pdf) and biomarker
guidelines17 is illustrated in Figure 3.
Immunotherapy
Immune checkpoint inhibitors are an emerging field for
oncology therapy. In 2015, patients with MSI tumors were
shown to benefit from immune checkpoint blockade with
pembrolizumab.33 Subsequently, the same group expanded
their study to various tumor types and showed the large pro-
portion of mutant neoantigens in MMR-deficient cancers make
them sensitive to immune checkpoint blockade, regardless of
the cancers’ tissue of origin.43 On the basis of this information,
the FDA-approved pembrolizumab for unresectable or meta-
static MSI-H solid tumors in 2017. In the same year, the FDA
also granted accelerated approval to Nibolumab for dMMR
and MSI-H metastatic CRC (http://www.fda.gov/drugs/drug-
approvals-and-databases).44 Another predictive marker for
immune checkpoint inhibitor therapy is tumor mutational
burden (TMB). A recent study suggests that TMB seems to be
an important independent biomarker within MSI-H mCRC to
stratify patients for likelihood of response to immune check-
point inhibitors.45 However, currently, there are no guidelines
for TMB estimation and reporting, which results in large var-
iability. Two organizations, Friends of Cancer Research
(Friends) and the Quality Assurance Initiative Pathology
(QuIP), initiated an effort to standardize the process and have
proposed evidence-based recommendations for achieving con-
sistent TMB estimation and reporting in clinical samples across
assays and centers.46
Recent preclinical and clinical studies suggest that the
checkpoint inhibitors can be expanded to microsatellite-
stable CRC in combinations with chemotherapy, molecular
targeted therapy, radiation, or novel immunomodulatory
agents.47 Many clinical trials are currently in progress,
which may open a door for new treatment options for
microsatellite-stable tumors. Duan et al48 demonstrated
effective immunotherapy of CRC through systemic delivery
of an immunostimulatory chemotherapeutic combination in
nanoscale coordination polymer core-shell particles, syner-
gizing with anti-PD-L1 antibody in murine model. As these
novel agents progress through the phases of clinical devel-
opment, novel predictive markers specific to these agents
Adv Anat Pathol  Volume 27, Number 1, January 2020
may become important to optimally select patients most
likely to benefit from them.47 Comprehensive analysis of
multiple markers to identify sensitive patients to immune
checkpoint blockade therapy and its standardization will be
needed.
Liquid Biopsy
Most tissue used in CRC biomarker testing is derived
from formalin-fixed paraffin-embedded (FFPE) tissue. How-
ever, adequate tumor cells may not be available in tissue block
for genomic profiling, especially in small biopsies in an
advanced-stage setting. Small biopsy samples may not be
representative when the tumor shows heterogeneity. In addi-
tion, repeat biopsies for recurrent or metastatic lesions or for
monitoring the response to the therapy are not practical. The
term “liquid biopsy” has been introduced to indicate the
sampling from nonsolid biological tissue, most commonly of
blood.49 Plasma cell-free DNA (cfDNA) or circulating tumor
DNA (ctDNA) has been widely accepted among oncologists
as a promising noninvasive biomarker for longitudinal mon-
itoring of a tumor. Recent prospective multicenter study of
patients with stage I to III CRC revealed that ctDNA-positive
patients at postoperative day 30 or after adjuvant chemo-
therapy were likely to relapse (7 times or 17 times, respec-
tively), and ctDNA status was independently associated with
relapse after adjusting for known clinicopathologic risk
factors.50 ctDNA analysis can potentially change the post-
operative management of CRC by enabling risk stratification,
adjuvant chemotherapy monitoring, and early relapse
detection.50 In a proper setting, ctDNA can be utilized as a
noninvasive biomarker to guide management of the patient.
However, there are disadvantages including high false-
positive and false-negative rates. Therefore, cfDNA is cur-
rently not generally recommended for initial disease diagnosis,
early detection, or screening. Detection of cfDNA mutations
(eg, TP53, KRAS, and APC) has limited diagnostic sensitivity
(40% to 60%).51 Testing for methylated DNA in gene such as
SEPT9, SFRP1, SDC2 has increased sensitivity (up to 90%)
for CRC.51 Improvements in technologies such as NGS and
digital droplet PCR are rapidly overcoming some of the issues
of analytical sensitivity, and it is likely that mutation and
methylation analysis of ctDNA will improve specificity for the
diagnosis of cancer in the future.52 Currently, there is no
standardization for the assay and preanalytical, analytical,
and postanalytical considerations were identified that need to
be addressed.52 Guidelines and recommendations for uti-
lization of ctDNA in clinical practice are needed.
CONCLUSIONS
This review discussed molecular mechanisms of color-
ectal carcinogenesis and current biomarker guidelines.
Current standard molecular workup includes evaluation of
MSI status and extended RAS and BRAF status. We also
discussed emerging technologies utilizing high throughput
sequencing for TMB and ctDNA. New consensus and
guidelines/recommendations are needed for these new tech-
nologies, and pathologists should be actively involved in the
process. The advancement in CRC research and tech-
nologies will allow better prognostication and therapy
stratification for the management of patients with CRCs.
Although not discussed in this review, early detection, and
prevention of CRC is an ultimate goal for precision medi-
cine and public health. Molecular pathologic epidemiology
(MEP) approach integrating genetic and environmental
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Molecular Pathology of Colorectal Cancer
information could provide etiological insights into tailored
strategies of disease prevention and treatment.53
REFERENCES
1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA
Cancer J Clin. 2018;68:7–30.
2. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer
statistics 2018: GLOBOCAN estimates of incidence and
mortality worldwide for 36 cancers in 185 countries. CA Cancer
J Clin. 2018;68:394–424.
3. Soreide K, Janssen EA, Soiland H, et al. Microsatellite
instability in colorectal cancer. Br J Surg. 2006;93:395–406.
4. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in
colorectal cancers. Nature. 1997;386:623–627.
5. Lynch HT, de la Chapelle A. Hereditary colorectal cancer.
N Engl J Med. 2003;348:919–932.
6. Gryfe R, Kim H, Hsieh ET, et al. Tumor microsatellite
instability and clinical outcome in young patients with color-
ectal cancer. N Engl J Med. 2000;342:69–77.
7. Toyota M, Ahuja N, Ohe-Toyota M, et al. CpG island
methylator phenotype in colorectal cancer. Proc Natl Acad Sci
U S A. 1999;96:8681–8686.
8. Markowitz SD, Bertagnolli MM. Molecular origins of cancer:
molecular basis of colorectal cancer. N Engl J Med. 2009;361:
2449–2460.
9. Fearon ER, Vogelstein B. A genetic model for colorectal
tumorigenesis. Cell. 1990;61:759–767.
10. The Cancer Genomic Atlas Network. Comprehensive molec-
ular characterization of human colon and rectal cancer. Nature.
2012;487:330–337.
11. Guinney J, Dienstmann R, Wang X, et al. The consensus
molecular subtypes of colorectal cancer. Nat Med. 2015;21:
1350–1356.
12. Aaltonen LA, Peltomaki P, Leach FS, et al. Clues to the
pathogenesis of familial colorectal cancer. Science. 1993;260:
812–816.
13. Ogino S, Nosho K, Kirkner GJ, et al. CpG island methylator
phenotype, microsatellite instability, BRAF mutation and
clinical outcome in colon cancer. Gut. 2009;58:90–96.
14. Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island
methylator phenotype underlies sporadic microsatellite insta-
bility and is tightly associated with BRAF mutation in
colorectal cancer. Nat Genet. 2006;38:787–793.
15. Fischer J, Walker LC, Robinson BA, et al. Clinical implications
of the genetics of sporadic colorectal cancer. ANZ J Surg. 2019.
Epub ahead of print.
16. Rhee YY, Kim KJ, Kang GH. CpG island methylator
phenotype-high colorectal cancers and their prognostic impli-
cations and relationships with the serrated neoplasia pathway.
Gut Liver. 2017;11:38–46.
17. Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular
biomarkers for the evaluation of colorectal cancer: Guideline
From the American Society for Clinical Pathology, College of
American Pathologists, Association for Molecular Pathology,
and American Society of Clinical Oncology. J Mol Diagn.
2017;19:187–225.
18. Funkhouser WK Jr, Lubin IM, Monzon FA, et al. Relevance,
pathogenesis, and testing algorithm for mismatch repair-
defective colorectal carcinomas: a report of the association for
molecular pathology. J Mol Diagn. 2012;14:91–103.
19. Boland CR, Goel A. Microsatellite instability in colorectal
cancer. Gastroenterology. 2010;138:2073.e3–2087.e3.
20. Murphy KM, Zhang S, Geiger T, et al. Comparison of the
microsatellite instability analysis system and the Bethesda panel
for the determination of microsatellite instability in colorectal
cancers. J Mol Diagn. 2006;8:305–311.
21. Ellegren H. Microsatellites: simple sequences with complex
evolution. Nat Rev Genet. 2004;5:435–445.
22. Boland CR, Thibodeau SN, Hamilton SR, et al. A National
Cancer Institute Workshop on Microsatellite Instability for
cancer detection and familial predisposition: development of
www.anatomicpathology.com | 25
Harada and Morlote
international criteria for the determination of microsatellite
instability in colorectal cancer. Cancer Res. 1998;58:5248–5257.
23. Zhang L. Immunohistochemistry versus microsatellite insta-
bility testing for screening colorectal cancer patients at risk for
hereditary nonpolyposis colorectal cancer syndrome: part II.
The utility of microsatellite instability testing. J Mol Diag.
2008;10:301–307.
24. Maertens G, De Craene B, Rondelez E, et al. Detection of
microsatellite instability (MSI) in colorectal cancer samples with
the automated Idylla MSI test. Ann Oncol. 2017;28(suppl 5):
v22–v42.
25. Zhu L, Huang Y, Fang X, et al. A novel and reliable method to
detect microsatellite instability in colorectal cancer by next-
generation sequencing. J Mol Diagn. 2018;20:225–231.
26. Waalkes A, Smith N, Penewit K, et al. Accurate pan-cancer
molecular diagnosis of microsatellite instability by single-
molecule molecular inversion probe capture and high-through-
put sequencing. Clin Chem. 2018;64:950–958.
27. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al.
A National Cancer Institute Workshop on Hereditary Non-
polyposis Colorectal Cancer Syndrome: meeting highlights
and Bethesda guidelines. J Natl Cancer Inst. 1997;89:
1758–1762.
28. Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda
Guidelines for hereditary nonpolyposis colorectal cancer
(Lynch syndrome) and microsatellite instability. J Natl Cancer
Inst. 2004;96:261–268.
29. Hampel H, Frankel WL, Martin E, et al. Screening for the
Lynch syndrome (hereditary nonpolyposis colorectal cancer). N
Engl J Med. 2005;352:1851–1860.
30. Hampel H. NCCN increases the emphasis on genetic/familial
high-risk assessment in colorectal cancer. J Natl Compr Cancer
Netw. 2014;12(suppl 5):829–831.
31. Malesci A, Laghi L, Bianchi P, et al. Reduced likelihood of
metastases in patients with microsatellite-unstable colorectal
cancer. Clin Cancer Res. 2007;13:3831–3839.
32. Ribic CM, Sargent DJ, Moore MJ, et al. Tumor microsatellite-
instability status as a predictor of benefit from fluorouracil-
based adjuvant chemotherapy for colon cancer. N Engl J Med.
2003;349:247–257.
33. Le DT, Uram JN, Wang H, et al. PD-1 blockade in tumors
with mismatch-repair deficiency. N Engl J Med. 2015;372:
2509–2520.
34. Cunningham D, Humblet Y, Siena S, et al. Cetuximab
monotherapy and cetuximab plus irinotecan in irinotecan-
refractory metastatic colorectal cancer. N Engl J Med. 2004;351:
337–345.
35. Allegra CJ, Jessup JM, Somerfield MR, et al. American Society
of Clinical Oncology provisional clinical opinion: testing for
KRAS gene mutations in patients with metastatic colorectal
carcinoma to predict response to anti-epidermal growth factor
receptor monoclonal antibody therapy. J Clin Oncol. 2009;27:
2091–2096.
36. Douillard JY, Rong A, Sidhu R. RAS mutations in colorectal
cancer. N Engl J Med. 2013;369:2159–2160.
37. Douillard JY, Oliner KS, Siena S, et al. Panitumumab-
FOLFOX4 treatment and RAS mutations in colorectal cancer.
N Engl J Med. 2013;369:1023–1034.
26 | www.anatomicpathology.com Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
Copyright r 2019 Wolters Kluwer Health, Inc. All rights reserved.
Adv Anat Pathol  Volume 27, Number 1, January 2020
38. Richman SD, Fairley J, Butler R, et al. How close are we to
standardised extended RAS gene mutation testing? The UK
NEQAS evaluation. J Clin Pathol. 2017;70:58–62.
39. Zekri J, Baghdadi MA, Alardati H, et al. Evaluation of the
Idylla KRAS and NRAS mutation test in colorectal cancer
tissue. Exp Mol Pathol. 2019;110:104270.
40. Prieto-Potin I, Montagut C, Bellosillo B, et al. Multicenter
evaluation of the Idylla NRAS-BRAF mutation test in
metastatic colorectal cancer. J Mol Diagn. 2018;20:664–676.
41. Solassol J, Vendrell J, Markl B, et al. Multi-center evaluation of
the fully automated PCR-based Idylla KRAS mutation assay
for rapid kras mutation status determination on formalin-fixed
paraffin-embedded tissue of human colorectal cancer. PloS
One. 2016;11:e0163444.
42. Franczak C, Dubouis L, Gilson P, et al. Integrated routine
workflow using next-generation sequencing and a fully-auto-
mated platform for the detection of KRAS, NRAS and BRAF
mutations in formalin-fixed paraffin embedded samples with
poor DNA quality in patients with colorectal carcinoma. PloS
One. 2019;14:e0212801.
43. Le DT, Durham JN, Smith KN, et al. Mismatch repair
deficiency predicts response of solid tumors to PD-1 blockade.
Science. 2017;357:409–413.
44. Zhao P, Li L, Jiang X, et al. Mismatch repair deficiency/
microsatellite instability-high as a predictor for anti-PD-1/PD-
L1 immunotherapy efficacy. J Hematol Oncol. 2019;12:54.
45. Schrock AB, Ouyang C, Sandhu J, et al. Tumor mutational
burden is predictive of response to immune checkpoint inhibitors
in MSI-high metastatic colorectal cancer. Ann Oncol. 2019. Epub
ahead of print.
46. Stenzinger A, Allen JD, Maas J, et al. Tumor mutational
burden standardization initiatives: recommendations for con-
sistent tumor mutational burden assessment in clinical samples
to guide immunotherapy treatment decisions. Genes Chromo-
somes Cancer. 2019;58:578–588.
47. Hermel DJ, Sigal D. The emerging role of checkpoint inhibition in
microsatellite stable colorectal cancer. J Pers Med. 2019:9:pii:E5.
48. Duan X, Chan C, Han W, et al. Immunostimulatory nano-
medicines synergize with checkpoint blockade immunotherapy
to eradicate colorectal tumors. Nat Commun. 2019;10:1899.
49. Castro-Giner F, Gkountela S, Donato C, et al. Cancer
diagnosis using a liquid biopsy: challenges and expectations.
Diagnostics (Basel). 2018:8:pii:E31.
50. Reinert T, Henriksen TV, Christensen E, et al. Analysis of
plasma cell-free DNA by ultradeep sequencing in patients with
stages I to III colorectal cancer. JAMA Oncol. 2019. Epub ahead
of print.
51. Molnar B, Galamb O, Kalmar A, et al. Circulating cell-free
nucleic acids as biomarkers in colorectal cancer screening and
diagnosis—an update. Expert Rev Mol Diagn. 2019;19:477–498.
52. Cree IA, Uttley L, Buckley Woods H, et al. The evidence base
for circulating tumour DNA blood-based biomarkers for the
early detection of cancer: a systematic mapping review. BMC
Cancer. 2017;17:697.
53. Hamada T, Nowak JA, Milner DA Jr, et al. Integration of
microbiology, molecular pathology, and epidemiology: a new
paradigm to explore the pathogenesis of microbiome-driven
neoplasms. J Pathol. 2019;247:615–628.