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Review Article
Colorectal carcinoma: Pathologic aspects
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California, Los Angeles, California, USA
*Equal contributions |
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Abstract
Colorectal carcinoma is one of the most common cancers and one of the leading causes of cancerrelated
death in the United States. Pathologic examination of biopsy, polypectomy and resection specimens
is crucial to appropriate patient managemnt, prognosis assessment and family counseling. Molecular testing
plays an increasingly important role in the era of personalized medicine. This review article focuses on the
histopathology and molecular pathology of colorectal carcinoma and its precursor lesions, with an emphasis
on their clinical relevance.
Key words
Colorectal carcinoma; pathology; adenoma; molecular; MSI; KRAS; BRAF
Submitted Apr 16, 2012. Accepted for publication Apr 23, 2012.
DOI: 10.3978/j.issn.2078-6891.2012.030 | ||||||||||||||||||||||||||||||||||||||||
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Introduction
Colorectal carcinoma is the third most common cancer in
the United States after prostate and lung/bronchus cancers
in men and after breast and lung/bronchus cancers in
women. It is also the third leading cause of cancer-related
death in the United States after lung/bronchus and prostate
cancers in men and after lung/bronchus and breast cancers
in women (1). In 2011, an estimated 141,210 new cases of
colorectal carcinoma were diagnosed in United States, with
an estimated 49,380 deaths, representing approximately 9%
of all newly diagnosed cancers and all cancer-related deaths
(excluding basal and squamous cell skin cancers).
With the rapid therapeutic advancement in the era
of personalized medicine, the role of pathologists in
the management of patients with colorectal carcinoma
has greatly expanded from traditional morphologists to
clinical consultants for gastroenterologists, colorectal
surgeons, oncologists and medical geneticists. In addition to
providing accurate histopathologic diagnosis, pathologists
are responsible for accurately assessing pathologic staging,
analyzing surgical margins, searching for prognistic
parameters that are not included in the staging such as
lymphovascular and perineural invasion, and assessing
therapeutic effect in patients who have received neoadjavant
therapy. Pathologists also play a central role in analyzing
histologic features of the tumors that are suggestive of
microsatellite instability (MSI), selecting appropriate tissue
sections for MSI testing and mutation analysis for KRAS
and BRAF, and interpreting the results of these important
therapeutic and prognostic tests (2).
This review article focuses on the histolopathology of
colorectal carcinoma and its precursor lesions. Recent
advances in molecular pathology and molecular tests are
discussed. Their clinical relevance is emphasized. | ||||||||||||||||||||||||||||||||||||||||
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Histopathologic diagnosis of colorectal carcinoma
More than 90% of colorectal carcinomas are adenocarcinomas
originating from epithelial cells of the colorectal mucosa
(3). Other rare types of colorectal carcinomas include
neuroendocrine, squamous cell, adenosquamous, spindle
cell and undifferentiated carcinomas. Conventional
adenocarcinoma is characterized by glandular formation,
which is the basis for histologic tumor grading. In well
differentiated adenocarcinoma >95% of the tumor is
gland forming. Moderately differentiated adenocarcinoma
shows 50-95% gland formation. Poorly differentiated
adenocarcinoma is mostly solid with <50% gland formation.
In practice, most colorectal adenocarcinomas (~70%) are
diagnosed as moderately differentiated (Figure 1). Well and
poorly differentiated carcinomas account for 10% and 20%,
respectively.
Figure 1 An example of moderately differentiated adenocarcinoma
showing complicated glandular structures in a desmoplastic stroma
(original magnification ×200)
It is apparent that the determination of tumor grade
is a subjective exercise. Many studies have demonstrated that a 2-tiered grading system, which combines well
and moderately differentiated to low grade (50% gland
formation) and defines poorly differentiated as high grade
(<50% gland formation), reduces interobserver variation
and improves prognostic significance (4,5). Though
controversial, tumor grade is generally considered as a
stage-independent prognostic variable, and high grade
or poorly differentiated histology is associated with poor
patient survival (6-8). It should be emphasized, however,
that histologic grading should apply only to conventional
adenocarcinoma. Some of the histologic variants, which will
be discussed later, may show high grade morphology but
behave as low grade tumors because of their MSI status.
The vast majority of colorectal carcinomas are initially
diagnosed by endoscopic biopsy or polypectomy. The key
aspect of microscopic examination is to look for evidence
of invasion. However, this can be difficult when the biopsy
is superficial or poorly oriented. If the muscularis mucosae
can be identified, it is important to determine whether
it is disrupted by neoplastic cells. Invasive carcinoma
typically invades through the muscularis mucosae into the
submucosa, and is sometimes seen in close proximity to
submucosal blood vessels. Another important feature of
invasion is the presence of desmoplasia or desmoplastic
reaction (Figure 2), a type of fibrous proliferation
surrounding tumor cells secondary to invasive tumor
growth. Invasive colorectal carcinoma also frequently shows
characteristic necrotic debris in glandular lumina, so-called
“dirty necrosis” (Figure 3). This unique feature can be quite
useful to suggest a colorectal primary when a metastasis of
unknown origin is encountered.
Figure 2 Desmoplastic reaction characterized by proliferation of
spindle cells surrounding an adenocarcinomatous gland (original
magnification ×400)
Figure 3 Necrotic debris (“dirty necrosis”) within the lumina of
adenocarcinomatous glands (original magnification ×400)
It should be noted that when a diagnosis of invasive
carcinoma is rendered, it means that carcinoma has at
least invaded into the submucosa of the colorectum. This
differs from the concept of invasion in other parts of
the gastrointestinal tract (esophagus, stomach and small
intestine), where the presence of mucosal invasion is
sufficient for the diagnosis of invasive carcinoma (pT1).
In the colorectum, submucosal invasion is required for
the diagnosis of a pT1 tumor. For reasons that are not
entirely clear but generally thought to be due to the
relative paucity of lymphatics, invasion confined to the
lamina propria and muscularis mucosae has no risk of
nodal or distant metastasis. Thus, intramucosal carcinoma
is preferably called high grade dysplasia (discussed later)
by pathologists in order to avoid unnecessary surgical intervention. In the American Joint Committee on Cancer
(AJCC) Cancer Staging Manual (9), mucosal invasion
is classified as carcinoma in situ (Tis). Nevertheless, the
term of intramucosal carcinoma may still be used by some
pathologists. No matter what term is used by pathologists,
the identification of high grade dysplasia or intramucosal
carcinoma in a biopsy or polypectomy specimen should
not affect the decision-making for patient management.
The decision to perform surgical resection should be
ultimately determined by the gross appearance of the
lesion, endoscopic ultrasound findings, and endoscopic
resectability. | ||||||||||||||||||||||||||||||||||||||||
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Histologic variants
In World Health Organization (WHO) classification, a
number of histologic variants of colorectal carcinomas
are listed, such as mucinous, signet ring cell, medullary,
micropapillary, serrated, cribriform comedo-type,
adenosquamous, spindle cell, and undifferentiated. Only the
first 3 variants are discussed here.
Mucinous adenocarcinoma
This special type of colorectal carcinoma is defined by
>50% of the tumor volume composed of extracellular
mucin (3). Tumors with a significant mucinous component
(>10%) but <50% are usually termed adenocarcinoma
with mucinous features or mucinous differentiation.
Mucinous adenocarcinoma typically shows large glandular
structures with pools of extracellular mucin (Figure 4).
A variable number of individual tumor cells, including
signet ring cells, may be seen. The prognosis of mucinous
adenocarcinoma in comparison with conventional
adenocarcinoma has been controversial among different
studies (10,11). Many mucinous adenocarcinomas occur
in patients with hereditary nonpolyposis colorectal cancer
(HNPCC or Lynch syndrome) and thus represent highlevel
MSI (MSI-H) tumors (12). These tumors are expected
to behave in a low grade fashion. In contrast, mucinous
adenocarcinomas that are microsatellite stable (MSS) are
expected to behave more aggressively, particularly when
detected at an advanced stage.
Figure 4 Mucinous adenocarcinoma showing abundant
extracellular mucin (original magnification ×200)
Signet ring cell adenocarcinoma
In contrast to that in the stomach, signet ring cell
adenocarcinoma is rare in the colorectum, representing
<1% of all colorectal carcinomas. Similar to mucinous
carcinoma, signet ring cell carcinoma is defined by the
presence of >50% of tumor cells showing signet ring cell
features characterized by a prominent intracytoplasmic
mucin vacuole that pushes the nucleus to the periphery
(Figure 5). Signet ring cells may show an infiltrative growth
pattern or are present within the pools of extracellular
mucin. By definition, signet ring cell carcinoma is poorly
differentiated (high grade) and carries a worse outcome than
conventional adenocarcinoma (11,13,14). However, some
signet ring cell carcinomas may be MSI-H tumors and thus
may behave as low grade tumors biologically (3).
Figure 5 Signet ring cell carcinoma (original magnification ×400)
Medullary carcinoma
Medullary carcinoma is extremely rare, constituting approximately 5-8 cases for every 10,000 colorectal cancers
diagnosed, with a mean annual incidence of 3.47 (±0.75)
per 10 million population (15). This tumor is characterized
by sheets of epithelioid neoplastic cells with large vesicular
nuclei, prominent nucleoli, and abundant cytoplasm. It
typically has a pushing border on resection specimens (Figure 6),
and is characteristically associated with marked tumorinfiltrating
lymphocytes (Figure 7). Medullary carcinoma is a
distinctive histologic subtype that is strongly associated with
MSI-H (16,17). It usually has a favorable prognosis despite
its poorly differentiated or undifferentiated histology.
Figure 6 Medullary carcinoma showing a pushing border at the
tumor edge (original magnification ×40)
Figure 7 Medullary carcinoma showing poorly differentiated
histology and tumor-infiltrating lymphocytes (original
magnification ×40
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Immunohistochemical phenotype
The most widely used immunohistochemical markers
for colorectal adenocarcinoma are cytokeratin (CK) 20,
CK7 and CDX2. The most common immunophenotype
of colorectal adenocarcinoma is positivity for CK20 and
negativity for CK7, which is a relatively specific staining
pattern for colorectal origin (18). However, up to 20% of
the tumors may exhibit a CK7-positive/CK20 negative
or CK7-negative/CK20-negative staining pattern. It has
been suggested that reduced or absent CK20 expression
in colorectal carcinoma is associated with MSI-H (19).
CDX2 is a marker of enteric differentiation and is positive
in >90% of colorectal adenocarcinomas (20,21). However,
CDX2 can be positive in any carcinoma that shows enteric
differentiation, and thus is not entirely colorectal-specific.
Interestingly, medullary carcinomas of the colorectum are
frequently CK20-negative and CDX2-negative, in line with
the concept of MSI (16,19). | ||||||||||||||||||||||||||||||||||||||||
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Pathologic staging
Tumor staging is by far the most important prognostic
predictor of clinical outcome for patients with colorectal
carcinoma. Histologic examination of surgically resected
specimens serves an irreplaceable role in determining
the depth of tumor invasion (T) and the extent of nodal
metastasis (N). The histologic determination of T1 (tumor
invades submucosa), T2 (tumor invades muscularis propria)
and T3 (tumor invades through the muscularis propria
into pericolorectal tissues) is usually straightforward
when using the AJCC TNM staging system (9). However,
determination of T4a (tumor penetrates to the surface of
the visceral peritoneum) and T4b (tumor directly invades
or is adherent to other organs or structures) can sometimes
be problematic. First, serosal surface (visceral peritoneum)
involvement can be missed if the specimen is not adequately
sampled for histologic examination. Second, the serosal
surface may be confused with the circumferential (radial)
or mesenteric margin, which is a nonperitonealized
surface created surgically by blunt or sharp dissection. A
T3 tumor may involve the radial margin and a T4 tumor
may have a negative radial margin. Third, a surgically
induced perforation at the tumor site may be confused
with true tumor perforation, which requires clarification
from surgeons. Fourth, adherence of other organs or
structures at the tumor site does not necessarily qualify
for T4b. Histologically, the adherent site may show only
inflammatory changes, abscess formation and/or fibrosis,
but without direct tumor involvement. Finally, there is
some confusion about the definition of visceral peritoneum
involvement. Clearly, the interpretation of T4a can be unequivocal if, (I) tumor cells are present at the serosal
surface with inflammatory reaction, mesothelial hyperplasia,
and/or erosion; or (II) free tumor cells are seen on the
serosal surface with underlying ulceration of the visceral
peritoneum. However, identification of tumor cells close
to, but not at, the serosal surface would be considered T4a
by some investigators if there are associated mesothelial
inflammatory and/or hyperplastic reactions (Figure 8) (22).
Apparently, the application of this third criterion is prone
to subjective judgment and lacks reproducibility. It is noted
that in the updated cancer protocols and checklists by
College of American Pathologists (CAP), only the first two
criteria are listed as the diagnostic features of T4a, and the
third criterion is deleted (23).
Figure 8 Tumor cells close to, but not at, the serosal surface, with
mesothelial inflammatory and hyperplastic reactions, which may be
considered T4a by some investigators (original magnification ×40)
It is pathologists’ obligation to retrieve as many
lymph nodes as possible from surgical specimens. The
vast majority of pathologists follow the guidelines of a
minimum of 12 nodes (24). Extra efforts will be made if
<12 nodes are retrieved, although this will increase the
turnaround time for pathology reports. The extra efforts
may include repeated manual searches, submitting more
sections, utilizing fat clearance techniques (25,26), or ex
vivo injection of methylene blue (27,28). The application of
fat clearance techniques has several potential disadvantages,
such as further delay in signout of the pathology reports,
cost, toxicity and disposal of clearing solutions, and
unknown effect on immunohistochemistry. As a result, fat
clearance has not become a standard practice in pathology
laboratories. Methylene blue injection is a relatively new
method for colorectal cancer. There have been only a few
publications in this area, mostly from the same study group
(27,28). Its clinical application needs further investigation.
It should be realized that the total number of nodes
retrieved is not only dissector-dependent, but also
influenced by a number of specimen and patient variables.
Studies have shown a positive correlation with the
specimen length, pericolorectal fat width, female gender
and tumor size; and a negative correlation with the age of
patient and the rectosigmoid location of tumors (29,30).
Not surprisingly, fewer than 12 nodes may be expected if
patients have received preoperative neoadjuvant therapies
(31,32). It is recommended that pathologists document the
degree of diligence of their efforts to find lymph nodes in a
specimen in pathology reports, if <12 nodes are retrieved.
One of the interesting issues in nodal staging is the
interpretation of discrete tumor deposits in pericolorectal
fat away from the main tumor but without identifiable
residual lymph node tissue. In AJCC Cancer Staging
Manual 5th edition, a tumor nodule >3 mm was counted
as a positive node, whereas a nodule ≤3 mm was classified
in the category of discontinuous extension (T3). In the 6th
edition, tumor deposits were considered as positive nodes
if they are round and have a smooth contour irrespective
of size, but classified in the T category as well as venous
invasion if they are irregular in shape. The current edition
(7th edition) recognizes the fact that tumor deposits may
represent discontinuous extension, venous invasion with
extravascular spread, or truly totally replaced lymph
nodes. Given their association with reduced disease-free
and overall survival (33,34), these tumor deposits are now
considered nodal metastasis, irrespective of size or contour,
and are designated N1c in the absence of regional lymph
node metastasis to favor additional postoperative treatment.
However, if a single positive lymph node is also identified,
the N stage will be changed from N1c to N1a. The
presence of discontinuous tumor deposits does not change
the T stage in the 7th edition (9).
The prognostic significance of isolated tumor
cells (ITCs), defined as single tumor cells or small
clusters of tumor cells ≤0.2 mm, detected by either
immunohistochemical staining or standard hematoxylin
and eosin staining in regional lymph nodes remains unclear
at present. In the absence of overt nodal metastasis, ITCs
are classified as N0 but annotated as N0 (i+) with “i”
standing for “isolated tumor cells”. On the other hand,
micrometastasis (>0.2 mm but ≤2.0 mm) is reported as
N1(mic). The number of lymph nodes involved by ITCs or
micrometastasis should be stated (9,23). | ||||||||||||||||||||||||||||||||||||||||
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Pathology reporting
Most pathologists use standardized synoptic report for
colorectal carcinoma following the checklist recommended by CAP (23). The details that should be included in
the report are specimen type, tumor site, tumor size,
macroscopic tumor perforation, histologic type, histologic
grade, microscopic tumor extension, margins (proximal,
distal and radial), treatment effect (for tumors treated with
neoadjuvant therapy), lymphovascular invasion, perineural
invasion, tumor deposits (discontinuous extramural
extension), TNM staging (including the total number of
lymph nodes examined and the total number of nodes
involved). Some pathology reports may also include leading
edge of the tumor (infiltrative or expansile), presence or
absence of tumor budding, and assessment of histologic
features that are suggestive of MSI such as tumorinfiltrating
lymphocytes, peritumoral Crohn-like lymphoid
response and the percentage of mucinous component. | ||||||||||||||||||||||||||||||||||||||||
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Specimen handling and sampling
In pathology laboratories, surgically resected specimens are
processed in a systematic manner to ensure completeness
and accuracy of pathology report. The external surface
of the specimen is inspected before opening for possible
serosal involvement, radial margin involvement, tumor
perforation, and distant tumor implants. For rectal
resections, the intactness of the mesorectum is examined.
Once the specimen is oriented and the specimen is
measured, the radial margin around tumor is inked. The
specimen is then opened, usually along the antimesenteric
border with an attempt to avoid cutting through the tumor.
The location and size of the tumor and its distance from the
closest margin are recorded. Small portions of fresh tumor
and nonneoplastic tissues may be procured for tissue bank,
but this should not compromise the quantity of tumor for
diagnosis.
The opened and cleaned specimen is pinned down on a
wax board and immersed in an adequate volume of formalin
for fixation overnight. The tumor is then sliced at 3-4 mm
intervals to assess the depth of invasion. The rest of the
specimen is also examined for additional lesions. Adequate
sections of the tumor (usually 5 sections depending on the
size of the tumor) should be submitted for microscopic
examination to include the area of deepest invasion and to
maximize the chance to find lymphovascular and perineural
invasion. Including both tumor and adjacent uninvolved
colorectum in the same sections is desirable because there
is always a possibility that the case may be used for research
in the future. Additional sections should include proximal
and distal margins, radial margin (if not included in tumor
sections), any additional polyps or lesions, and random
uninvolved colorectum.
After taking the above sections, the mesenteric fat
or pericolorectal soft tissue is stripped off and dissected
for lymph nodes. All grossly negative lymph nodes are
entirely submitted for microscopic examination. Grossly
positive lymph nodes may be submitted in part or entirely
depending on their size.
A polypectomy specimen is inked at the cauterized base,
but the stalk may retract and thus be difficult to identify.
The specimen is either bisected or serially sectioned
depending on its size, and entirely submitted. Sectioning
should follow the vertical plane of the stalk to maximize
the histologic evaluation of polypectomy margin and
submucosal involvement. If the specimen is received in
multiple pieces, however, margin evaluation may become
impossible. | ||||||||||||||||||||||||||||||||||||||||
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Precursor lesions
It has been well established that the vast majority of colorectal
adenocarcinomas derive from precursor lesions such as
adenomas and dysplasia. Residual adenoma is a common
finding in colorectal adenocarcinomas. Endoscopic
polypectomy decreases the incidence of colorectal cancers
in treated population and prevents death from colorectal
cancer (35,36). Some of the common precursor lesions are
discussed here.
Adenomas
At least half of adults in Western countries will have an
adenomatous polyp in their lifetime and one-tenth of these
lesions will progress to adenocarcinoma (37). The risk
increases after the age of 50. Endoscopically, adenomas
can be pedunculated or sessile. By definition, adenomas
are clonal lesions that show at least low grade dysplasia
characterized by enlarged, hyperchromatic and elongated
(pencillate) nuclei arranged in a stratified configuration
along the basement membrane. The adenomatous cells
may show mucin depletion and increased apoptotic activity.
Interestingly, adenomatous polyps appear to develop
through a “top-down” mechanism (38). As such, small
lesions will often only have adenomatous epithelium in their
superficial portions.
Conventional adenomas are subclassified as tubular,
tubulovillous and villous based on their architectural features
(Figure 9). Tubular adenomas are composed of simple cryptlike
dysplastic glands and contain <25% villous component.
Villous adenomas consist of >75% villous component that
resemble finger-like projections. Tubulovillous adenomas
are intermediate lesions with 25-75% villous component.
Adenomas that are large in size (>1 cm) or predominantly
villous, or contain high grade dysplasia (discussed below) are considered “advanced adenomas” (39), which require
more aggressive endoscopic surveillance.
Figure 9 Examples of tubular adenoma (A: original magnification ×200), and tubulovillous adenoma (B: original magnification ×100)
Serrated polyps
Serrated polyp is a general term for any polyp that shows a
serrated (sawtooth or stellate) architecture of the epithelial
compartment. It is a heterogeneous group of lesions that
mainly include hyperplastic polyp, sessile serrated adenoma/
polyp, and traditional serrated adenoma (40).
Hyperplastic polyps (HP) are the most common serrated
lesions that are more likely to be found in the distal colon
and generally small in size (<5 mm). Only rare HPs are
>1 cm. Endoscopically, HPs can be difficult to distinguish
from adenomas (41). Histologically, HPs are characterized
by a simple tubular architecture with elongated and straight
crypts and by luminal serration that is more pronounced
in the upper portions of the crypts with an appearance of
surface maturation (Figure 10). The proliferation zone is
limited to the basal portion of the crypts, which remains
narrow and is not serrated (42). HPs can be further divided
into microvesicular, goblet cell and mucin-poor subtypes
(43), but this histologic subclassification does not appear to
have any clinical relevance.
Figure 10 Hyperplastic polyp (original magnification ×100)
Sessile serrated adenoma (SSA) and sessile serrated polyp
(SSP) refer to the same serrated lesion and are currently
used interchangeably. SSA/Ps are more commonly seen
in the proximal colon and are usually larger than HPs
(44). Histologic diagnosis of SSA/Ps are entirely based
on architectural features characterized by exaggerated
crypt serration, serration throughout the crypt length,
hypermucinous epithelium, crypt dilatation, crypt
branching, horizontal crypt extensions at the crypt base, and
aberrant proliferation (45). Despite the name, SSA/Ps lack
the dysplastic nuclear changes that characterize conventional
adenomas. It should be remembered that SSA/P is a relatively
new entity that used to be classified as HP in the past. Thus,
pathologists may have difficulty to separate between SSA/
P and HP on histologic ground (46-48). In cases where the
separation is not easy, a descriptive diagnosis of “serrated
polyp” with a comment may be rendered.
Nevertheless, the separation of SSA/P from HP
appears important because SSA/P is now thought to be the
precursor lesion for colorectal carcinomas with MSI and
probably also for CpG island methylated MSS carcinomas
(40), whereas HP is generally believed to be innocuous.
The most reliable features for SSA/P to distinguish from
HP are dilation of the crypts at the base, often assuming a
L, inverted T, or anchor-shaped configuration (Figure 11).
These unusual shapes (“architectural dysplasia”) are often
observed in two or more contiguous crypts and are thought
to result from abnormal proliferation and/or decreased
apoptosis (42-44). Given the presumed premalignant
potential, it is probably warranted for patients with SSA/
Ps to undergo endoscopic surveillance similar to those
with conventional adenomas. In addition, a subset of
these lesions may potentially progress to carcinoma more
rapidly than conventional adenomas. Patients with these
lesions may thus need an even more aggressive endoscopic
surveillance (49,50).
Figure 11 Low power (A: original magnification ×40) and high power (B: original magnification ×200) views of sessile serrated polyp. Note
the presence of basal serration
Traditional serrated adenoma (TSA) is a unique and
uncommon type of true adenoma that exhibits low grade
nuclear dysplasia similar to that seen for conventional
adenoma, and also shows a serrated architecture similar
to that seen for HP and SSA/P. Prominent cytoplasmic
eosinophilia and a villous growth pattern are characteristic
(Figure 12).
Figure 12 An example of traditional serrated adenoma (original
magnification ×400). Note the presence of luminal serration, low
grade cytologic dysplasia and cytologic eosinophilia
Dysplasia in inflammatory bowel disease
Inflammatory bowel disease (IBD) is a well-known risk
factor for the development of dysplasia and carcinoma.
Dysplastic lesions in the setting of IBD can be flat
(endoscopically invisible) or raised (51,52), which are both
graded as indefinite for dysplasia, low grade dysplasia or
high grade dysplasia. Raised lesions are commonly termed
dysplasia-associated lesions or masses (DALMs) and can
be difficult or impossible to distinguish from sporadic
adenomas. However, several studies have shown that
adenoma-like lesions in IBD patients, regardless of whether
it represents an IBD-associated DALM lesion or a sporadic
adenoma, can be adequately managed by polypectomy and
continued endoscopic surveillance if there is no coexisting
flat dysplasia (53-55).
G iven the treatment implications, it is recommended
that the diagnosis of dysplasia in the setting of IBD
be confirmed by an experienced pathologist (56). The
diagnosis of indefinite for dysplasia should not become a waste basket, and should be reserved for cases showing
worrisome cytologic and architectural changes but also
showing surface maturation or abundant inflammation. The
diagnosis is also appropriate if the mucosal surface cannot
be evaluated due to tangential sectioning of the tissue, the
presence of marked cautery effect, or the presence of other
processing artifacts.
Lynch syndrome
Lynch syndrome is the most common inherited colorectal
cancer syndrome (57). It is characterized by increased
lifetime cancer risks primarily in the gastrointestinal
and gynecologic tracts, with colorectal and endometrial
carcinomas being most common. The cumulative lifetime
risk for colorectal cancer is estimated to be 66% for men and
43% for women (58). Patients with Lynch syndrome tend to
develop mucinous, poorly differentiated, undifferentiated,
or medullary carcinomas in the right colon at a relatively
young age. Tumor-infiltrating lymphocytes and Crohn-like
peritumoral lymphoid reaction may be prominent.
Lynch syndrome results from germline mutation in one
of the four DNA mismatch repair (MMR) genes (MLH1,
MSH2, MSH6, PMS2), and is inherited in an autosomal
dominant mood. Almost 90% of patients have mutations in
either MLH1 or MSH2 gene (57,59). Mutations in MSH6
and PMS2 genes are much less frequent. The diagnosis is
established by following Amsterdam Criteria II (Table 1) (60)
and MSI testing following the revised Bethesda guidelines
(Table 2) (61). Patients with a MSI tumor but without an
identifiable germline defect in a MMR gene may still have
Lynch syndrome if other causes of MSI, such as methylation
of the MLH1 promoter, are excluded.
Familial adenomatous polyposis
Familial adenomatous polyposis (FAP) is a rare autosomal
dominant inherited colorectal cancer syndrome (62,63),
characterized by early development of hundreds to
thousands of adenomatous polyps in the colorectum
(Figure 13). If left untreated, there is an almost inevitable
progression to colorectal cancer at an average age of 35-40
years (63,64). These patients are also at risk of developing
adenomatous polyps in the small bowel (65) and fundic
gland polyps in the stomach (66). Although syndromic
fundic gland polyps more frequently show low grade
dysplasia than sporadic counterparts (67-69), the likelihood
to progress to high grade dysplasia or invasive carcinoma is
exceedingly low.
Figure 13 A case of familial adenomatous polyposis. Note the
presence of innumerable polyps in the colon
The diagnostic criteria for FAP include: (I) 100 colorectal adenomatous polyps; (II) germline mutation of
the adenomatous polyposis coli (APC) gene; or (III) family
history of FAP and any number of adenomas at a young age
(70). Patients with attenuated FAP have <100 colorectal
adenomatous polyps, usually averaging approximately 30.
Their lifetime risk to develop colorectal cancers drops to
roughly 70% and most patients tend to develop cancers
later in life (63,71). Gardner syndrome is a variant of FAP.
Patients with this syndrome also have epidermoid cysts,
osteomas, dental anomalies and desmoid tumors. Turcot
syndrome is another variant which includes brain tumors,
typically medulloblastoma (70).
The APC tumor suppressor gene is a large gene that
contains 21 exons spanning a region of 120 kb and encoding
a 2,843 amino-acid protein. Most of the germline mutations
are nonsense and frameshift mutations and cluster within
a “hot spot” in the largest exon 15 (72,73), leading to the
synthesis of a truncated protein, which, in turn, leads to
aberrant nuclear accumulation of β-catenin and subsequent
activation of the β-catenin/Tcf transcription factor complex
to promote uncontrolled activation of the Wnt signaling
pathway of tumorigenesis (74).
Peutz-Jeghers syndrome
This is an autosomal dominant inherited cancer syndrome
characterized by hamartomatous polyps in the gastrointestinal
tract, pigmented mucocutaneous lesions, and an increased risk
of gastrointestinal and extragastrointestinal malignancies (75).
The cumulative lifetime risk for colorectal cancer approaches
40% (76). It remains questionable, however, whether
the malignancies occurring in the intestines derive from
direct transformation of hamartomatous polyps because
dysplasia is exceedingly rare in these polyps. Patients with
Peutz-Jeghers syndrome have germline mutations in the
LKB1/STK11 gene (77-79). The hamartomatous polyps in
Peutz-Jeghers syndrome are most commonly seen in the
small intestine, but can also occur in the colon. They are
composed of proliferative epithelium, stroma and smooth
muscle arranged in an arborizing pattern (Figure 14).
Figure 14 Peutz-Jeghers polyp in the colon. Note the lobular
pattern of colonic crypts divided by smooth muscle bundles
(original magnification ×100)
Juvenile polyposis syndrome
This is also an autosomal dominant inherited cancer
syndrome diagnosed if, (I) 5 juvenile polyps in the
colorectum; (II) juvenile polyps throughout the
gastrointestinal tract; or (III) any number of juvenile
polyps and a family history of juvenile polyposis (80).
Similar to Peutz-Jeghers syndrome, the cumulative
lifetime risk to develop colorectal cancer in patients
with juvenile polyposis syndrome also approaches 40%
(80,81). In contrast to Peutz-Jeghers syndrome, however,
colorectal cancers in patients with juvenile polyposis
syndrome are believed to develop directly from neoplastic
transformation within a juvenile polyp because dysplasia is
a frequent finding in these polyps. Approximately 50-60%
of the patients have germline mutations in the SMAD4 or
BMPR1A genes (82). Histologically, juvenile polyps feature
cystically dilated crypts with edematous and inflamed
stroma (Figure 15). The surface of the polyp may be
eroded, with granulation tissue and epithelial regenerative
changes.
Figure 15 Juvenile polyp showing dilated crypts and inflamed
stroma (original magnification ×40)
It should be pointed out that syndromic juvenile polyps
cannot be distinguished from sporadic counterparts, and
can be confused with inflammatory polyps on histologic
ground. Despite the name, juvenile polyps can occur in
adults or even elderly. Patients with sporadic juvenile polyps
do not have an increased risk for malignancy (83).
MUTYH-associated polyposis
MUTYH-associated polyposis (MAP) is an autosomal
recessive polyposis syndrome that carries an increased
risk for colorectal cancers (84,85). It is caused by biallelic
germline mutations in the MUTYH gene (also known as
MYH gene) that encodes a base excision repair (BER)
enzyme responsible for preventing mutations following
oxidative DNA damage. The most common mutations
are missense variants Y165C and G382D, accounting
for >70% of all mutant alleles (886,87). MAP patients
usually have >10 synchronous colorectal adenomas and
can have several hundreds or even up to 1,000 polyps.
Most patients have <100 polyps at the time of diagnosis,
however (88). Therefore, MAP is often phenotypically
indistinguishable from attenuated FAP. In contrast to
FAP, however, there is a lack of APC gene mutations in
MAP patients. In addition, serrated polyps (hyperplastic
and sessile serrated polyps) are a common finding in
MAP patients (89), which can be confused with serrated
polyposis (described below). Furthermore, due to its
recessive mode of inheritance, MAP has a tendency to
skip generations, which makes identification of MAP
patients more difficult since many patients seemingly
present as sporadic cases.
Serrated polyposis
Serrated polyposis is a new term used by WHO, which
was historically called hyperplastic polyposis (40). It
is defined by: (I) at least 5 serrated polyps proximal to
the sigmoid colon with 2 or more polyps >1 cm; (II)
any number of serrated polyps proximal to the sigmoid
colon in an individual who has a first-degree relative with
serrated polyposis; or (III) >20 serrated polyps of any size
throughout the colon. The polyps can be either SSA/Ps or
HPs. | ||||||||||||||||||||||||||||||||||||||||
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High grade dysplasia
Pathologic evaluation of an adenomatous polyp and
dysplasia includes the determination of the presence or
absence of high grade dysplasia, which represents the
immediate precursor to invasive colorectal adenocarcinoma.
High grade dysplasia manifests as a constellation of
architectural complexity and cytologic atypia that are more
malignant-appearing than those seen in a conventional
adenoma (Figure 16). Architecturally, high grade areas
typically show increased glandular density with crowded
glands that have a cribriform or back-to-back growth
pattern. Cytologically, cells with high grade dysplasia exhibit
rounded nuclei, coarse chromatin, prominent nucleoli, and
loss of nuclear polarity with nuclei no longer being oriented
perpendicular to the basement membrane. Necrotic debris
within the lumina of dysplastic glands may be seen.
Figure 16 High grade dysplasia showing complex architecture and
marked nuclear atypia (original magnification ×400)
High grade dysplasia is usually focal and situated on
the superficial portion of the polyp, and thus requires no
additional treatment beyond polypectomy if the polyp is
completely removed endoscopically. As discussed earlier,
high grade dysplasia in the colorectum is synonymous with
carcinoma in situ or intraepithelial carcinoma. Intramucosal
adenocarcinoma, defined by lamina propria invasion
including invasion into (but not through) the muscularis
mucosae, still belongs to the category of high grade dysplasia
because of its negligible potential of metastasis and can still
be successfully managed by polypectomy alone (90). | ||||||||||||||||||||||||||||||||||||||||
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Malignant polyp
The term malignant polyp is used to describe a polyp that
contains invasive adenocarcinoma in the submucosa.
Prior studies have suggested a prevalence of 2-5% in
endoscopically removed adenomas (91). When a malignant
polyp is encountered, several critical histologic features
need to be assessed, which include the status of the resection
margin, histologic grade, and the presence or absence of
lymphovascular invasion. These factors are related to the
risk of adverse outcomes such as nodal metastasis and/
or local recurrence following polypectomy. Polyps with
a negative polypectomy margin, low grade histology, and
no lymphovascular invasion can be safely treated with
endoscopic polypectomy. An increased risk of adverse
outcomes has been shown to be associated with positive
margin (defined as <2 mm from deep cauterized margin)
(Figure 17), high grade (poorly differentiated) histology,
and lymphovascular invasion. If any of these features is
present, surgical resection is indicated (91-93). Therefore, it
is important that polypectomy specimens be received in one
intact piece in order for margins to be accurately evaluated
by pathologists. Inability to assess margin status because
of piecemeal resection should also be considered as a risk
factor (91,93), and surgical resection may be recommended
in clinically fit patients.
Figure 17 A malignant polyp showing adenocarcinomatous
glands present within 1 mm of polypectomy margin (original
magnification ×100)
A pitfall in the assessment of an adenomatous polyp
is pseudoinvasion where adenomatous elements are
misplaced or herniated into the submucosa, usually
secondary to traumatization such as twisting and
torsion of the stalk (Figure 18). Histologic features that
help distinguish from true invasion include a lobular
configuration of herniated elements, lack of overt high
grade architectural and cytologic atypia, presence of a rim
of lamina propria inflammatory cells around entrapped
elements, lack of desmoplastic reaction, lack of direct
contact with submucosal muscular vessels, and presence
of hemosiderin or hemorrhage. Occasionally, herniated
adenomatous glands exhibit high grade histology, which
can be even more difficult to distinguish from invasive
adenocarcinoma. However, other histologic features that
favor pseudoinvasion may still be present. For rare cases
in which a definitive distinction cannot be made, complete
polypectomy or surgical resection may be considered based
on the clinical and endoscopic circumstances of the patient.
Figure 18 Low power (A: original magnification ×40) and high power (B: original magnification ×200) views of pseudoinvasion in a tubular
adenoma. Note the presence of hemorrhage and hemosiderin
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Pathogenesis and molecular classification
Colorectal cancer is a heterogeneous group of diseases with
distinctive genetic and epigenetic background (94). In order
to improve clinical management and better predict patient
outcome, attempts have been made to classify colorectal
cancers based on location, histology, etiologic factors,
and molecular mechanisms of tumorigenesis. As early as
in the 1980’s, it has been recognized that cancers arising
in the proximal colon and distal colon involve different
genetic mechanisms (95,96). For instance, Lynch syndrome
preferentially involves the proximal colon whereas FAP
tends to show more polyps in the left colon. These familial
forms of colorectal cancer have served as prototypes
for understanding distinct molecular mechanisms of
tumorigenesis. As discussed earlier, Lynch syndrome results
from loss of function in one of the MMR genes and follows
the MSI pathway (“mutator” pathway). In contrast, FAP
arises in patient with inherited mutations in the APC gene,
which has been the center of the original Fearon-Vogelstein
model of colorectal tumorigenesis (97) that forms the basis
of chromosomal instability (CIN) pathway (“suppressor”
pathway).
Both MSI and CIN pathways describe colorectal cancer
pathogenesis based on genetic abnormities that lead to
loss of function of tumor suppressor genes and/or gain
of function of oncogenes. In the last decade, epigenetic
instability has gained considerable attention and is now
believed to be implicated in the pathogenesis of almost
one third of colorectal cancers (49). In addition to DNA
sequence and structure, gene expression is controlled by
a number of epigenetic modifications that include DNA
methylation, histone alterations and chromatin remodeling
(98). One of the best characterized epigenetic modifications
associated with colorectal tumorigenesis is silencing of
genes (tumor suppressor and/or MMR genes) through
hypermethylation of their promoter regions. Although it was debated whether the phenomenon of epigenetic
instability represents an adaptive cellular mechanism during
carcinogenesis aimed to abort cellular proliferation, a
secondary alteration to yet unidentified genetic mutations,
a phenomenon expected to occur during tumor cell
senescence, or simply an artifact (99-104), transcriptional
silencing of certain genes by hypermethylation has
undoubtedly shown to result in tumor development
(105-110). In particular, promoter hypermethylation of
the MLH1, one of the MMR genes, is demonstrated in
the majority of sporadic colorectal cancers with a MSI
phenotype (108,111,112). Since many genes are rich in
cytosine and guanine dinucleotides (CpG islands) in their
promoters, methylation of the cytosine residues in CpG
islands is a common phenomenon, which leads to alterations
of the chromosomal structure and suppression of gene
expression. Colorectal cancers with CpG island methylator
phenotype (CIMP) are characterized by epigenetic loss
of function of tumor suppressor genes without mutations
(49,113).
Figure 19 summaries the current understanding of the
molecular pathways involved in colorectal tumorigenesis.
CIN pathway is implicated in both sporadic and syndromic
colorectal cancers. CIN tumors are characterized by
karyotypic abnormalities and chromosomal gains and
losses, which can be assessed by DNA ploidy or loss of
heterozygosity (LOH) analyses. These tumors almost
always harbor APC mutations, frequently show KRAS and
p53 mutations, and often have 18q allelic loss (3,94). MSI
pathway is also implicated in both sporadic and syndromic
colorectal cancers and tends to be mutually exclusive with
CIN. As discussed earlier, MSI tumors are characterized
by loss of the DNA mismatch repair function. In sporadic
colorectal cancers, the loss of function is primarily due
to methylation of the MLH1gene promoter that leads
to epigenetic inhibition of protein expression of MLH1
and its binding partner PMS2. These tumors often show
BRAF mutation but only rarely KRAS mutations. In Lynch
syndrome, the loss of function usually results from germline
mutations in one of the MMR genes. These tumors never
harbor BRAF mutations. Finally, CIMP pathway represents
a unique molecular mechanism in colorectal tumorigenesis,
which can occur in either MSI-H or MSS tumors.
Figure 19 Molecular pathways in colorectal tumorigenesis. CRC, colorectal cancer; MSS, microsatellite stable; MSI-H, high level
microsatellite instability; FAP, familial adenomatous polyposis; AFAP, attenuated FAP; PJS, Peutz-Jeghers syndrome; JPS, juvenile polyposis
syndrome; MAP, MUTYH-associated polyposis; CIN, chromosomal instability pathway; MSI, microsatellite instability; CIMP, CpG island
methylator phenotype; ?, pathways yet undefined
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Molecular genetic testing
With rapid advances in the understanding of colorectal
tumorigenesis and pharmacogenetics, more and more
molecular and genetic tests are demanded in order to
optimally design personalized therapies for individual
patients, to better predict patient prognosis, and to more
accurately determine the necessity for family counseling.
Currently, MSI, KRAS and BRAF are the most commonly
performed tests in pathology laboratories.
MSI testing
As discussed earlier, MSI tumors account for ~15% of
colorectal adenocarcinomas. These tumors tend to show
unique clinicopathologic features, tend to have a better stageadjusted
prognosis when compared with MSS tumors, and
appear to be resistant to treatment with 5-fluorouracil (114).
Microsatellites are repetitive DNA sequences that are
prone to errors during DNA replication if the MMR system
is defective. MSI is defined as alterations in the length of the
microsatellite sequences. It is typically assessed by analyzing
two mononucleotide repeats (BAT-25 and BAT-26) and three
dinucleotide repeats (D2S123, D5S346, and D17S250), known as the Bethesda panel (61,115), by comparison
between DNA samples extracted from normal and tumor
tissues from the same patient. The test is polymerase chain
reaction (PCR)-based, and can be performed on formalinfixed
paraffin-embedded tissues. A tumor is designated as
MSI-H if two or more (>40%) of the five microsatellite
markers show instability, MSI-L (low-level) if only one
marker shows instability, or MSS if none of the markers
show instability. The clinical significance of MSI-L remains
unclear and controversial (116), and it may be helpful if
additional microsatellite markers are tested in order to
increase the accuracy of MSI classification.
An indirect analysis of MSI status can be achieved by
immunohistochemical stains for MMR proteins. These
proteins are ubiquitously present in normal cells but show
loss of expression in MSI tumor cells. Several staining
patterns may be observed based on the underlying genetic
or epigenetic abnormalities (Table 3). It is interesting to note
that loss of MLH1 protein expression is always accompanied
by the loss of its binding partner PMS2 (Figure 20), but loss
of PMS2 expression can occur by itself. The same holds
true for MSH2 and its binding partner MSH6.
Figure 20 A MSI tumor showing loss of MLH1 (A) and PMS2 (D) protein expression, and normal expression of MSH2 (B) and MSH6 (C).
Note the presence of positive staining in benign colonic crypts and inflammatory cells, which serve as good internal controls for the stains
(original magnification ×200)
The sensitivity of PCR-based MSI test using the Bethesda
panel ranges from 55% to 84% for the detection of mutations
in different MMR gene. The sensitivity is increased if
three or more mononucleotide repeat markers are used.
The specificity of MSI test is 90%. Immunohistochemistry
has been accepted as a reliable substitute for MSI with
a concordance rate of >90%. It also provides additional
information over PCR-based MSI test in that it allows
gene-specific DNA sequence analysis based on the staining
pattern. However, immunohistochemistry may miss rare MSI
cases that are caused by germline mutations by other genes
and does not discriminate germline mutation from epigenetic
alteration when loss of MLH1 protein expression is detected.
Thus, the most recent recommendation is to perform both PCR-based MSI test and immunohistochemistry in order
to minimize the chance of missing the diagnosis of Lynch
syndrome (117). It is also recently advocated to test all newly
diagnosed colorectal cancers regardless of patient’s age and
family history because ~25% of the patients with Lynch
syndrome do not meet Amsterdam Criteria II or Bethesda
guidelines (117). In that setting, only one test, either
immunohistochemistry or MSI analysis, may be performed
because the cost of the tests will become an issue.
KRAS testing
Mutations in the KRAS (Kirsten rat sarcoma viral oncogene
homolog) gene lead to expression of a constitutively activated
KRAS protein, which are detected in ~40% of colorectal
cancers (2,118). As a critical downstream molecule in the
epidermal growth factor receptor (EGFR) signaling pathway,
mutant KRAS renders tumors resistant to EGFR-targeted
therapies (2,119-121). As a result, the American Society for
Clinical Oncology (ASCO) and the National Comprehensive
Cancer Network (NCCN) have recommended mutation
analysis of the KRAS gene for candidate patients who will
receive anti-EGFR therapies (122,123).
Greater than 95% of KRAS mutations occur in codons
12 and 13 in exon 2 (118,124,125), and thus PCR-based
methodologies designed to detect KRAS mutations are
primarily for these mutations. Mutations can also occur in
other loci such as codons 61 and 146 (126), but they are
generally not screened because of rarity. Clinically available
real-time PCR-based methods include allele-specific
amplification assay and post-PCR melting curve analysis.
The allele-specific real-time PCR technique detects seven
most common mutations in the codons 12 and 13 (127),
whereas the melting-curve analysis detects all possible
mutations in these two codons (128,129).
Another detection method is DNA sequencing, which
can detect all possible mutations in the KRAS gene, not
just limited to codons 12 and 13. In comparison to the
traditional Sanger sequencing method, the pyrosequencing
technology offers a higher analytical sensitivity and is more
advantageous for the analysis of DNA samples extracted
from paraffin-embedded tissue blocks (130,131).
BRAF testing
In addition to KRAS, mutations in other members of
the EGFR signaling pathway can also cause resistance
to anti-EGFR therapy. A good example is BRAF (v-raf
murine sarcoma viral oncogene homolog B1) gene
mutation, which has been reported in ~10% of colorectal
cancers (132-134). There are several interesting facts
about BRAF mutation in colorectal cancers. First,
activating BRAF and KRAS mutations are almost
always mutually exclusive (135,136), and thus mutation
testing of the BRAF gene should be considered
following a negative KRAS mutation analysis. In fact,
many laboratories offer reflex BRAF test if no KRAS
mutation is detected in a specimen. Second, almost all
BRAF mutations are identical V600E point mutation
(134), which can be readily detected by a number of
commercially available PCR-based assays (137). Third,
BRAF mutation is almost exclusively seen in sporadic
MSI tumors that are presumed to develop through
the serrated tumorigenic pathway, but has never been
reported in Lynch syndrome (138). More specifically,
activating mutation of the BRAF gene is associated with
a high level of global DNA methylation and epigenetic
silencing of the MLH1 gene, found in 70-90% of
sporadic colorectal tumors with a microsatellite unstable
phenotype (136,139). Therefore, further testing BRAF
mutation in a MSI tumor will help clarify the sporadic
or syndromic nature of the tumor (140). Fourth, the
impact of BRAF mutation on prognosis appears MSIdependent.
As expected, BRAF wild-type MSI-H tumors
have the best prognosis, whereas BRAF-mutated MSS
tumors are associated with the worst outcome. BRAF-mutated
MSI-H tumor and BRAF wild-type MSS
tumor are intermediate in terms of prognosis (132,133).
Therefore, testing for both MMR abnormalities
and BRAF mutations offers additional prognostic
information. | ||||||||||||||||||||||||||||||||||||||||
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Conclusions
Colorectal adenocarcinoma is a heterogeneous disease that
involves multiple tumorigenic pathways. Pathologic analysis
provides histologic and molecular information critical to
appropriate patient treatment, prognosis assessment, and
family counseling. Further understanding the molecular
mechanisms in tumorigenesis will certainly lead to the
development of new targeted therapies and new molecular
tests, which will ultimately benefit the patients and their
families. | ||||||||||||||||||||||||||||||||||||||||
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Acknowledgements
Disclosure: The authors declare no conflict of interest.
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References
Cite this article as: Fleming M, Ravula S, Tatishchev
SF, Wang HL. Colorectal carcinoma: Pathologic aspects.
J Gastrointest Oncol 2012;3(3):153-173. DOI: 10.3978/
j.issn.2078-6891.2012.030
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