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Regular Article |
From the Conjoint Gastroenterology Laboratory,*
Royal
Brisbane Hospital Foundation Clinical Research Centre, Bancroft Centre,
Herston; the Genetic Services of Western
Australia,
King Edward Memorial Hospital,
Subiaco; the Queensland Health Pathology Services and the Department of
Surgery,
Gold Coast Hospital, Southport; the
Department of Surgery,
Royal Brisbane
Hospital, Herston; the Queensland Health Pathology
Service,¶
Herston; the Department of
Pathology,||
University of Queensland, Herston; and the
Queensland Institute of Medical Research,**
Bancroft Centre, Herston, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSI-H cancers comprise the balance of colorectal adenocarcinomas, representing 10 to 15% of sporadic colorectal cancers and virtually all cancers occurring in the familial syndrome hereditary nonpolyposis colon cancer (HNPCC). MSI-H cancers are defined by the presence of MSI in mononucleotide repeats as well as higher order repeats and can also be distinguished from MSI-L cancers by a set of clinical and pathological features that includes proximal location, mucinous histology, poor differentiation and the presence of tumor-infiltrating lymphocytes, and the demonstration of mismatch repair gene deficiency, most of which have diagnostic utility.2 After the recognition of MSI-H cancers in 1993, HNPCC and sporadic MSI-H cancers came to be regarded as the familial and sporadic counterparts of the same pathway of tumorigenesis. It may therefore be difficult to distinguish between MSI-H cancers occurring as part of the HNPCC syndrome and sporadic MSI-H cancers, especially when the patient is of intermediate age and is uncertain of family history details.
Family history is considered the most useful indicator of HNPCC in
individual patients in the clinical setting. Counterintuitively, no
association between MSI-H status and a family history of colorectal
cancer (CRC) is seen at the population level.5-7
It has therefore been proposed that MSI status is not a useful clinical
indicator of genetic predisposition.7
The lack of
association of MSI-H phenotype and family history is readily explained.
First, the frequency of CRC because of HNPCC is low. Indeed,
population-based surveys in high-risk areas for CRC suggest a figure as
low as 1%, indicating that only 
7% of MSI-H cancers would occur in
the context of HNPCC.8
Second, a proportion of individuals
with HNPCC will lack a strong family history.9,10
Finally,
up to 17% of patients with CRC will have an affected first-degree
relative, yet few of these family clusters of CRC will be because of
HNPCC.11,12
Endoscopic surveillance of patients with HNPCC
reduces mortality due to colorectal cancer.13
With this
fact in mind, criteria for instituting selective MSI testing that
reflect clinical (notably family history and young age at onset of
malignancy) and pathological features indicative of HNPCC have been
proposed.14
The availability of monoclonal antibodies to
the DNA mismatch repair proteins hMLH1, hMSH2, hPMS2, and hMSH6 not
only simplifies testing for a DNA repair deficiency but also pinpoints
the underlying causative gene. Nevertheless, selective testing will
miss instances of HNPCC presenting at older than the age of 45 years
and lacking a strong family history. Some of the cancers presenting in
these individuals may be suspected as being MSI-H on histopathological
grounds, because proximal mucinous or poorly differentiated cancers and
cancers showing intraepithelial or tumor-infiltrating lymphocytes or a
Crohn-like reaction are more likely to be MSI-H.2,15
Confirmation of this suspicion will not serve as a useful diagnostic
aid if the majority of MSI-H cancers detected in this way is sporadic
and there is no reliable method of distinguishing the small subset
occurring in a background of HNPCC.
Because the discrimination of MSI-H cancers has clinical implications for prognosis16 in sporadic disease and for risk assessment of the relatives of the HNPCC patient, this article examines the feasibility of distinguishing familial (HNPCC) and sporadic MSI-H CRC on the basis that these are not merely the bimodally distributed representatives of a single pathway of tumorigenesis.7 Apart from differences in age at presentation, up to 50% of HNPCC cancers are caused by a germline mutation in hMSH2 whereas virtually all sporadic MSI-H cancers are associated with loss of expression of hMLH1.17 Occasional sporadic hPMS2 mutations have been reported as the primary cause of colorectal cancers.18 Additionally, sporadic MSI-H cancers have been linked with the serrated pathway of tumorigenesis19 and a high frequency of mucinous differentiation with gastric mucinous metaplasia,20 whereas HNPCC cancers are associated with traditional adenomas.21 The recognition of fundamental differences between sporadic and familial MSI-H CRC may not only translate into diagnostic algorithms but may also account for inconsistencies in the literature regarding the molecular genetic profile, natural history, and responsiveness to adjuvant chemotherapy of the MSI-H subset of CRC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was performed on a selected group of 57 MSI-H sporadic colorectal cancers derived from patients with no evidence of family history of colorectal cancer and in whom germline mutations in hMLH1 had been excluded by denaturing HPLC analysis. The patients that were chosen represented all of the MSI-H cases lacking family history from serial colectomies in two large public hospitals in Queensland, Australia. Age was not a criterion for ascertaining patients with sporadic MSI-H cancer. A further series of 112 MSI-H colorectal cancers from patients belonging to 73 families was studied. Of these, 49 fulfilled the original or modified Amsterdam criteria22 and the remaining 24 either satisfied the minimal Bethesda criteria,14 or were previously found to carry germline mutations in the mismatch repair genes hMLH1, hMSH2, or hMSH6. Patients provided informed consent in writing. Patient sex and age at onset of cancer were recorded. DNA was extracted from all fresh samples by a salt precipitation technique23 and from all archival samples by a simple proteinase K digestion24 followed by a phenol/chloroform purification step.
Microsatellite Instability Testing
MSI status was determined under the current recommendations of the National Cancer Institute Co-operative Family Registry for Colorectal Cancer Studies Biospecimens Subcommittee. Specimens were tested with a panel of 10 microsatellites (mononucleotides BAT-26, BAT-25, BAT-40, BAT-34C4, and higher order repeats MYCL, D10S197, D18S55, D5S346, D17S250, and ACTC) using techniques described in a previous report.2
Immunohistochemistry
Paraffin sections were affixed to Superfrost Plus adhesive slides
(Menzel-Gläser; Braunschweig, Germany) and air-dried overnight at
37°C. After dewaxing and rehydration to dH2O,
sections for immunostaining for mismatch repair proteins were subject
to heat antigen retrieval in 0.001 mol/L of ethylenediaminetetraacetic
acid, pH 8.0, at 121°C in an autoclave on wet cycle. Those for
staining for p53 and ß-catenin were antigen retrieved in 0.01 of
mol/L citric acid, pH 6.0. After cooling, the slides were thoroughly
washed in Tris-buffered saline (TBS; 0.05 mol/L Tris, 0.15 mol/L NaCl),
pH 7.2 to 7.4. Endogenous peroxidase activity was blocked using 1.0%
H2O2, 0.1%
NaN3 in TBS. Nonspecific antibody binding was
inhibited by incubating the sections in 4% skim-milk powder in TBS
with the exception of slides for ß-catenin staining. After transfer
to a humidified chamber, the sections were incubated with 10% normal
(nonimmune) goat serum (Zymed Corp., San Francisco, CA) or in the case
of slides for ß-catenin staining, nonimmune horse serum (Silenus
Laboratories, Melbourne, Australia). Excess normal serum was decanted
and the sections incubated overnight with primary antibody. The various
primary antibodies are summarized in Table 1
.
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Assessment of altered ß-catenin expression was undertaken at the deep tumor margin and was based on: loss of lateral membrane expression (score 0 for present and 1 for absent), cytoplasmic staining (score 0, 1, or 2 for none, weak, and strong, respectively), and nuclear staining (score 0, 1, or 2 for none, weak, or strong, respectively). The final total of 0 to 5 was collapsed into ß-catenin grade 1 (total 0 or 1), grade 2 (total 2 or 3), and grade 3 (total 4 or 5).25 Sections stained for p53 protein were scored by two independent observers (MDW and JRJ). The proportion of positive cancer cell staining was graded as follows: 0, negative; 1, <10%; 2, 11 to 25%; 3, 26 to 50%; 4, 51 to 75%; and 5, and >75%. Only tumors scoring 3 or more were considered positive for p53 overexpression. Normal epithelium and stromal cells provided a positive internal control for mismatch repair proteins and ß-catenin, whereas known p53-positive colorectal adenocarcinomas were stained with each batch.
Methylation Studies
A subset of tumors was screened for methylation in the promoter region of hMLH1 using methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA) assays.26,27
Bisulfite Modification
Genomic DNA (300 ng) was diluted in 20 µl of H2O, denatured by treatment with 0.3 N NaOH, incubated at 75°C for 20 minutes, and quenched on ice. Freshly prepared hydroquinone (14 µl of 10 mmol/L) and NaHSO3 (250 µl of 4.8 mol/L) were added to each denatured DNA sample. All samples were incubated under mineral oil at 55°C for 5 hours. The NaHSO3-treated DNA samples were purified using Wizard DNA purification resin (Promega, Madison, WI), denatured with 0.3 N NaOH, precipitated with ethanol, and resuspended in 20 µl of H2O.
Methylation-Specific PCR (MSP)
MSP analysis was performed using primer pairs previously reported.27 The polymerase chain reaction (PCR) mixture included: 10x reaction buffer IV (AB gene), deoxynucleotide triphosphates (200 µmol/L), MgCl2 (1.5 mmol/L), primers (16 pmol each per reaction), 2.5 µl of bisulfite-modified DNA, and 1.25 U of Red Hot DNA polymerase (AB gene) in a final volume of 40 µl. Amplification was performed in an MJ Research PTC-200 thermal cycler (MJ Research, Inc., Watertown, MA) for 35 cycles (1 minute at 95°C, 1 minute at 59°C, and 1 minute at 72°C) followed by a final 5-minute extension at 72°C. Fourteen µl of each PCR reaction product was loaded onto a 10% nondenaturing polyacrylamide gel and visualized by ethidium bromide staining.
Combined Bisulfite Restriction Analysis (COBRA)
Primary PCR reactions were performed in a volume of 25 µl containing 10x reaction buffer IV (AB gene), dNTPs (250 µmol/L), MgCl2 (1.5 mmol/L), primers (10 pmol each per reaction), 2 µl of bisulfite-modified DNA and 0.5 units of Red Hot DNA polymerase (AB gene). The amplification conditions consisted of a touchdown protocol with annealing temperatures decreasing from 59°C to 50°C every two cycles, followed by cycling at 50°C annealing temperature for 30 cycles. A nested PCR reaction was performed in a 50-µl volume using 1- to 2-µl PCR product from the primary reaction as template. The nested PCR products were digested with 8000 U of RsaI overnight at 37°C. Digested samples were analyzed on 15% nondenaturing polyacrylamide gels and visualized by ethidium bromide staining. The amount of hMLH1 methylation in each sample was determined using ImageQuant software and expressed as a percentage. AluI digests were performed to confirm that the sodium bisulfite conversion was complete. Cleavage with AluI will only occur if the recognition sequence (AGCT) has not been destroyed by bisulfite conversion. The primer sequences used for the primary COBRA assay were those reported elsewhere.28 The sequences for the nested reaction were 5'-GATTTAGTAATTTATAGAGT (sense) and 5'-AATACCTTCAACCAATCAC (antisense). The primers for the MSP assay correspond to the CpG sites in the region -716 to -601 and the primers for the COBRA assay correspond to the CpG sites in the region -221 to -28.
Pathology Review
Data relating to cancer site and stage, specifically depth of invasion, nodal spread, and distant spread, were obtained from the pathological and clinical records of each patient. Sections from the primary tumor were reviewed by a single pathologist (JRJ) to assess additional pathological parameters according to published criteria and without knowledge of the status of the specimen (familial or nonfamilial origin). In specimens with two or more synchronous cancers, only the largest was assessed. The features included: 1) tumor margin classified as either expanding or infiltrating29 ; 2) presence of poor differentiation whether or not this was the major component. Criteria for poor differentiation included poor gland development with epithelial cells being arranged in small and irregular clusters, or as single cells as in signet ring cell carcinoma, or as solid sheets (medullary pattern) or in the form of trabeculae or islands30 ; 3) mucinous carcinoma in which at least 50% of the tumor comprised lakes of mucin30 ; 4) tumor-infiltrating lymphocytes based on the finding in a hematoxylin and eosin-stained section of at least four unequivocal intraepithelial lymphocytes in a single x40 field31 ; 5) peritumoral lymphocytes based on the finding of a cap or mantle of chronic inflammatory cells at the deepest point of direct spread30 ; 6) Crohn’s-like infiltrate scored on the basis of finding within a single x4 field of at least three nodular aggregates of lymphocytes deep to the advancing margin of the tumor32 ; 7) presence of residual adenoma classified as tubular, tubulovillous, villous, or serrated30 ; and 8) presence of tumor heterogeneity as defined by the finding of two or more distinct subclones distinguished on the basis of tumor type, grade of differentiation, or distinctive architectural property and described by others as a mixed or mosaic pattern.15
Loss of Heterozygosity Studies and Mutation Detection
A subset of tumors was examined for early traditional colorectal cancer mutations essentially as previously reported.25 Loss of heterozygosity was analyzed on chromosome 5q using PCR loss of heterozygosity and restriction fragment length polymorphism (RFLP) markers within the APC gene to avoid the interference MSI would contribute to the reading of the assay. K-ras mutations in codons 12 were sought using mutation-specific RFLP analysis,33 and tumors from both groups underwent mutation analysis in the coding repeats of TGF-ßRII.34
Statistical Analyses
Means and percentages of the various demographic, clinical, molecular, and pathological features of tumors were compared between HNPCC and sporadic MSI-H patient groups using t-tests and Pearson’s chi-square. Where numbers for percentages were small, a Fisher’s exact test was performed to compare groups. To evaluate the independent effects of the demographic, clinical, molecular, and pathological factors on patient group, a binary logistic regression was used. Those factors whose likelihood ratio statistic for inclusion into the model had a corresponding P value of >0.20 were removed from the final model.
Data were analyzed using the STATXACT package from Borland Inc. and SPSS for Windows Release 10.0 (SPSS Inc., Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mean age of cancer onset in patients with sporadic MSI-H tumors was significantly different, 74.5 years (range, 57 to 96 years) compared with 46.7 years (range, 18 to 80 years) for HNPCC patients (P < 0.001, t-test). Gender distribution also differed between the groups with females comprising 68% of sporadic MSI-H tumors and 50% of HNPCC (P = 0.023, chi-square test). Within HNPCC, the mean age of onset differed slightly between males and females but this was not significant (45.8 years for males and 48.0 years for females; P = 0.45 t-test).
Microsatellite Instability Testing
A summary of molecular and immunohistochemistry findings is given
in Table 2
. Examples of markers used in
the study are shown in Figure 1
. Sporadic
MSI-H tumors were characterized by a very high level of MSI with a mean
positive yield of 87% of markers (range, 50 to 100%) whereas HNPCC
tumors scored significantly less (72%; range, 17 to 100%)
(P < 0.001, t-test for independent
samples). The mononucleotide marker BAT26 detected 57 of 57 (100%)
sporadic tumors and 108 of 112 (96%) HNPCC tumors. Four HNPCC tumors,
which did not show mutations in BAT26, did not express hMSH2 protein
and showed levels of MSI ranging from 30 to 50% of markers.
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Examples of immunohistochemistry staining for MMR proteins are
given in Figure 2
. All sporadic tumors
tested showed loss of expression of hMLH1 (47 of 47, 100%). In
contrast, loss of both major mismatch repair genes was seen in the
HNPCC group, with 51 (48%) losing hMLH1 and 47 (45%) losing hMSH2. Of
the cancers losing neither hMLH1 nor hMSH2, two lost hMSH6, five lost
hPMS2, and one lost both hMSH6 and hPMS2. A further single tumor showed
no loss of any of the four MMR proteins tested. In 47 tumors lacking
hMLH1 from both groups, which were stained for hPMS2, there was
concomitant loss of hPMS2 in all cases. Of 32 HNPCC tumors lacking
hMSH2, 27 (84%) showed simultaneous loss of hMSH6. The average
proportion of positive microsatellite markers did not vary between
tumors deficient in hMLH1 and hMSH2. Tumors with immunohistochemistry
indicating germline mutation of hPMS2 or hMSH6
were indistinguishable with respect to microsatellite status from those
with results indicative of hMLH1 or hMSH2
mutations. The mean scores for ß-catenin differed significantly
between the two groups with HNPCC tumors showing higher levels of
nuclear staining (1.9 versus 0.8, P <
0.0001, t-test). Low levels of nuclear p53 staining were
seen in both groups.
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Methylation of hMLH1 was examined in a subset of tumors
from both groups. Representative results are shown in Figure 3
. The majority of sporadic tumors so
tested (20 of 23, 87%) showed methylation of hMLH1 by both
MSP and COBRA. Three cases, all males, had tumors which were
unmethylated at hMLH1. In the HNPCC group, 11 tumors of 30
tested were methylated at hMLH1. Of these 30 tumors, 20 were
hMLH1-negative and all 11 tumors methylated at hMLH1 fell
into this subgroup (55% of hMLH1-negative tumors). Nine tumors of 11
methylated at hMLH1 were proximal (P
= 0.02, Fisher’s exact test).
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Loss of heterozygosity at 5q was seen in 0 of 20 sporadic and 1 of 13 HNPCC tumors informative for at least one polymorphic marker in the region of the APC gene. K-ras mutations in codon 12 were present in 0 of 23 sporadic and 2 of 20 HNPCC tumors examined. TGF-ßRII mutations were found in similar proportions across both groups with 18 of 27 (66%) sporadic and 14 of 20 (70%) HNPCC tumors showing mutations.
Pathology Review
Table 3
shows pathology features
analyzed between the two tumor groups, and the features are illustrated
in Figure 4
. Both tumor types commonly
showed an expanding rather than infiltrative margin. Tumor-associated
lymphocytes (Crohns-like, peritumoral, and tumor-infiltrating) were
also common but more frequently seen in HNPCC cancers. Although both
groups were more likely to display proximal location, mucinous
histology, and poor differentiation level than common colorectal
cancer, these featured more often in the sporadic MSI-H tumors,
reaching statistical significance in all three. The presence of
subclones within the tumor (heterogeneity) was also more common in the
sporadic group. A notable feature in subclones associated with sporadic
MSI-H cancers was a serrated pattern reminiscent of serrated adenoma.
Contiguous adenoma was observed in 30 cases (17 in HNPCC and 13 in
sporadic MSI-H tumors). There was a striking difference in the
histology of these contiguous lesions with 16 of 17 (94%) in HNPCC
lesions comprising tubular or tubulovillous (traditional) adenomas
contrasting with only 1 of 13 (8%) in sporadic cancers
(P < 0.001). Conversely, 12 of 13 (92%) MSI-H
sporadic cancers were adjacent to serrated adenomas. Adjacent serrated
adenoma was observed in only 1 of 17 (6%) HNPCC cancers
(P < 0.001).
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A multivariate logistic regression was used to establish a predictive model for familial and sporadic tumors. Variables considered for inclusion in the model were age, sex, histological type of tumor (mucinous or nonmucinous), location of tumor in the colon (proximal or distal), the presence of tumor-infiltrating lymphocytes, of peritumoral lymphocytes, of subclones, of contiguous adenoma, and whether the tumor was poorly differentiated as well as the ß-catenin score and the absence of hMSH2 staining. Those variables found to independently significantly predict sporadic tumors were increasing age (P < 0.001) and presence of subclones (P = 0.025). Presence of peritumoral lymphocytes significantly predicted HNPCC tumors (P = 0.025), and there was a tendency toward tumor-infiltrating lymphocyte presence predicting HNPCC tumors (P = 0.149). Based on the classification of these four features, the model was able to classify 94.5% of tumors as either sporadic or HNPCC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found the MSI-H sporadic cancer to be predominantly a late onset disorder of females and its familial counterpart to occur significantly earlier and to affect both sexes equally. HNPCC colorectal cancer has been shown to be more common in males in a previous report, although females were more likely to develop sporadic MSI-H colorectal cancer35 despite the reported association with cigarette smoking.36 Females were also more likely to show methylation of hMLH1, a key mechanism in the genesis of sporadic MSI-H colorectal cancer.35 Although familial and sporadic MSI-H cancers showed an overlapping age distribution, no sporadic cancers were seen in patients younger than 57 years. Hence patients presenting at 55 years and younger are highly likely to have HNPCC. Although HNPCC colorectal cancer occurred in young patients, sporadic MSI-H colorectal cancer was more age-related than either MSS or MSI-L colorectal cancer. The demographic data indicate a fundamental distinction of familial and sporadic MSI-H colorectal cancer.
MSI testing is used to define MSI-H colorectal cancer. In this study, all sporadic cancers were characterized by instability in at least 50% of markers, whereas HNPCC cancers showed significantly less extensive instability. All sporadic cancers lacked hMLH1 staining, whereas absent staining for a range of mismatch repair proteins was seen in HNPCC. All tumors lacking hMLH1 showed absence of hPMS2, whereas five lacked hPMS2 alone. The relation between hMLH1 and hPMS2 has been reported previously,18,37 and suggests that hPMS2 may be degraded in the absence of its binding partner, hMLH1, as hPMS2 mRNA is still abundant in tumors despite the lack of protein. Similarly, many tumors lacking hMSH2 had no staining with hMSH6, consistent with the findings of others.38 Methylation seemed to be the predominant form of silencing of hMLH1 in sporadic disease. However, it was not absent from HNPCC with more than half of hMLH1 tumors showing methylation presumably of the wild-type allele.39 In HNPCC, this process was not restricted to females or to older patients, although it was more likely to be found in the proximal colon (data not shown).
Significant differences are apparent in the spectrum of molecular
alterations in the two tumor sets. The most striking difference was
seen in the wnt pathway where nuclear staining for
ß-catenin was a common feature in HNPCC. K-ras mutations
in codon 12 were rare in both groups. Both groups displayed TGF-ßRII
mutations at rates of 70%. Loss of 5q was not common in either
group, despite the increased nuclear staining for ß-catenin in
familial tumors. It is likely that direct mutations in ß-catenin
itself or other genes in its signaling pathway such as axin2 may be
responsible for this observation in familial tumors.40
Given the differences in the early morphogenesis of familial and
sporadic MSI-H colorectal cancer, one would expect to observe matching
differences in the molecular profiles of the two pathways. Studies
including HNPCC cancers and/or MSI-H cell lines show evidence of
disruption of the wnt signaling pathway as indicated by
APC mutation,41,42
ß-catenin
mutation43
or abnormal cytoplasmic and nuclear staining of
ß-catenin. Studies that focus on sporadic MSI-H colorectal cancer
show little evidence of APC mutation,44,45
ß-catenin mutation,44
or abnormal immunolocalization of
ß-catenin.25
Most studies show a trend toward or
significant reduction of K-ras mutation in sporadic MSI-H
cancers25,46
whereas codon 13 K-ras mutation
occurs at a higher frequency in HNPCC.47
These data are
consistent with the differing routes of morphogenesis of familial
versus sporadic MSI-H colorectal cancer, specifically the
traditional adenoma-carcinoma sequence in the former and an alternative
(serrated) route in the latter.
Although HNPCC has long been known to show a predilection for the
proximal colon, up to 40% of cancers present in the distal bowel.
Rectal cancers are a significant complication after total colectomy and
ileorectal anastomosis.48
By contrast, 90% of sporadic
MSI-H cancers occur in the proximal colon.49
In using
histological features to distinguish MSI-H cancers (regardless of
whether these were sporadic or familial), Alexander and
colleagues15
found the subjective interpretation of the
overall histopathological appearance was more discriminating than any
individual feature. No such attempt to apply an overall diagnosis
of sporadic versus familial MSI-H cancer was used in this
study. Nevertheless, experience derived in retrospect allowed such
a global impression to be formed. Overall, HNPCC cancers were more like
traditional colorectal cancers apart from the higher frequency of
lymphocytic infiltration. This was a feature of both familial and
nonfamilial groups but peritumoral and tumor-infiltrating lymphocytes
were more frequent in HNPCC cancers. Although the most distinctive
feature of HNPCC cancers was the pronounced lymphocytic infiltrate,
sporadic MSI-H cancers showed several additional features
distinguishing them from both HNPCC and common sporadic colorectal
cancer. The frequency of poor differentiation, proximal location,
and mucinous histology was higher in these cancers, and even
nonmucinous carcinomas were likely to include fields containing pools
of extracellular mucin (Figure 4, A
-I).
Sporadic MSI-H cancers were more likely to show two or more
subclones. These were distinguished on the basis of grade of
differentiation, for example moderate and poor or type of cancer, for
example mucinous and nonmucinous (Figure 4G)
. The mucinous areas in
sporadic MSI-H cancers were more likely to be poorly differentiated and
composed of ribbons, irregular cell clusters, or laciform structures.
Mucinous change in HNPCC, by contrast, usually featured a
well-differentiated columnar epithelium similar to the epithelium of
villous adenoma. Areas showing obvious foci of serration occurred in
many sporadic MSI-H cancers (Figure 4, H and I)
. In these fields and
also in nonserrated areas, cytoplasm is typically abundant and
eosinophilic whereas nuclei are vesicular and contain a prominent
nucleolus. Residual serrated adenoma occurred in 12 sporadic MSI-H
cases (Figure 4F)
, whereas residual traditional adenoma featured in 16
HNPCC cancers. In only one instance were remnants of traditional
adenoma observed adjacent to a sporadic MSI-H and serrated adenoma
adjacent to an HNPCC cancer. This finding adds further evidence to the
case for a serrated precursor for these lesions. These findings are
also supported by a recent report50
that examined a series
of 466 colorectal cancers for the presence of remnant serrated adenoma.
These were found in 5.8% of cases and were significantly associated
with the presence of MSI.
Previous reports that looked at the question of distinctive features in MSI-H cancers may have overlooked differences because of more permissive definitions of MSI-H cancers (which included some MSI-L cases) and because the cohorts studied pooled MSI-H sporadic cancers with those occurring in HNPCC. In this report, we show that MSI-H cancers occurring in HNPCC more closely resemble cancers developing by traditional pathways, both in morphology and mutation spectrum, whereas those in the sporadic setting show strong evidence of evolution from serrated precursors. Although this report does not separate the two types of cancer into nonoverlapping groups, and finds that no single assay can be used to unequivocally partition the two groups, the combination of age at diagnosis and three pathology features (tumor heterogeneity, peritumoral lymphocytes, and tumor-infiltrating lymphocytes) allowed 94.5% of MSI-H cancers to be classified as sporadic or HNPCC.
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Footnotes |
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Supported by the National Health and Medical Research Council of Australia, the Walter Paulsen Memorial Tumor Bank, the National Institutes of Health (grant no U-01-CA74778), and the Royal Brisbane Hospital Foundation. During this work, J. Y. was supported by the Department of Pathology at Royal Brisbane Hospital.
Accepted for publication August 17, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 B. Halvarsson, W. Muller, M. Planck, A. C. Benoni, P. Mangell, J. Ottosson, M. Hallen, A. Isinger, and M. Nilbert Phenotypic heterogeneity in hereditary non-polyposis colorectal cancer: identical germline mutations associated with variable tumour morphology and immunohistochemical expression J. Clin. Pathol., July 1, 2007; 60(7): 781 - 786. [Abstract] [Full Text] [PDF]
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L. J. Mead, M. A. Jenkins, J. Young, S. G. Royce, L. Smith, D. J. B. St. John, F. Macrae, G. G. Giles, J. L. Hopper, and M. C. Southey Microsatellite Instability Markers for Identifying Early-Onset Colorectal Cancers Caused by Germ-Line Mutations in DNA Mismatch Repair Genes Clin. Cancer Res., May 15, 2007; 13(10): 2865 - 2869. [Abstract] [Full Text] [PDF]
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K. Trautmann, J. P. Terdiman, A. J. French, R. Roydasgupta, N. Sein, S. Kakar, J. Fridlyand, A. M. Snijders, D. G. Albertson, S. N. Thibodeau, et al. Chromosomal Instability in Microsatellite-Unstable and Stable Colon Cancer. Clin. Cancer Res., November 1, 2006; 12(21): 6379 - 6385. [Abstract] [Full Text] [PDF]
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J. L. Westra, M. Schaapveld, H. Hollema, J. P. de Boer, M. M.J. Kraak, D. de Jong, A. ter Elst, N. H. Mulder, C. H.C.M. Buys, R. M.W. Hofstra, et al. Determination of TP53 Mutation Is More Relevant Than Microsatellite Instability Status for the Prediction of Disease-Free Survival in Adjuvant-Treated Stage III Colon Cancer Patients J. Clin. Oncol., August 20, 2005; 23(24): 5635 - 5643. [Abstract] [Full Text] [PDF]
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S. B. Hatch, H. M. Lightfoot Jr., C. P. Garwacki, D. T. Moore, B. F. Calvo, J. T. Woosley, J. Sciarrotta, W. K. Funkhouser, and R. A. Farber Microsatellite Instability Testing in Colorectal Carcinoma: Choice of Markers Affects Sensitivity of Detection of Mismatch Repair-Deficient Tumors Clin. Cancer Res., March 15, 2005; 11(6): 2180 - 2187. [Abstract] [Full Text] [PDF]
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T Kambara, L A Simms, V L J Whitehall, K J Spring, C V A Wynter, M D Walsh, M A Barker, S Arnold, A McGivern, N Matsubara, et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum Gut, August 1, 2004; 53(8): 1137 - 1144. [Abstract] [Full Text] [PDF]
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 A. Goel, C. N. Arnold, D. Niedzwiecki, J. M. Carethers, J. M. Dowell, L. Wasserman, C. Compton, R. J. Mayer, M. M. Bertagnolli, and C. R. Boland Frequent Inactivation of PTEN by Promoter Hypermethylation in Microsatellite Instability-High Sporadic Colorectal Cancers Cancer Res., May 1, 2004; 64(9): 3014 - 3021. [Abstract] [Full Text] [PDF]
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Y. Mori, J. Yin, F. Sato, A. Sterian, L. A. Simms, F. M. Selaru, K. Schulmann, Y. Xu, A. Olaru, S. Wang, et al. Identification of Genes Uniquely Involved in Frequent Microsatellite Instability Colon Carcinogenesis by Expression Profiling Combined with Epigenetic Scanning Cancer Res., April 1, 2004; 64(7): 2434 - 2438. [Abstract] [Full Text] [PDF]
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A. E. de Jong, M. van Puijenbroek, Y. Hendriks, C. Tops, J. Wijnen, M. G. E. M. Ausems, H. Meijers-Heijboer, A. Wagner, T. A. M. van Os, A. H. J. T. Brocker-Vriends, et al. Microsatellite Instability, Immunohistochemistry, and Additional PMS2 Staining in Suspected Hereditary Nonpolyposis Colorectal Cancer Clin. Cancer Res., February 1, 2004; 10(3): 972 - 980. [Abstract] [Full Text] [PDF]
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M. G. Daidone, A. Costa, M. Frattini, D. Balestra, L. Bertario, M. A. Pierotti, B. M. Boman, T. Zhang, and J. Z. Fields Correspondence re: T. Zhang et al., Evidence That APC Regulates Survivin Expression: A Possible Mechanism Contributing to the Stem Cell Origin of Colon Cancer. Cancer Res., 61: 8664-8667, 2001. Cancer Res., January 15, 2004; 64(2): 776 - 779. [Full Text] [PDF]
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G. Deng, I. Bell, S. Crawley, J. Gum, J. P. Terdiman, B. A. Allen, B. Truta, M. H. Sleisenger, and Y. S. Kim BRAF Mutation Is Frequently Present in Sporadic Colorectal Cancer with Methylated hMLH1, But Not in Hereditary Nonpolyposis Colorectal Cancer Clin. Cancer Res., January 1, 2004; 10(1): 191 - 195. [Abstract] [Full Text] [PDF]
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N. Matsumoto, T. Yoshida, and I. Okayasu High Epithelial and Stromal Genetic Instability of Chromosome 17 in Ulcerative Colitis-associated Carcinogenesis Cancer Res., October 1, 2003; 63(19): 6158 - 6161. [Abstract] [Full Text] [PDF]
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A. Goel, C. N. Arnold, D. Niedzwiecki, D. K. Chang, L. Ricciardiello, J. M. Carethers, J. M. Dowell, L. Wasserman, C. Compton, R. J. Mayer, et al. Characterization of Sporadic Colon Cancer by Patterns of Genomic Instability Cancer Res., April 1, 2003; 63(7): 1608 - 1614. [Abstract] [Full Text] [PDF]
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J R Jass, M Barker, L Fraser, M D Walsh, V L J Whitehall, B Gabrielli, J Young, and B A Leggett APC mutation and tumour budding in colorectal cancer J. Clin. Pathol., January 1, 2003; 56(1): 69 - 73. [Abstract] [Full Text] [PDF]
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V. L. J. Whitehall, C. V. A. Wynter, M. D. Walsh, L. A. Simms, D. Purdie, N. Pandeya, J. Young, S. J. Meltzer, B. A. Leggett, and J. R. Jass Morphological and Molecular Heterogeneity within Nonmicrosatellite Instability-High Colorectal Cancer Cancer Res., November 1, 2002; 62(21): 6011 - 6014. [Abstract] [Full Text] [PDF]
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S. A. Kuismanen, A.-L. Moisio, P. Schweizer, K. Truninger, R. Salovaara, J. Arola, R. Butzow, J. Jiricny, M. Nystrom-Lahti, and P. Peltomaki Endometrial and Colorectal Tumors from Patients with Hereditary Nonpolyposis Colon Cancer Display Different Patterns of Microsatellite Instability Am. J. Pathol., June 1, 2002; 160(6): 1953 - 1958. [Abstract] [Full Text] [PDF]
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