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J. Pathol. 188: 252?257 (1999)
?. ?????1,2*, ?. ????1, ?. ???????????2, ?. ??????3, ?. ?????1, ?. ?????3, ?. ??????2, ?. ???????1, ?. ??????3 ???
?. ?????-???????1,2
Servi鏾 de Gastrenterologia, Instituto Portugu阺 de Oncologia Francisco Gentil, 1093 Lisboa Codex, Portugal
Centro de Investiga玢o de Patobiologia Molecular, Instituto Portugu阺 de Oncologia Francisco Gentil,
1093 Lisboa Codex, Portugal
Departamento de Patologia Morfol骻ica, Instituto Portugu阺 de Oncologia Francisco Gentil, 1093 Lisboa Codex, Portugal
Microsatellite instability (MSI) is present in most colorectal cancers (CRC) associated with hereditary nonpolyposis colorectal cancer
(HNPCC). MSI testing in so-called sporadic forms of CRC may become a useful tool in identifying new HNPCC kindred. The aim of
this study was to analyse the utility of BAT-26 as a marker to identify CRCs with MSI and to investigate whether sporadic CRCs with
MSI have a phenotypic expression similar to HNPCC cases. MSI was detected using two methods, an association of 7 poly(CA) repeats
and a poly(A) repeat alone, BAT-26, in a series of 62 patients with apparently sporadic forms of CRC. Germ-line and somatic mutations
in the hMSH2, hMLH1, and hMSH6 genes were analysed in patients with MSI+ tumours. Patients with MSI+ at poly(CA) loci and
at BAT-26 were younger (p=0�4 and p=0�2), had tumours more frequently right sided (p=0�7 and p=0�01) and more often
mucinous (p=0�7 and p=0�5, respectively) than patients with MSI negative tumours. Mutation analysis allowed the identification
of two patients carrying germ-line mutations in the hMLH1 gene (both were BAT-26+) and two other patients who had somatic
mutation in the hMSH2 and in hMLH1 genes. In conclusion, the detection of MSI using poly(CA) repeats or BAT-26 alone allowed
the identification of a subset of patients with clinico-pathological characteristics similar to those associated to HNPCC. BAT-26 has
the advantage of being a simple and less expensive method that might be used as a screening procedure before mutation analysis.
Copyright 1999 John Wiley & Sons, Ltd.
KEY WORDS?sporadic
colorectal cancer; microsatellite instability; BAT-26
Studies performed in the past two decades have
identified two well-defined pathways for colorectal
carcinogenesis:1?3 the suppressor pathway, involving
mutations of key oncogenes and inactivation of tumour
suppressor genes; and the mutator pathway, in which
mutational inactivation of mismatch repair (MMR)
genes leads to an increased rate of mutations in tumour
suppressor genes and other cancer genes which control
cell growth (e.g. TGF�-RII or BAX).4?7 Tumours originating through the latter pathway accumulate hundreds
of thousands of somatic mutations in simple repeated
sequences called microsatellites which, because of their
repeating nature, are particularly prone to slippage
errors during DNA replication.
*Correspondence to: Dr M. Cravo, Servic?o de Gastrenterologia, Instituto Portugue?s de Oncologia Francisco Gentil, Rua
Professor Lima Basto, 1093 Lisboa Codex, Portugal. E-mail:
Contract/grant sponsor: Junta Nacional de Investigac?a?o Cient??fica e
Tecnolo?gica; Contract/grant numbers: Project PRAXIS/PSAU/C/
CCC 0022?3417/99/080252?06$17.50
Copyright 1999 John Wiley & Sons, Ltd.
Microsatellite instability (MSI) is the hallmark of
most colorectal cancers (CRC) associated with hereditary non-polyposis colorectal cancer (HNPCC),8 but it
has also been detected in 12?16 per cent of so-called
sporadic forms of CRC.9?12 Nonetheless, non-familial
cases were not systematically excluded from most of
these previous studies. Some of these studies suggest that
sporadic MSI+ tumours show phenotypic similarities to
HNPCC cases; MSI testing could further contribute to
the identification of a subset of colorectal tumours with
different biological behaviour, or even new HNPCC
families which do not comply with the previously
defined Amsterdam criteria.13 However, there is no
consensus either regarding the number of microsatellite
markers to be examined, or the percentage of unstable
microsatellites required to consider a tumour as being
MSI+. One previous study suggested that the use of
BAT-26, a specific poly(A) repeat located in an intron of
hMSH2, could be as useful as a panel of multiple
poly(CA) markers for MSI assessment.14
The aims of the present study were to investigate the
utility of BAT-26 as a marker for the identification of
tumours with MSI; to ascertain whether MSI+ sporadic
CRCs have a clinico-pathological profile similar to that
Received 10 August 1998
Revised 19 October 1998
Accepted 17 February 1999
found in HNPCC cases; and to correlate MSI status
with mutations in MMR genes.
Patient selection
The study was approved by the ethics and scientific
local committees and informed written consent was
obtained from all live patients entering the study. Sixtytwo patients with colorectal cancers were included in the
present series, 35 male and 27 female with a mean age of
6412 (range: 34?83) years. A familial history of colorectal cancer in first degree relatives was present in 13 per
cent (8/62) of patients. Cases with criteria for a diagnosis
of HNPCC13 or familial adenomatous polyposis, as well
as cases associated with idiopathic inflammatory bowel
disease, preoperative radiation and chemotherapy, were
excluded from the present series. The mean follow-up
time was 3622 months; 19/62 patients had died at the
time of performing this study; 5/62 were alive but with
disease recurrence; and 6/62 were lost for follow-up.
Thirty-two were alive and free of disease.
Histopathological analysis
Fifteen tumours were located in the proximal colon,
25 in the distal colon, and the remaining 22 in the
rectum. Histopathological characteristics were specifically evaluated by two independent pathologists who
were not aware of the MSI status. The TNM system of
UICC15 was used for tumour staging. Histological diagnosis and grading of differentiation were performed
according to WHO criteria16 and mucin secretion was
evaluated using the criteria of Wiggers et al.17 as follows:
absent (no extracellular mucin production), focal (area
of extracellular mucin smaller than 50 per cent) or
predominant (area of extracellular mucin greater than
50 per cent). Peri-tumoural lymphocytic infiltration
was classified in four grades: 0?absent; 1?mild;
2?moderate; and 3?severe. Grade 3 corresponds to a
Crohn?s-like lymphoid reaction with lymphoid aggregates, some of them presenting germinal centres. Venous
invasion and lymphatic permeation were assessed as
MSI analysis
DNA was extracted from fresh frozen tissue from
colorectal tumours and their corresponding normal
mucosa using a previously described method, and PCR
amplified at seven loci containing dinucleotide repeated
sequences (CA)n: D1S216, D2S118, D3S1611, D5S404,
D8S260, D17S783, D22S282, using primers specific for
each locus and obtained from Research Genetics
(Huntsville, AL, U.S.A.). PCR was performed in a total
volume of 12�靗, using 200 m? concentration of each
deoxynucleotide triphosphate, 10 pmol of each primer,
75 ng of DNA, 0�U of Taq polymerase (BRL), and
1 霤i of 32P-dCTP. Samples were processed through 35
cycles of amplification with annealing temperatures
Copyright 1999 John Wiley & Sons, Ltd.
which ranged from 55 to 60C. PCR products were
diluted 1:1 with loading buffer containing 95 per cent
formamide, denatured and electrophoresed on polyacrylamide gels containing 6�M urea and 32�per cent
formamide. Gels were fixed in 10 per cent acetic acid,
dried, and exposed overnight to MP film (Amersham
Corp., U.K.) at 70C. Tumour MSI was defined as
changes of size bands observed in neoplastic DNA, but
not visible in the corresponding non-neoplastic DNA.
PCR amplification of BAT-26 was performed in a
similar way using previously published primers,14 but
because it is monomorphic or quasi-monomorphic, only
DNA from tumour was analysed. Whenever there was a
minor size variation not exceeding 2 bp, comparison
with DNA from non-neoplastic mucosa was performed,
and in all instances these alterations were also present in
constitutional DNA. Therefore, for this poly(A) marker,
only the presence of small unstable alleles allowed us to
consider the tumour as BAT-26+, with changes in
length less than 2 bp being interpreted as negative.
Mutation analysis
Mutations in hMSH2 and hMLH1 MMR genes were
analysed using denaturing gradient gel electrophoresis
(DGGE) with previously described primers,18,19 with
one of the primers containing at its 5 end a GC-rich
sequence (GC-clamp) to increase screening efficiency.
The amplified product was then loaded on a 6 per cent
polyacrylamide gel containing a linearly increasing
denaturing gradient (100 per cent denaturant=7 M and
40 per cent formamide (v/v); acrylamide: bisacrylamide=39:1). The electrophoresis was performed at
160 V in TAE buffer (40 m? Tris-acetate pH 7� 20 m?
sodium acetate, 1 m? Na2EDTA) at a constant temperature of 60C during 4 h and 30 min. Finally the gel
was stained with ethidium bromide and visualized under
UV light.
For hMSH6 mutation analysis, In Vitro Synthesized
Protein (IVSP) assay was used as described by
Papadopoulos et al.20 Briefly, RNA was obtained from
lymphoblastoid cells infected with Epstein?Barr virus
(ATCC-USA) and cDNA was generated using random
hexamers and reverse transcription (RT-PCR) as
described. According to Powel et al.21 primers used for
amplification of these segments had in 5 end the promotor sequence T7 used for transcription, as well as a
sequence for translation onset. In vitro translated proteins were then separated in a 4?20 per cent gradient
SDS-polyacrylamide gel and visualized under fluorography. Whenever there was a pattern corresponding to the
presence of homo- or heteroduplexes for a specific exon
on DGGE, or a truncated protein on IVSP assay, we
then proceeded to sequencing using the same primers
but without the GC-rich sequence, and f-mol sequencing
kit (Promega Corportation, U.S.A.).
Statistical analysis
Correlation between clinical and pathological variables and MSI status were analysed using either the
chi-squared test or analysis of variance (ANOVA) for
J. Pathol. 188: 252?257 (1999)
eral blood, whereas somatic mutations were analysed in
DNA from tumour. Germ-line mutations in MMR
genes were detected in two patients, both of whom had
BAT-26+tumour loci (Fig. 2). These mutations were
both located on the hMLH1 gene: one in exon 1, codon
26, GAG to TAG (Glu to STOP) and the other on exon
16, codon 618, AAG to ACG (Lys to Thr). Additionally,
we also found two somatic mutations, one in hMSH2
(exon 2, codon 93, CTT to CCT, Leu to Pro) and the
other in hMLH1 (exon 1, codon 26, GAG to TAG, Glu
to STOP). The first of these patients was BAT-26
negative, while the one carrying the nonsense somatic
mutation in the hMLH1 gene was BAT-26 positive. No
germ-line or somatic mutations were found in the
hMSH6 gene.
Fig. 1?Microsatellite instability in loci: D8S260, D22S282, and
BAT-26. T=tumour; N=normal mucosa. The arrows represent
additional alleles that are present when there is microsatellite
discontinuous or continuous variables, respectively.
Survival differences and curves were analysed using the
log-rank and Kaplan-Meier tests, respectively. Considering that more than 20 correlations are being carried
out, the significance threshold was established at 2 per
Somatic alterations in at least one poly(CA) locus
(Fig. 1) were detected in 17/62 (27 per cent) patients, but
these patients did not differ significantly from MSI
negative patients in terms of clinical and pathological
characteristics. MSI at d2 poly(CA) was present in 9/62
(15 per cent) patients. Clinical and pathological characteristics of these patients are shown in Table I. patients
with MSI+tumours were significantly younger (p=0�);
the tumours were more frequently right-sided (p=0�),
and presented a peri-tumoural lymphoid infiltrate of
moderate/marked grade in a greater proportion of cases
(p=0�). MSI+ tumours more often exhibited a mucinous component greater than 50 per cent, but the
differences did not reach statistical significance (p=0�).
With regard to BAT-26, 6/62 (10 per cent) patients
exhibited a shortened, unstable BAT-26 allele (Fig. 1).
Correlation between MSI status and clinical and pathological variables was more striking and specific than the
poly(CA) method (Table I). Thus, patients with BAT-26
positive tumours were significantly younger (p=0�2);
83 per cent of BAT-26+ lesions were located on the right
side (p=0�01), and 50 per cent of BAT-26+ tumours
exhibited some extracellular mucin production, whereas
this occurred solely in 21 per cent of BAT-26 negative
cases (p=0�5). Furthermore, BAT-26 alone was able
to identify 6/7 (86 per cent) patients whose tumours
displayed MSI at d3 poly(CA), whereas none of the
patients with MSI at one single locus was BAT-26+.
Germ-line or somatic mutations in hMSH2, hMLH1
and hMSH6 genes were analysed in 8/9 patients with
tumours exhibiting MSI at d2 poly(CA). Germ-line
mutations were analysed in DNA extracted from periphCopyright 1999 John Wiley & Sons, Ltd.
While it has become widely accepted that malignant
transformation in the colon and rectum can evolve
through two different pathways, the suppressor and the
mutator pathways,7 it is still unclear whether these
pathways correspond to distinct entities from a clinical
and pathological point of view. As the hallmark of the
mutator pathway is the presence of MSI at di- or
mono-nucleotide repeats in tumour DNA, in the present
study we compared some clinical and pathological features of tumours with and without MSI, in an attempt to
identify differences in the respective profiles.
Two previous studies have approached this type of
comparison22,23 in patients with non-familial CRC and
have found that tumours displaying MSI shared some
clinical and histological features with colorectal carcinomas arising in the context of HNPCC. Tumours with
MSI occurred at a younger age, were mainly located on
the right colon, and there was a preponderance of
lesions with a high mucinous component, as well as of
peri-tumoural lymphocytes. In contrast to these results,
a recent study by Senba et al.,24 in a Japanese series of
sporadic CRCs, did not find a correlation between the
MSI status and any clinical and pathological features,
except for tumour location in the proximal colon. However, it is worth noting that in the latter study, only 4/16
patients considered to have MSI+ tumours had lesions
with instability at two or more loci. Jass et al.23 and
Aaltonen et al.9 previously reported that colorectal
tumours with MSI at one single locus do not differ
substantially from MSI negative lesions and for this
reason, in the present study, only tumours with instability at two or more loci were classified as MSI+. Similarly to Jass et al.,23 we have also observed in the present
study that, as opposed to patients harbouring tumours
with MSI at d2 loci which exhibited a number of
phenotypic similarities with HNPCC cases, the 17
patients exhibiting MSI at more than one locus did not
differ significantly from MSI negative cases. These
clinico-pathological similarities between sporadic CRCs
with MSI and tumours from patients belonging to
HNPCC families, raise the question of whether MSI
testing might aid in the identification of new HNPCC
families. In fact, although the Amsterdam criteria13
J. Pathol. 188: 252?257 (1999)
Table I?Clinico-pathological characteristics according to MSI status: MSI at d2 poly(CA) loci or at the
BAT-26 locus
Mean age (years)
c45 years
>45 years
Proximal colon
Distal colon
TNM stage
Mucin secretion*
Venous invasion
Lymphatic permeation
Lymphoid cell infiltrate?
4 (8%)
49 (92%)
3 (33%)
6 (67%)
4 (7%)
52 (93%)
3 (50%)
3 (50%)
30 (57%)
23 (43%)
5 (56%)
4 (44%)
32 (57%)
24 (43%)
3 (50%)
3 (50%)
47 (89%)
6 (11%)
7 (78%)
2 (22%)
50 (89%)
6 (11%)
4 (67%)
2 (33%)
10 (19%)
23 (43%)
20 (38%)
5 (56%)
2 (22%)
2 (22%)
10 (17%)
24 (43%)
22 (39%)
5 (83%)
1 (17%)
0 (0%)
41 (77%)
10 (19%)
2 (4%)
6 (67%)
1 (11%)
2 (22%)
44 (79%)
10 (18%)
2 (3%)
3 (50%)
1 (17%)
2 (33%)
8 (15%)
42 (79%)
3 (6%)
3 (33%)
5 (56%)
1 (11%)
9 (16%)
44 (79%)
3 (5%)
2 (33%)
3 (50%)
1 (17%)
49 (92%)
4 (8%)
8 (89%)
1 (11%)
52 (93%)
4 (7%)
5 (83%)
1 (17%)
41 (77%)
12 (23%)
7 (78%)
2 (22%)
43 (77%)
13 (23%)
5 (83%)
1 (17%)
34 (64%)
19 (36%)
2 (22%)
7 (78%)
34 (61%)
22 (39%)
2 (33%)
4 (67%)
FHCCR=familial history of colorectal cancer; NS=no statistical significance.
*Considering two groups: absent or <50 per cent and tumours with >50 per cent of mucin production.
?Considering two groups: absent/mild and tumours with moderate/severe infiltrate.
define a homogeneous population suitable for research,
in clinical practice these criteria appear to be too restrictive and, as previously suggested, some HNPCC kindred
with a less flagrant history of inherited colon cancer can
be easily missed.25 Thus, MSI testing followed by mutation analysis in sporadic CRCs displaying certain clinical and pathological features could help in identifying
new HNPCC kindred.
Liu et al.26 have suggested that the probability of
finding a germ-line mutation in MMR genes in patients
harbouring an apparently sporadic MSI+ tumour
would mainly depend on the patients? age. It could be as
high as 42 per cent in patients younger than 35 years and
it would probably be less than 10 per cent in patients
older than that. In the present series, 25 per cent (2/8) of
patients with MSId2 loci were found to carry germ-line
mutations in hMLH1 gene. The higher percentage found
in the present study might be explained by the methodCopyright 1999 John Wiley & Sons, Ltd.
ology used for mutation analysis. IVSP assay, as used by
Liu et al.,26 is a rapid screening method but it has the
disadvantage of not detecting non-truncating mutations,
which can account for as much as 50 per cent of
mutations reported in the hMLH1 gene and 30 per cent
of those found in the hMSH2 gene.27 According to a
recent paper by Wijnen et al.,28 who performed mutation analysis in 184 families with clustering of CRC,
DGGE might become one of the most useful and
sensitive tests for mutation analysis in MMR genes. It is
also important to note that the two patients found to
carry germ-line mutations in the hMLH1 gene, with no
previous family history, had tumours displaying instability at the BAT-26 poly(A) marker. Considering that
there was a strong correlation between positivity at the
BAT-26 locus and clinico-pathological characteristics
typical of HNPCC, and that none of the patients
with tumours exhibiting MSI at one single locus was
J. Pathol. 188: 252?257 (1999)
a quite sensitive and rather specific tool for identifying
tumours with a mutator phenotype, some of them
occurring in a context of yet unknown hereditary syndromes. Amplification of this marker can be performed
on DNA extracted from paraffin-embedded specimens32
and, in most instances, it does not require nonneoplastic DNA for comparison purposes because it
has a quasi-monomorphic behaviour, which makes it
particularly suitable to use on a routine basis.
Fig. 2?DGGE analysis for the hMLH1 gene, exon 1. N=normal
control. Lanes 3 and 4 exhibit a characteristic four band pattern,
consisting of two homoduplex (h) and two heteroduplex (H) bands. In
the remaining samples (lanes 1 and 2), no mutation was detected
BAT-26+, these results further strengthen the value of
BAT-26 as an efficient marker to identify new HNPCC
kindred. In the present series of apparently sporadic
CRCs, 33 per cent of BAT-26+ cases proved to be new
unrecognized hereditary syndromes; this favours the
hypothesis that, besides the patients? age,26 the proper
selection of patients with adequate markers for MSI
testing is also important in identifying new HNPCC
In another 2/8 patients with MSI at d2 loci, somatic
mutations in the hMSH2 and hMLH1 genes were found.
In the remaining patients whose tumours exhibited MSI
but no germ-line or somatic mutations in MMR genes, a
recent study may help to shed some light on the underlying genetic defects of these subsets of colorectal
tumours. When analysing hMLH1 expression in 66
sporadic colorectal tumours, Kane et al.29 found that
four tumours did not express hMLH1 by immunohistochemistry, despite the absence of mutations in its coding
sequence. Cytosine methylation of the hMLH1 promoter region was found in these four cases and this
could be an additional way of silencing these MMR
genes, gives rise to the MSI phenotype.
As suggested by a number of studies, the identification
of colorectal tumours with the MSI phenotype might
have prognostic and treatment implications, both for the
patient and for at-risk relatives.11,30 Considering the
recently published Bethesda Guidelines,31 according to
which MSI testing is probably indicated in as many as
15?20 per cent of all colorectal cancers, it would be of
great interest to have a simple and practical method to
analyse MSI status, that could be routinely used by
pathology departments. In the present study, the use of
the BAT-26 poly(A) marker alone allowed us to identify
86 per cent (6/7) of tumours exhibiting MSI at 3 or more
poly(CA) loci and there was a highly significant correlation with clinical and pathological characteristics now
considered to be typical of tumours with MSI. Furthermore, in 33 per cent (2/6) of these cases, we were able to
identify germ-line mutations which corresponded to new
HNPCC kindred. These observations strongly suggest
that the use of this single marker alone might constitute
Copyright 1999 John Wiley & Sons, Ltd.
This research was supported by Junta Nacional de
Investigac?a?o Cient??fica e Tecnolo?gica (Project PRAXIS/
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