Laser microdissection and microsatellite analysis of colorectal adenocarcinomas.

Abstract

Microsatellite instability (MSI) is an important marker in colorectal cancer. The analysis may be difficult if the tumour is heterogeneous or only scarce material is available. The aim of this study was to apply laser microdissection (LMD) to MSI analysis in an attempt to allow diagnosis in these situations.
Twenty-two primary tumours and eight lymph node metastases from twenty patients were laser microdissected and MSI analysis was performed with an optimised multiplex PCR. Differences in allelic size between tumour and blood were evaluated to determine the MSI status.
The method proved efficient in as little as 4,000 microm3 formalin-treated and paraffin-embedded tumour tissue. The result of microsatellite analysis was independent of sample location in the primary tumour and its metastasis.
LMD followed by a multiplex PCR is a useful method for MSI analysis in cases of tumour heterogeneity and scarce tumour material.

Full text

Page 1

Abstract. Background: Microsatellite instability (MSI) is an
important marker in colorectal cancer. The analysis may be
difficult if the tumour is heterogeneous or only scarce material
is available. The aim of this study was to apply laser
microdissection (LMD) to MSI analysis in an attempt to allow
diagnosis in these situations. Materials and Methods: Twenty-
two primary tumours and eight lymph node metastases from
twenty patients were laser microdissected and MSI analysis was
performed with an optimised multiplex PCR. Differences in
allelic size between tumour and blood were evaluated to
determine the MSI status. Results: The method proved efficient
in as little as 4,000 Ìm3formalin-treated and paraffin-
embedded tumour tissue. The result of microsatellite analysis
was independent of sample location in the primary tumour and
its metastasis. Conclusion: LMD followed by a multiplex PCR
is a useful method for MSI analysis in cases of tumour
heterogeneity and scarce tumour material.
Two distinct pathways in the development of colorectal
(CR) adenocarcinomas have been described. One is the
chromosomal instability pathway and the other is the
microsatellite instability (MSI) pathway (1, 2). The first is
characterised by aneuploidy with gains and losses of
chromosomal regions. The latter is characterised by an
impaired DNA repair system resulting in instable DNA
tandem repeats. This is called MSI.
CR adenocarcinomas with a high level of MSI (MSI-H)
appear to have a better prognosis than microsatellite stable
(MSS) tumours (3). Ten to 15% of sporadic CR cancers and
most cases of hereditary non-polyposis colon cancer
(HNPCC) exhibit MSI-H (4). MSI analysis is, therefore,
important as a prognostic parameter and also as an efficient
screening method before further genetic testing is
considered (5).
MSI analysis is readily performed by DNA extraction
from macroscopic amounts of tumour tissue followed by
PCR with specific microsatellite markers. However,
difficulties arise when a tumour is heterogeneous and the
distinction between tumour and normal tissue is less clear.
Another problem can arise if only a very small amount of
tumour is available for analysis or if the tissue has been
stored for an extended period.
Laser microdissection (LMD) combined with laser
pressure catapulting (LPC) is a new method for capturing
specific cells identified by microscope (6). LMD can
separate neoplastic tissue from normal tissue and may, thus,
improve the quality of subsequent MSI analysis. However,
the amount of tissue is much smaller in LMD samples,
challenging the subsequent DNA purification and PCR.
The aim of the present study was to apply LMD to MSI
analysis in CR adenocarcinomas and to clarify,
methodologically, aspects of analysing small numbers of
cells. Focus centred on detection limits with respect to the
amount of tissue and fraction of tumour cells in a sample,
and the effects of formalin fixation and long-term storage.
Materials and Methods
Patients and samples. Twenty patients were included from a cohort
of CR patients already tested for MSI. Ten patients had MSI-H
tumours and ten patients had MSS tumours. Five patients in each
group had lymph node metastases and, in each group, four of these
metastases were available for MSI analysis.
Patients gave informed consent prior to surgery, accepting
blood sample and tumour analyses. An experienced pathologist
collected samples for MSI analysis from the fresh tumour
specimen. The specimens were then fixed in formalin for 2 days
and processed according to standard pathology procedure.
Tissues from all patients were initially analysed following our
standard procedure, i.e., DNA was extracted from blood with a
standard desalting method and from a macroscopic amount of
fresh tumour with a commercial kit (NucleoSpin Tissue, Macherey-
Nagel, Düren, Germany). Amplification was done by PCR with five
fluorescently-labelled microsatellite markers, BAT25, BAT26,
2069
Correspondence to: Henrik Jensen, Department of Oncology, Vejle
Hospital, 25 Kabbeltoft, DK-7100 Vejle, Denmark. Tel: +45
79406832, Fax +45 79406856, e-mail laheje@vgs.vejleamt.dk
Key Words:
microdissection, metastasis, PCR.
Microsatellite repeats, colorectal neoplasm,
ANTICANCER RESEARCH 26: 2069-2074 (2006)
Laser Microdissection and Microsatellite
Analysis of Colorectal Adenocarcinomas
LARS HENRIK JENSEN, DORTHE G. CRUGER, JAN LINDEBJERG,
LENE BYRIEL, GERT BRUUN-PETERSEN and ANDERS JAKOBSEN
Danish Colorectal Cancer Group South, University of Southern Denmark, Vejle Hospital, Vejle, Denmark
0250-7005/2006 $2.00+.40

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D17S250, D2S123 and D5S346, followed by detection on an ABI
310 Gene Analyzer (Applied Biosystems, Foster City, CA, USA).
The tumour was classified as MSS if comparison of blood and
neoplastic tissue showed no length mutations. If one or more
markers were mutated, the tumour was classified as low-grade MSI
(MSI-L) or MSI-H, respectively (7). To assess whether the time of
formalin treatment interferes with MSI-analysis, samples of normal
colonic tissue from two patients were treated with formalin for
varying lengths of time up to 14 days and compared to fresh-frozen
samples. Also the effect of long-term storage was investigated by
analysing up to 17-year-old samples randomly selected from the
archives.
LMD and LPC. Five-Ìm sections of formalin-fixed and paraffin-
embedded tumour and metastasis were mounted on glass slides and
stained with HE. From each section of primary tumour, three
samples were isolated by LMD and LPC using PALM MicroBeam
(P.A.L.M. Microlaser Technologies AG, Bernried, Germany). Two
samples were dissected from the base of the tumour (central and
peripheral) and one near the luminal surface. From each section
of lymph node metastasis, one sample was dissected. The area of
every sample was between 100,000 and 200,000 Ìm2. Figure 1
illustrates the usefulness of LMD in lymph node metastasis.
From the group of ten MSI-H patients, the five first tumours
were selected and, for each, a series of increasing amount of tissue
(from 6,400 Ìm2to 625,000 Ìm2) was microdissected. From the
same five patients, samples of malignant cells and samples of
normal epithelial cells were obtained and mixed in a series of
decreasing ratio of normal vs. neoplastic cells. The total number of
cells in these suspensions was estimated to be approximately 100.
DNA preparation and PCR. The laser-microdissected samples were
collected directly into 20 Ìl proteinase K buffer (Picopureì DNA
Extraction Kit) and incubated for at least 16 hours at 65ÆC,
followed by heat inactivation at 95ÆC for 10 minutes. Three Ìl of
this sample were added directly to 3.75 Ìl Mastermix (Qiagen
Multiplex PCR Kit) and 0.75 Ìl primermix (0.1 ÌM D5S346 and
BAT26, 0.2 ÌM D17S250, BAT25 and D2S123). The primer
sequences were according to Umetani et al. (8). The PCR reaction
took place in a thermocycler with enzyme activation at 95ÆC for
15 minutes followed by 36 cycles of denaturation at 94ÆC for
30 seconds, annealing at 56ÆC for 3 minutes and extension at 72ÆC.
A final extension step at 72ÆC for 30 minutes was added. One Ìl
of DNA extracted from blood and 2 Ìl water were amplified under
the same conditions, except 20 instead of 36 cycles were used.
The fluorescently-labelled PCR products were detected and
interpreted as described for standard MSI analysis. If there was no
detectable PCR product, up to two extra amplifications were
carried out. If that failed, another sample was obtained, and only if
both amplification and double amplification failed, was the sample
considered non-amplifiable.
Results
Patient characteristics. The MSI-H tumours included nine
right-sided colon cancers and one rectal cancer. One patient
had three synchronous tumours. The control group of ten
right-sided MSS tumours were matched with the MSI group
regarding lymph node metastases. The metastases in lymph
nodes from one patient in each group were not available.
Table I summarizes the patient characteristics.
Detection limits. The detection limit for MSI analysis is closely
related to the amount of good quality DNA for amplification
of the five microsatellite markers. Table II lists the MSI
results of different sample sizes. The number of instable
markers as a ratio of the number of informative markers is
given for each sample. At least two instable markers were
required for the tumour to be scored as MSI-H. All samples
of 4,000 Ìm3or above were scored correctly. This volume is
equivalent to approximately four cells per PCR. Three out of
five samples were also scored correctly for the smallest
amount of 1,600 Ìm3. All five markers were successfully
amplified for samples of more than 25,000 Ìm3. This volume
of tissue is equivalent to approximately 25 cells per PCR. The
most frequent non-amplifiable markers for lower volumes of
tissue were D17S250 (seven instances) and D2S123 (four
ANTICANCER RESEARCH 26: 2069-2074 (2006)
2070
Figure 1. Neoplastic cells (N) and lymphocytes (L) are shown in a lymph
node metastasis before (top) and after (bottom) laser microdissection.

Page 3

instances), whereas BAT25, BAT26 and D5S346 only failed
in two instances each.
The detection limit of MSI in tumour tissue mixed with
normal tissue was examined in five different MSI-H
tumours. The total number of cells in each reaction was
approximately 100 with different percentages of tumour
cells. Table III shows the results of MSI analysis in these
samples. All the tumours were correctly classified when 60%
or more of the cells came from tumour tissue.
Formalin treatment and storage. Fresh-frozen tissue from two
patients was analysed and compared to neighbouring samples
Jensen et al: Laser Microdissection and Microsatellite Analysis
2071
Table I. Patient characteristics are shown for ten consecutive MSI-H tumours and ten right-sided MSS tumours (matched by lymph node metastasis).
Patient GenderAge LocalisationDukes’ stageNodesNode met. DifferentiationStandard MSI
1
2
3
4
5
6
7
8
9
M
F
F
F
F
F
F
F
M
F
M
F
F
F
M
F
M
F
M
F
67
70
81
85
83
73
69
72
80
71
67
67
87
77
73
65
70
69
50
79
R
R
R
R
R
C
A
C
C
C
33
11
32
23
29
32
23
16
21
15
1
0
3
2
3
0
0
5
0
0
1
0
0
0
1
3
0
0
5
lowMSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSS
moderate
moderate
moderate
low
low
low
low
moderate
low
low
moderate
low
moderate
moderate
moderate-low
moderate
moderate
moderate
low
R R L*
R
R
R
R
R
R
R
R
R
R
R
R
R
R
B A B*
B
C
B
B
D#
B
B
B
C
C
B
B
C
D#
10
11
12
13
14
15
16
17
18
19
20
9
20
14
14
9
13
37
14
5
2510
R=right-sided, L=left-sided, nodes=total number of lymph nodes, node met.=number of lymph nodes with metastasis.
*three synchronous tumours. #Liver metastasis discovered during surgery.
Table II. Number of instable microsatellite markers/number of informative markers is given for each volume of microdissected sample. Bold numbers
indicate that the sample is interpreted as MSI-H.
Patient1,600 Ìm3
4,000 Ìm3
10,000 Ìm3
25,000 Ìm3
62,500 Ìm3
156,250 Ìm3
1
2
3
4
5
0/1
3/3
3/3
4/4
1/2
3/5
3/5
3/3
5/5
3/5
2/4
3/4
3/4
4/5
4/5
3/5
2/5
4/5
4/5
4/5
4/5
5/5
5/5
5/5
4/5
4/5
5/5
4/5
5/5
4/5
Table III. Number of instable microsatellite markers/number of informative markers for different percentages of tumour tissue mixed with normal tissue.
Bold numbers indicate that the sample is interpreted as MSI-H.
Patient 0%10%20%30%40%50%60%70%80%90%100%
1
2
3
4
5
1/5
0/5
0/5
0/5
0/5
0/5
2/5
0/5
1/5
1/5
0/4
1/5
2/5
0/5
0/5
2/4
1/5
1/5
1/5
2/5
3/5
1/5
2/5
2/5
2/5
1/5
2/5
1/5
3/5
3/5
3/5
2/5
2/5
3/5
3/5
3/5
4/5
3/5
3/5
3/5
4/5
2/5
4/5
2/5
3/5
4/5
4/5
4/5
5/5
4/5
4/5
4/5
5/5
5/5
4/5

Page 4

treated with formalin for one, four or 14 days. All markers
were amplified in all samples. Furthermore, no differences in
intensity of fluorescence signal were seen (data not shown).
A series of ten archive samples, ranging from four to 17
years of age, were analysed. All five markers were amplified
in the first run for samples up to six years of age and also in
one ten-year-old sample. For samples more than eight to ten
years old, not all five markers were successfully amplified but,
by repetitive PCR, one to four markers were informative (data
not shown).
Efficiency of PCR. With LMD of formalin-fixed and paraffin-
embedded CR tumours, 100,000 to 200,000 Ìm2 of 5-Ìm
sections were added into 20 Ìl buffer. Three Ìl were used in a
subsequent multiplex PCR as described and 83% of the
samples were successfully amplified for all five markers
initially. Twelve % and 1% had to be repeated once and twice,
respectively. Recollection because of evaporation had to be
done in 4% of the samples. Multiplex PCR of DNA extracted
from blood was efficient, all markers being informative in the
initial analysis.
Regional reproducibility of MSI. All primary tumours were
sampled at three pre-defined locations to test for the regional
reproducibility of MSI. In addition, when available a sample
was taken from a lymph node metastasis. The three samples
obtained from the nine solitary primary MSI-H tumours were
all also MSI-H. There were, though, differences in the
number of instable markers and the size of the PCR
product. Three samples from each of the three synchronous
primary tumours from patient 6 were all instable except in
one instance. Table IV shows all the results of the
intratumoral and metastasis samples.
Six of the ten MSS tumours had one instable marker in
one of the regional samples. In four cases minor shifts were
found in D17S250, in one case a minor shift in D2S123,
while the last instance was a two-base pair deletion in
BAT25. One tumour (patient 17) was perforated and
standard MSI analysis showed MSS, but repeated LMD
samples showed MSI-H. Immunohistochemistry of the four
major mismatch repair enzymes, MLH1, PMS2, MSH2 and
MSH6, demonstrated loss of the former two and focal
expression of the latter two in the tumour. It is likely that
the perforation led to a sampling error with the fresh
tumour, which could explain why MSI-H was not picked up
by the standard method.
All lymph node metastases from MSI-H tumours were also
MSI-H, while MSS lymph node metastasis was found from
MSS tumours (Table IV). As seen within the MSI-H primary
tumour, there were differences in the number of instable
markers and the size of the PCR product. In two cases (one
MSI-H and one MSS), it was not possible to find any tumour
cells in the sections of lymph nodes prepared for LMD.
Discussion
The MSI status of a CR tumour is important information
for the clinical management of this disease, primarily
because of the assumed, more favourable prognosis (3), but
also because of the linkage to HNPCC and the possibility of
early intervention for at-risk relatives in these families (5).
The patients with MSI-H tumours in this study were
characterised by a predominantly right-sided localisation
with only one rectal cancer. Five of the patients had local
lymph node metastasis but none had distant metastasis. This
is in concordance with initial observations characterising
MSI-H tumours as predominantly right-sided with a
reduced tendency to distant metastases (9). None had a
known family history of HNPCC.
The MSI status is traditionally found by a PCR-based
method on DNA isolated from a macroscopic specimen.
The use of a macroscopic piece of tissue for MSI analysis
has the obvious advantage that there is no scarcity of
material, making the assay robust even for low quality
specimens (10). The disadvantage is, however, that the piece
of tissue may not contain very many tumour cells due to
tumour heterogeneity and an admixture of normal tissue. It
may very well be that the inconsistent results we obtained
by routine methods in patient 17 were due to this
phenomenon. Table III illustrates the problems with MSI
ANTICANCER RESEARCH 26: 2069-2074 (2006)
2072
Table IV. Results of microsatellite analysis in three different locations in
the primary tumour and its lymph node metastasis. Bold numbers indicate
that the sample was interpreted as MSI-H. N/A=not available.
Patient Standard Central
basal
Basal
periphery
LuminalLymph
nodeMSI
1
2
3
4
5
6.1
6.2
6.3
7
8
9
10
11
12
13
14
15
16
17
18
19
20
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSS
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSS
MSS
MSS
MSS
MSI-L
MSS
MSI-H
MSI-L
MSS
MSS
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-L
MSS
MSS
MSS
MSI-L
MSS
MSI-H
MSS
MSS
MSS
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-H
MSI-L
MSI-H
MSI-H
MSI-H
MSI-H
MSI-L
MSS
MSS
MSS
MSS
MSS
MSI-H
MSS
MSS
MSS
MSI-H
N/A
MSI-H
N/A
MSI-H
N/A
N/A
N/A
N/A
MSI-H
N/A
N/A
MSS
N/A
N/A
N/A
MSS
N/A
N/A
N/A
MSS
MSI-L

Page 5

and low fractions of tumour cells. It was found that when
the ratio of tumour cells to normal cell was below 60%,
false-negative results were seen. Other groups have been
able to detect even lower fractions of tumour DNA in a
multiplex PCR (11). However, this level of detection is only
reproducible with larger amounts of template DNA, where
chance amplification (12) does not need to be considered.
Several groups have tried to improve the quality of the
tissue sampled for MSI analysis by microdissecting sections
of the tumour. The advantage of this approach is that the
tissue for MSI analysis has been verified to be tumour by
visual inspection. However, several problems can arise by
using this technique prior to MSI analysis. One problem is
the reduced sensitivity of the MSI assay due to a much
lower volume of tissue employed for the analysis. The
other, theoretical, problem is that there might be regional
differences in degree of MSI between different regions
within a specific tumour. If this is true, then using very
small samples of tumour could result in variable results,
depending on the sample size and the actual biopsy site of
the tumour.
In the present paper, these two issues were addressed.
With regard to the question of sensitivity, a decreasing
series of very small samples from sections of tumour
material were analysed. In these series, we estimated the
minimum amount of tissue needed for MSI analysis. All
markers were amplified for volumes of 25,000 Ìm3and
above and, therefore, we recommend this volume to be the
minimum for MSI analysis. This volume is equivalent to
25 cells assuming that one mg of tumour tissue contains
106cells and that the density of the tissue is 1 mg/mm3.
Quite often, both alleles are mutated in MSI-H tumours
and, since most of the markers are likely to be instable, MSI
analysis is less sensitive to failure of amplification of both
alleles or of all markers compared to, for example, allelic
imbalance analysis. For studies of allelic imbalance, a
minimum of 100 cells is, therefore, recommended (12). Our
results showed that the correct classification of a tumour as
MSI-H was obtained for even smaller amounts of tissue
than 25,000 Ìm3(Table II). This is also an indicator of the
robustness, even in sub-optimal settings, of the widely used
Bethesda guidelines from 1998 for MSI analysis (7).
The most frequent instable
mononucleotide repeats BAT25 and BAT26, which are
easily amplified. The dinucleotide markers D17S250 and
D2S123 were less consistent in our study but, in contrast,
they were less likely to be instable. Mutated dinucleotide
markers were also found to be the most frequent cause of
MSI-L in presumed MSS tumours. The MSI-H and MSS
phenotypes are well characterised, unlike MSI-L. MSI-L may
be regarded as a subgroup of MSS (13) caused by
insignificant microsatellite mutations or, as our results could
indicate, an insufficient amount or quality of template DNA.
markers are the
The question of possible differences in MSI status
between small samples taken at different locations was also
addressed. All tumours were systematically sampled at
three predefined locations and, in addition, one sample was
taken from local lymph node metastasis, if available. The
samples were analysed for MSI in parallel. We found that
with our microdissection method, the same degree of MSI
was found at all three locations in the primary tumour and
lymph node metastasis. This is in agreement with previous
findings from analysis of larger samples (14-18), and
verifies that the microdissection method is not influenced
by the sampling location.
The basic requirements of a MSI assay thus seem to be
met by our method. In order to apply it to clinical settings,
the effects of formalin treatment and long-term storage
were also examined. Formalin treatment up to 14 days
before paraffin embedding had no influence at all on the
amplification success or strength of the electrophoresis
fluorescence signal. However, long-term storage did have
some influence on the PCR product for samples more than
eight years old. Formalin treatment, storage and the
combination may cause DNA fragmentation and, perhaps
more interestingly, cross linking to proteins. Other groups
have found a strong negative effect of formalin treatment
on successful DNA amplification (19), but they did not
mention the use of proteinase K digestion, which was
applied in our method. The primer sets we used (8) were
designed for shorter PCR products than most standard
procedures, thus minimising the risk of interrupted
replication due to cross-linked proteins. To enhance the
effects of proteinase K all samples were treated for at least
16 hours.
MSI is a favourable prognostic factor, whereas its ability
to predict the effect of chemotherapy is still controversial
(3). Currently, the most important clinical implication for
MSI testing is the identification of HNPCC-like families for
further genetic testing and surveillance of at-risk relatives
(5). Further studies will clarify the value of MSI analysis as
a prognostic and predictive marker in sporadic CR cancers.
In conclusion, the method presented here for LMD and
multiplex PCR allows MSI analysis in selected cases with
tumour heterogeneity or scarce tumour material such as
lymph node metastasis.
Acknowledgements
This study was supported by grants from Vejle County, Denmark.
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tumor patients.
Received February 9, 2006
Accepted March 20, 2006
ANTICANCER RESEARCH 26: 2069-2074 (2006)
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