[CANCER RESEARCH 63, 5738 –5744, September 15, 2003]
Microsatellite Instability Is a Predictive Factor of the Tumor Response to Irinotecan
in Patients with Advanced Colorectal Cancer1
David Fallik,2 Francesco Borrini,2 Valérie Boige, Jérôme Viguier, Sandrine Jacob, Catherine Miquel,
Jean-Christophe Sabourin, Michel Ducreux, and Françoise Praz3
Departments of Medicine [D. F., V. B., M. D.] and Pathology [F. B., C. M., J-C. S.], Institut Gustave Roussy, Centre National de la Recherche Scientifique Unité Mixte de
Recherche 1598 [D. F., F. P.], and Unité Propre de Recherche 2169 [J. V., S. J., F. P.], Villejuif, France
ABSTRACT
The aim of our study was to assess the relationship between colorectal
tumor responsiveness to irinotecan and microsatellite instability (MSI), a
feature of colorectal tumors with DNA mismatch repair defect. Seventytwo patients with metastatic colorectal cancer were included in our retrospective study. A complete response to irinotecan was observed in 1
patient and a partial response in 10 patients, whereas 61 patients did not
respond to this treatment. We analyzed the protein expression of hMLH1,
hMSH2, and BAX by immunohistochemistry, determined the MSI phenotype, and looked for mutations in the coding repeats located in the
transforming growth factor ␤-RII, BAX, hMSH3, and hMSH6 genes. All
44 tumors analyzed expressed detectable levels of hMLH1; 1 tumor lacked
hMSH2 staining, whereas 4 tumors showed a marked decrease in BAX
expression. A better response to irinotecan was observed in the patients
whose tumors have lost BAX expression (P < 0.001). Among the 7 tumors
that displayed a MSI-H phenotype, 4 responded to irinotecan, whereas
only 7 of the 65 MSI-L/ microsatellite stable tumors did (P ⴝ 0.009). Seven
of the 72 tumors had inactivating mutations in the coding repeats of the
target genes. Three tumors displayed a mutation in the poly-A10 tract of
the transforming growth factor ␤-RII gene, associated with a 1-bp deletion in the poly-A8 tract of hMSH3 in one tumor and with a 1-bp deletion
in the poly-G8 tract of BAX in another. Four tumors displayed mutations
in the poly-G8 repeat of BAX, whereas 2 mutations in hMSH6 and hMSH3
were characterized. Among the 7 tumors with mutations in these target
genes, 5 responded to irinotecan, whereas only 6 of the other 65 tumors
did (P < 0.001), indicating that MSI-driven inactivation of target genes
modifies tumor chemosensitivity. Our observations allowed us to define
the first useful predictive criteria for irinotecan response in patients with
colorectal cancer.
INTRODUCTION
CRC4 is one of the most common adult malignant tumors affecting
1 person of 20 in Northern America and Western Europe. Although
about half of the patients may be cured with surgery, many will
develop metastatic disease, which necessitates chemotherapeutic
treatments (1). Fluorouracil modulated with folinic acid has been the
most extensively used first-line treatment for metastatic CRC, but
objective response to 5-FU is observed in only 20 –30% of the patients
Received 11/12/02; revised 6/26/03; accepted 7/8/03.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by grants from the “Institut Fédératif de Recherche 54, CNRS-Institut
Gustave Roussy” (#74538), the “Association pour la Recherche sur le Cancer” (#5366),
the “Ligue Nationale contre le Cancer-Comité de Seine-et-Marne,” and the “Groupement
des Entreprises Françaises dans la Lutte contre le Cancer.” D. F. and J. V. received
support from the “Association pour la Recherche sur le Cancer.” S. J. was supported by
the Ministère de l’Education Nationale, de la Recherche et de la Technologie.
2
D. F. and F. B. contributed equally to the work.
3
To whom requests for reprints should be addressed, at CNRS UPR 2169, “Genetic
Instability and Cancer,” Institut Fédératif de Recherche 54, Institut Gustave Roussy, 39
rue Camille Desmoulins, 94 800 Villejuif, France. Phone: 33 (1) 42 11 49 58; Fax:
33 (1) 42 11 50 08; E-mail: [email protected].
4
The abbreviations used are: CRC, colorectal cancer; MSI, microsatellite instability;
cMNR, coding mononucleotide repeat; CPT, camptothecin; DSB, double-strand break;
TGF, transforming growth factor; HNPCC, hereditary nonpolyposis colorectal cancer;
IHC, immunohistochemistry; IRI, Irinotecan; MMR, mismatch repair; MSI-L, low-level
microsatellite instability; MSI-H, high-level microsatellite instability.
(2). The antitumor activity of IRI, an analogue of CPT, has been
documented in 15–30% of patients with metastatic CRC after 5-FU
failure (3, 4).
Like CPT, IRI interferes with the catalytic cycle of the nuclear
enzyme topoisomerase I by stabilizing the covalent complex formed
between the DNA and enzyme (5). This results in an increase in the
number of single-strand breaks, as well as an inhibition of both
replication and transcription. The single-strand breaks may be converted into DSBs after replication fork collision (5). In a recent study,
we have investigated the possible involvement of the DNA MMR
system in the cytotoxicity of topoisomerase inhibitors and have shown
that CRC cell lines defective in MMR exhibit increased sensitivity to
CPT (6).
The MMR system is best known for its role in postreplicative repair
where it recognizes and repairs misincorporated bases, as well as
small insertion or deletion loops arising during DNA replication
(reviewed in Refs. 7–9). In addition to mutation avoidance, some of
the MMR components participate in various DNA repair processes,
including DSB repair and recombination (reviewed in Ref. 10). Furthermore, some MMR proteins have been involved in cell cycle
regulation and the induction of apoptosis in response to a variety of
DNA lesions (11–13). In human cells, mismatch recognition is performed by hMSH2 heterodimerized either with hMSH6 for base– base
mismatches and loops of one or a few nucleotides or with hMSH3 for
insertion/deletion of two or more extrahelical bases. Once bound to
mismatches, these complexes interact with another heterodimeric
complex, composed of hMLH1 and hPMS2 (14).
Germ-line alterations of MMR genes, usually hMSH2 or hMLH1,
cause susceptibility to HNPCC, a genetic disorder that accounts for
⬃5% of all cases of CRC (Refs. 15–17 and reviewed in Refs. 9,
18 –20). In HNPCC tumors, inactivation of the wild-type allele of the
inactive MMR gene most often results from loss of heterozygosity or
somatic mutation (21, 22). These tumors which display biallelic
inactivation of one of the MMR genes are characterized by high levels
of MSI, defined by the accumulation of mutations, mostly insertions
or deletions in short tandem repeats throughout the genome. In addition, the MSI phenotype is not confined to HNPCC tumors but also
occurs in ⱕ15% of sporadic CRC (23–25). Most of the mutations
arising in MSI tumor cells are located in untranslated intergenic or
intronic sequences. Yet, a number of genes whose sequence contains
short cMNRs have been reported to be frequently affected in colorectal MSI tumors. Among the possible targets are genes that encode
proteins involved in signal transduction (TGF␤-RII), apoptosis (BAX),
DNA repair (hMSH3 and hMSH6), transcription regulation, or inflammatory response (reviewed in Refs. 20 and 26).
Given the incidence of the MSI phenotype among CRCs, the fact
that a defect in MMR results in hypersensitivity to CPT may be
particularly relevant to the treatment of CRC (6). Specific predictive
criteria for IRI activity are cruelly lacking. The aim of our study was
to further investigate the relationship between the MSI phenotype, the
inactivation of target genes, the loss of MMR protein expression, and
the response to IRI in patients with metastatic CRC.
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MSI AND IRINOTECAN RESPONSE IN COLORECTAL CANCER
MATERIALS AND METHODS
Patients and Tumors. Seventy-two patients with advanced metastatic
CRC, whose disease had progressed under first-line 5-FU-based therapy, were
included in the present study. All patients were given IRI (Campto威, Aventis,
Antony, France) until disease progression or unacceptable toxic effects came
out. The chemotherapy regimen consisted of 300 –350 mg/m2 IRI infused
alone once every 3 weeks or 180 mg/m2 IRI combined with fluorouracil once
every 2 weeks. The end point was the tumor response to chemotherapy with
IRI defined according to the WHO criteria (27). The “responder” group
included the patients with either a complete response (disappearance of the
disease) or a partial response to the treatment (reduction of tumor volume of
ⱖ50%). The “nonresponder” group included patients with stabilized tumors
(volume reduction ⬍ 50% or enlargement ⱕ25%), as well as progressive
tumors (size enlargement ⬎ 25% or appearance of new lesions). The tumor
was located either proximal (ascending and transverse colon) or distal (descending and sigmoid colon) to the splenic flexure or in the rectum. The
clinicopathological characteristics of the patients included in our study are
shown in Table 1. The grading of differentiation was performed on the primary
tumors according to the WHO criteria. Tumors were classified as mucinous
when an area of extracellular mucin secretion was ⬎50%. The tumor samples
analyzed have been obtained from patients after failure of the first-line 5-FUbased therapy, before the second-line therapy.
Immunohistochemical Analysis. BAX immunostaining was performed on
the first 44 tumors included in our series. hMLH1 and hMSH2 was analyzed
on the same 44 tumors and the two additional MSI tumors (#115 and #150).
IHC for hMSH6 and hPMS2 was restricted to the MSI tumors. Four-micrometer sections from tissues fixed either in formalin or in Bouin and embedded in
paraffin were mounted onto glass slides, deparaffinized in xylene, and rehydrated through a graded alcohol series to distilled water. Antigen retrieval was
performed by immersing the slides in a 10 mM citrate buffer (pH 6 for hMLH1
and BAX and pH 7 for hMSH2, hMSH6, and hPMS2), and heating them in a
microwave oven for 30 min at 95°C. Sections were then incubated with mouse
monoclonal antibodies against hMLH1 (clone G168-15, 1:50 dilution; BD
PharMingen, San Diego, CA), hMSH2 (clone NA27, 1:30 dilution; Oncogene
Research Products, Darmstadt, Germany), hMSH6 (clone 44, 1: 200; BD
Transduction Laboratories), PMS2 (clone 37, 1:250 dilution; BD PharMingen),
or with rabbit polyclonal antibodies against BAX (A3533, 1:150 dilution;
DAKO, Glostrup, Denmark). Immunoreactivity was revealed with the DAKO
EnVision system® (DAKO) according to the manufacturer’s instructions and
using diaminobenzidine as a chromogen. Definite staining of adjacent nontumoral tissue served as an internal positive control.
DNA Extraction. Genomic DNA was extracted from 7-␮m-thick paraffinembedded tissue sections from primary tumors or metastatic lesions after
fixation either in a Bouin solution or in formalin or from frozen samples. Areas
of tumor tissue containing a ratio of ⬎90% neoplastic cells were selected
previously by microscopic examination of a reference slide stained with H&E.
Tumor tissue corresponding to ⬃1 cm2 was incubated for 2 h at 37°C in a 10
mM Tris buffer (pH 8.3) containing 50 mM KCl, 2.5 mM MgCl2, 0.5% Tween
20, 0.5% Triton X-100, and 120 ␮g/ml proteinase K (200 ␮l/cm2 slide).
Proteinase K was inactivated by a 10-min incubation at 95°C. Undigested
remnant debris was removed by centrifugation.
cMNR-MSI Phenotype Analysis. Frameshift mutations were screened in
the mononucleotide repeats located in the coding region of the TGF␤-RII,
BAX, hMSH3, and hMSH6 genes. The primers used to amplify the 82-bp region
(nucleotide 661–742, sequence accession no. M85079) encompassing the
poly-A10 tract (nucleotide 649 – 658) of TGF␤-RII were 5⬘-ATGACTTTATTCTGGAAGATGCTG-3⬘ and 5⬘-CACATGAAGAAAGTCTCACCAGGC3⬘. The region containing the poly-G8 repeat in the coding sequence of BAX
was amplified using 5⬘-TTCATCCAGGATCGAGCAGGGCGA-3⬘ and 5⬘CACTCGCTCAGCTTCTTGGTGGAC-3⬘ as primers. The oligonucleotides,
used to amplify the regions encompassing the poly-A8 and poly-C8 tracks in
hMSH3 and hMSH6, respectively, were as described previously (28). A first
round of PCR was performed on 50 –200 ng of genomic DNA with unlabeled
primers in a total volume of 20 ␮l using 200 ␮M deoxynucleoside triphosphate
(Amersham Pharmacia Biotech) and 0.5 unit of TaqDNA polymerase (Amersham Pharmacia Biotech). After an initial 10-min denaturation step at 95°C,
amplification was achieved by 35 cycles consisting of 1-min denaturation at
95°C, 1-min annealing at 55°C, and 1-min elongation at 72°C, followed by a
final 5-min elongation step at 72°C. A second round of PCR comprising 5–10
cycles was performed on 1/20 PCR1 product using sense primer labeled with
6-FAM, NED, or HEX fluorescent dye. After a 5-min heating at 95°C,
mixtures of PCR products (1 ␮l), formamide-loading buffer, and ROX-labeled
molecular weight markers (GS-400HD-ROX; Applied Biosystems) were
loaded onto a denaturating 4.75% polyacrylamide/8 M urea gels and run at
1600 V for 6 h, using an ABI373 automated fluorescent DNA sequencer.
Fragment sizes were determined using the GeneScan Analysis software. PCR
detecting abnormal products was repeated twice to confirm the results.
Analysis of hMSH2 Exons. PCR was performed on genomic DNA using
primers located at the intron-exon boundaries of the 16 hMSH2 exons, as
described (29).
MSI Phenotype Analysis. The two noncoding quasi-monomorphic BAT25
and BAT26 mononucleotide microsatellites were analyzed using the primers
published previously labeled with 6-FAM and NED, respectively (30, 31). The
three dinucleotide markers D2S123, D5S346, and D17S250 were analyzed as
described using primers labeled with 6-FAM, HEX, and HEX, respectively
(32). Tumors were classified as MSI-H, if two or more of the five markers
showed MSI, and MSI-L, if only one of the five markers did. A single round
of PCR was achieved by a 15-min denaturation step at 95°C, which allowed
Taq polymerase activation, followed by 50 cycles of 30 s at 96°C, 30 s at 50°C,
and 30 s at 72°C using 1 unit of HotStarTaq DNA Polymerase (Qiagen).
Fluorescent PCR products were mixed with formamide and GS-HD400-ROX
molecular weight standards and run on a short capillary containing GS Performance Optimized Polymer 4, at a voltage of 15 kV on the ABI 310 Genetic
Analyzer.
Statistical Analysis. The associations between the tumor response to IRI,
the defect in BAX expression, and the cMNR-MSI or MSI phenotypes were
assessed using Fisher’s exact test.
RESULTS
Table 1 Clinical and histological characteristics of the patients with advanced
colorectal cancer treated with irinotecan
Clinical parameters
Age at diagnosis (yr)
ⱖ50 yr
⬍50 yr
Mean ⫾ SE (range)
Gender
Male
Female
Primary tumor site
Rectum
Distal colon
Proximal colon
Degree of differentiationa
Well
Moderate
Poor
Mucinous
a
n
Percentage
45
27
53.6 ⫾ 10.9 (26–80)
62.5
37.5
41
31
56.9
43.1
14
36
22
19.4
50
30.6
19
17
3
5
43.2
38.6
6.8
11.4
The degree of differentiation is only indicated for the 44 primary tumors.
Patient Selection and Tissue Samples. Seventy-two patients with
CRC were included in the present series, 41 males and 31 females
with a mean age of 53.6 ⫾ 10.9 year (range 26 – 80). Patient clinical
characteristics are given in Table 1. All these patients received IRI as
second- or third-line treatment after disease progression on 5-FUbased chemotherapy. In our series, the responder group was constituted by the only patient (1.4%) who showed a complete response to
IRI and the 10 patients (13.9%) with a partial response. The nonresponder group included the 29 patients (40.3%) with stabilization and
the 32 patients (44.4%) with progressive disease. Twenty-two (30.6%)
tumors were located in the proximal colon, 36 (50%) in the distal
colon, and the remaining 14 (19.4%) in the rectum. Tumor specimens
were obtained from 44 surgically resected large bowel primary tumors, 9 local peritoneal metastasis, and 19 hepatic metastases. Of the
44 primary CRC, 19 were well differentiated (G1), 17 were moderately differentiated (G2), and 3 were poorly differentiated (G3); 5
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MSI AND IRINOTECAN RESPONSE IN COLORECTAL CANCER
Fig. 1. Immunohistochemical analysis of BAX
protein expression in colorectal tumors. A, primary
tumor (#28) with a strong cytoplasmic BAX expression (3 T) in the apical portions of normal
colonic epithelial cells (3 C) and in the lymphocytes (3 L). B, no detectable BAX immunoreactivity in a liver metastasis of an MSI tumor (#34)
with a BAX frameshift mutation (3 T), with positively stained infiltrating lymphocytes (3 L) and
endothelial cells (3 E) serving as controls. A and
B, magnification: ⫻200.
tumors were mucinous. Because the tumor samples analyzed have
been obtained from patients after failure of the first-line 5-FU-based
therapy, before the second-line therapy, they may be considered as
resistant to 5-FU.
Expression of hMLH1, hMSH2, hMSH6, and hPMS2 and Response to Chemotherapy with IRI. Protein expression for hMLH1
and hMSH2 was examined in 46 tumors. Both hMLH1 and hMSH2
were normally expressed in the nuclei of normal colonic mucosa and
confined in crypt epithelial cells, stromal cells, and lymphocytes. One
tumor exhibited an absence of nuclear staining of hMSH2 (#34) with
positively stained stromal cells in the surroundings (data not shown).
This tumor was poorly differentiated and arose in the proximal colon
in a young patient who had a familial history of CRC. This hMSH2negative tumor responded to the treatment with IRI. The expression of
hMLH1 was detectable in all 46 tumors analyzed, but two tumors
displayed low hMLH1 immunostaining intensity; one corresponded to
a primary lesion, and the other was a hepatic metastasis. Both tumors
primarily arose in the distal colon and were moderately differentiated.
These 2 hMLH1-low tumors were observed in patients over 50 years
and did not respond to IRI. The immunohistochemical detection of
hMSH6 and hPMS2 has been performed for tumors that displayed
MSI. The quality of staining with these antibodies was suboptimal for
two archival samples (#31 and #34), most probably because of poor
preservation. Among the six tumors that could be assessed, none
displayed an unambiguous complete loss of expression of either
hMSH6 or hPMS2 (data not shown).
Detection of hMSH2 Exonic Deletion. To determine whether
hMSH2 loss of expression in tumor #34 resulted from an hMSH2
genomic deletion, we have performed PCR on genomic DNA using
primers located at the intron– exon boundaries, as described (29). No
genomic deletion could be identified through the screening of all 16
exons of hMSH2 (data not shown).
Expression of BAX and Response to Chemotherapy with IRI.
BAX was normally expressed in the apical portion of cytoplasm of
normal colonic epithelial cells and cytoplasm of lymphocytes. BAXpositive immunostaining of tumor cells was detected in 40 of the 44
cases analyzed by IHC (Fig. 1A). Yet, both the ratio of positively
stained cells and intensity of BAX staining were heterogeneous on the
same slide. Four tumors definitely lacked detectable expression of
BAX, with the adjacent nontumoral tissue being positively stained.
The staining pattern of one of the BAX-negative tumors (#34) is
shown in Fig. 1B. Two of them corresponded to hepatic metastases
and one was a peritoneal metastasis, whereas the fourth specimen was
a primary rectal lesion. Interestingly, a partial response to IRI was
achieved in all 4 BAX-negative tumors, whereas among the 40 tumors
that expressed normal levels of BAX, only 3 patients experienced
tumor regression (Table 2). Statistical analysis indicated that the loss
of BAX expression is a predictive marker for the response to IRI
chemotherapy in metastatic CRC (P ⬍ 0.001).
Association of MSI with Response to Chemotherapy with IRI.
Tumor MSI analysis was first performed using two quasi-monomorphic mononucleotide repeats, BAT25 and BAT26 (Table 3). The
tumors that displayed instability in either mononucleotide markers
were further analyzed using the three dinucleotide repeats of the
Bethesda panel and classified according to the National Cancer Institute recommendations (31). Among the 72 tumors, 7 (9.7%) displayed
Table 2 Relationship among BAX protein expression, frameshift mutation in the BAX-poly G8 tract, and the response of metastatic colorectal cancer to the treatment
with irinotecana
Case
Primary tumor
site
Tumor specimen
studied
Irinotecan tumor
response
21
31
34
37
150
Rectum
Distal
Proximal
Distal
Proximal
Primary tumor
Metastasis
Metastasis
Metastasis
Primary tumor
Partial regression
Partial regression
Partial regression
Partial regression
Stable disease
Bax
Immunohistostaining
Not
Not
Not
Not
nd
detectable
detectable
detectable
detectable
BAX-G8 mutation
Wild-typeb
Mutant
Mutant
Mutant
Mutant
a
The primary tumor site is proximal for tumor originating in the ascending and transverse colon, or distal for the descending and sigmoid colon. The response to irinotecan was
scored according to the WHO criteria.
b
Wild-type, both BAX alleles have a normal poly-G8 tract; Mutant, deletion or insertion of G in the poly-G8 tract; nd, not determined.
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MSI AND IRINOTECAN RESPONSE IN COLORECTAL CANCER
Table 3 Relationship between MSI phenotype, frameshift mutation in cMNR of target genes, and the response of metastatic colorectal cancer to the treatment with irinotecana
Case
Primary tumor site
17
31
34
37
43
46
115
150
Proximal
Distal
Proximal
Distal
Distal
Proximal
Proximal
Proximal
Tumor specimen studied
Irinotecan tumor response
MSI phenotype
TGF␤-RII
BAX
hMSH3
hMSH6
Primary tumor
Metastasis
Metastasis
Metastasis
Primary tumor
Primary tumor
Primary tumor
Primary tumor
Partial regression
Partial regression
Partial regression
Partial regression
Progression
Partial regression
Progression
Stabilization
High
Low
High
High
High
High
High
High
mutb
—
—
—
—
mut
—
mut
—c
mut
mut
mut
—
—
—
mut
mut
—
—
—
—
—
mut
—
—
—
—
—
—
—
mut
mut
a
The primary tumor site is proximal for tumor originating in the ascending and transverse colon or distal for the descending and sigmoid colon. The response to irinotecan was
scored according to the WHO guidelines. The MSI phenotype is defined according to the Bethesda recommendations.
b
mut, presence of a mutation in the target gene mononucleotide repeats.
c
—, no mutation.
biallelic size variations of BAT25 and/or BAT26 that were characteristic profile in MSI tumors. The observed sizes were shortened by
5–10 bp for BAT25 and 6 –12 bp for BAT26, which is far outside the
range of normal allelic sizes. In four cases (#17, #34, #46, and #150),
both BAT25 and BAT26 were unstable, whereas two tumors (#43 and
#115) displayed instability at the BAT26 locus, with a normal BAT25
profile. In tumor #37, BAT26 could not be amplified, whereas BAT25
was stable. Using the complete Bethesda panel, this tumor, like the
tumors that displayed instability in BAT25 and/or BAT26, could be
unambiguously classified as MSI-H because at least two additional
markers displayed instability (Table 3). Among the 44 primary colorectal tumors, 5 (11.4%) were MSI-H, whereas 2 of the 28 (7.1%)
metastatic lesions analyzed displayed high MSI, indicating that in our
series, the incidence of MSI tumors did not differ significantly between primary colorectal tumors and metastases (P ⫽ 0.7, nonsignificant). Among these 7 MSI-H tumors, 5 arose in the proximal colon
and 2 in the distal colon; no cases of rectal cancer were found in this
group. Four (57.1%) of these MSI-H tumors partially regressed on
treatment with IRI, whereas 1 was stabilized, and 2 progressed.
Among the 65 MSI-L/microsatellite stable tumors, 7 (10.8%) tumors
responded to IRI, 28 were stabilized, and 30 continued to progress
under treatment. The relationship between the MSI phenotype and
response to IRI chemotherapy is statistically significant (P ⫽ 0.009;
Table 4).
Relationship between Mutations in cMNRs and the Response to
IRI. We looked for frameshift mutations in the cMNRs contained in
four genes, TGF␤-RII, BAX, hMSH3, and hMSH6 (Table 3). We
analyzed the TGF␤-RII poly-A10 microsatellite and found three primary tumors displaying a frameshift mutation because of a 1-bp
deletion (#17, #46, and #150). All three tumors with mutations in
TGF␤-RII exhibited high levels of instability at the Bethesda markers
and occurred in the proximal colon. Two of them (#17 and #46)
partially regressed on treatment with IRI; one (#17) of these tumors
also carried a 1-bp deletion in the poly-A8 tract of the hMSH3 gene.
The third tumor carrying a mutation in TGF␤-RII (#150) also displayed a 1-bp insertion in the poly-C8 of hMSH6 and a 1-bp deletion
in the poly-G8 repeat of BAX and remained stable on treatment (Table
Table 4 Relationship between the response of patients with advanced colorectal cancer
to chemotherapy with irinotecan and tumor alterationsa
a
Responder
Nonresponder
BAXmutant
BAXwt
3
8
1
60
P ⫽ 0.01
MSI-H
MSI-L/MSS
4
7
3
58
P ⫽ 0.009
MNR-MSI⫹
cMNR-MSI⫺
5
6
2
59
P ⬍ 0.001
The associations between the tumor response to irinotecan and the mutations in the
BAX-polyG8 tract, the MSI, or cMNR-MSI phenotypes were assessed using Fisher’s
exact test.
3). Three unrelated tumors (#31, #34, and #37) displayed a 1-bp
deletion in the poly-G8 tract of BAX, which resulted in the extinction
of BAX expression as assessed by IHC. No other mutation in the
target genes studied could be found in these three BAX-negative
tumors. Interestingly, all these three cases corresponded to hepatic
metastases and partially responded to IRI. Using the Bethesda panel,
two of them (#34 and #37) could be classified MSI-H, whereas the
third tumor (#31) was MSI-L. In one of the BAX-mutated MSI-H
tumors (#34), the expression of hMSH2 was undetectable by IHC.
Two tumors with mutations in hMSH6 could be detected in our series
(#115 and #150); both these mutations resulted from a 1-bp addition
in the poly-C8 tract and occurred in MSI-H tumors. One of them
(#150) also had a 1-bp deletion in both TGF␤-RII and BAX genes,
whereas the other had a 1-bp deletion in MSH3 (#115).
Interestingly, 5 of the 7 (71.4%) tumors displaying inactivation of
TGF␤-RII, BAX, or the hMSH3 gene responded to IRI, versus 6 of the
65 (9.2%) tumors without any mutation in the cMNRs of these genes
(P ⬍ 0.001; Table 4).
DISCUSSION
Numerous studies have reported that tumors with or without MSI
display different clinicopathological features. Indeed, MSI CRC are
more likely to be of high histological grade, located in the proximal
colon and associated with improved overall survival (33, 34). There is
increasing evidence of a relation between the MMR status of tumor
cells and their response to various chemotherapeutic drugs (reviewed
in Refs. 35 and 36). In particular, in vitro studies have shown that
MMR-deficient cell lines display moderate levels of resistance to
methylating agents and low level resistance to cisplatin (37, 38). More
recently, the hMLH1-deficient HCT116 CRC cell line, a prototype of
MSI cell lines, was found to be slightly more resistant to the cytotoxicity of 5-FU (39, 40). Unfortunately, the few clinical studies that
have been performed addressing this issue have come to contradictory
conclusions. A recent study reported survival benefits in patients with
MSI tumors who received adjuvant treatment with 5-FU, but these
results have been challenged (41, 42). Yet, the analysis of survival
benefit in a similar group of patients led these authors to opposite
findings (41).
To define molecular criteria predictive for tumor responsiveness to
chemotherapy, we have investigated previously the role of MMR in a
panel of MMR-defective colorectal cell lines and shown that a defect
in MMR results in increased sensitivity to CPT (6). Although neither
p53 status nor endogenous topoisomerase I levels could predict the
cellular sensitivity to CPT, we could establish that MMR status of the
cells is a critical determinant for chemosensitivity of colorectal cell
lines (6). In addition, using a model of CRC xenografts in nude mice,
it has also been shown that the MSI phenotype moderately increases
sensitivity to IRI (43). This is in agreement with our preliminary data
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MSI AND IRINOTECAN RESPONSE IN COLORECTAL CANCER
obtained in a previous study in which we evaluated the impact of
MSI-driven mutations on the tumor sensitivity to IRI (44).
These observations prompted us to further evaluate the impact of
the MSI phenotype on the response of CRC to the treatment with IRI.
All 72 patients included in our series had a metastatic CRC with
progressive disease on treatment with fluorouracil-based regimen.
One patient had personal and family cancer history reminiscent of
HNPCC. Among the tumors analyzed by IHC, only the tumor that
occurred in the young patient with a family history of CRC exhibited
a defect in hMSH2 expression, whereas none had a complete absence
of hMLH1. Although hMSH2 is frequently inactivated by exonic
rearrangements in HNPCC tumors, we have not detected any gross
gene deletion that could account for the loss of hMSH2 expression
observed in tumor #34 (45, 46). Given that we were unable to detect
loss of immunostaining of either hMSH6 or hPMS2, it is rather
unlikely that a defect in these genes accounts for the MSI phenotype.
Yet, in a recent study that assessed the expression pattern of all four
MMR proteins in a series of MSI tumors, it was reported that loss of
hMSH6 only or hPMS2 only occurred in rare instances (47). Abnormal expression of hMSH6 was mainly observed in hMSH2-negative
tumors, whereas loss of hPMS2 expression was concomitant with loss
of hMLH1 expression, confirming that in the absence of their partner,
these proteins are unstable (47). Given that hMLH1 inactivation is
responsible for the majority of MSI sporadic CRC, this phenomenon
is expected to occur in ⬃10 –15% of unselected primary CRC (23, 24,
48). Whether the occurrence of hMLH1 inactivation in metastatic
CRC is comparable has not yet been reported. Yet the loss of hMLH1
expression appears to be less frequent among the 5-FU-resistant
metastatic CRC cases that we have analyzed. Several explanations
may account for this observation: (a) cells that had lost hMLH1
expression in the primary tumor may have been counter-selected on
the treatment with 5-FU; (b) the selection of tumor cells with high
metastatic potential that occurs during tumor progression may apply
to cells expressing hMLH1; and (c) several drugs are known to induce
demethylation of DNA resulting in the re-expression of genes when
silencing is caused by promoter hypermethylation. In particular, this
phenomenon has been documented for hMLH1 both in in vitro experiments and in a model of human colorectal tumor xenografts (24,
49). Thus, it is conceivable that re-expression of hMLH1 has taken
place in a subset of tumor cells, particularly when patients are treated
with DNA-damaging drugs.
We have determined the MSI phenotype using both BAT26 and
BAT25 microsatellites, because their sensitivity and specificity are
very similar to those of the Bethesda panel, allowing to establish the
tumor MSI status with ⬎99% accuracy, with no need for normal
matched DNA (31, 32, 34, 50, 51). Interestingly, because BAT25 and
BAT26 are mononucleotide repeats, they display instability not only
in tumors with a defect in either hMSH2 or hMLH1 but also in
hMSH6-deficient tumors (32, 52, 53). Given that we had observed that
colorectal cells with a defect in hMSH6 also displayed increased
sensitivity to CPT, we decided to use both BAT25 and BAT26 as
phenotypical markers of the MSI phenotype to efficiently screen the
tumors whose MSI was restricted to mononucleotide repeats (6). We
have further performed MSI analysis using the Bethesda panel on the
cases that displayed instability at either BAT25 or BAT26 mononucleotide repeats or in the coding repeats. As expected, we have
observed that tumors displaying MSI at BAT26 and/or BAT25 loci
were MSI-H tumors, confirming that the use of BAT26 and BAT25
markers allows unambiguous identification of MSI tumors. It is worth
noting that the rate of MSI tumors in our series of metastatic CRC is
within the range reported for unselected familial and sporadic CRC,
challenging the idea that MSI colorectal tumors have a reduced risk of
liver metastasis (54 –56).
We further investigated the presence of inactivating mutations in
coding repeats of genes whose role in colorectal carcinogenesis had
been suspected. These included TGF␤-RII, a potent inhibitor of cell
growth and tumor progression, BAX, a proapoptotic member of the
Bcl-2 family, as well as hMSH3 and hMSH6, two DNA MMR
components (28, 57, 58). Seven tumors displayed a mutation in at
least one of the genes analyzed. Three primary CRC tumors displayed
a frameshift mutation in the TGF␤-RII gene. The inactivation of the
TGF␤-RII gene has been reported in 70 –90% of MSI CRC and is
believed to occur at an early stage, during the transition from colon
adenoma to carcinoma (30, 59 – 62). In our series, the TGF␤-RII
mutations are underrepresented and restricted to primary tumors,
indicating that they may be counter-selected during the metastatic
process. Conversely, all MSI-driven mutations of BAX were observed
in metastatic lesions. Although the inactivation of the BAX gene
occurs in approximately half of the MSI primary CRC, data concerning hepatic metastasis are not available (58, 60, 63). BAX inactivation
is apparently not required for the initiation step of the tumorigenic
process but rather confers a selective advantage during clonal evolution (60, 64). It is remarkable that the expression level of BAX is
significantly lower in the metastases compared with the primary
colorectal tumors (65). The tumors with the lowest expression of BAX
displayed a more infiltrative growth pattern and more distal metastases (65). In this context, our results showing that BAX inactivation was
predominantly observed in hepatic metastases indicate a possible role
of BAX in the metastatic progression of CRC.
The molecular mechanisms underlying the hypersensitivity of MSI
tumors to IRI are not yet clear. In most tumors, the MSI phenotype
results from the inactivation of either hMSH2 or hMLH1, two components of MMR that have been shown to participate in recombination. Given that IRI acts by generating DSB in DNA, a decrease in
recombinational repair efficiency resulting from a defect in MMR
could account for the higher chemosensitivity of MSI tumors, a
hypothesis that is currently under investigation in our laboratory.
Moreover, a link between MMR deficiency and loss of normal cell
cycle control, particularly G2 arrest, has been established. Because
DSBs are lethal lesions if not repaired before mitosis, a defect in G2
checkpoint in response to IRI-induced damage may also contribute to
increase its cytotoxicity. In addition, any gene that contains a microsatellite repeat is a potential target for MSI-driven insertion/deletion
mutations. Consequently, MSI tumors accumulate widespread mutations, not only in genes that participate in tumor initiation and progression but also in genes that are involved in various DNA repair
pathways, e.g., several reports have shown that both MRE11 and
RAD50 are frequently inactivated in MSI tumors (66, 67). These
genes being part of the MRE11-NBS1-RAD50 complex, which plays
a key role in DSB repair, it is reasonable to speculate that their defect
contributes to enhance IRI-induced cytotoxicity. Other DNA repair
genes contain coding microsatellite coding repeats and may therefore
be inactivated in MSI tumors. It follows that the sensitivity of MSI
CRC to IRI may not be a direct consequence of MMR deficiency itself
but may rather be the result of the impairment of a crucial DNA repair
pathway.
In conclusion, this study allowed us to establish that the MSI
phenotype and loss of BAX expression are thus far the best criteria for
selecting patients who could benefit from chemotherapy with IRI.
Therefore, provided that these results are confirmed on a larger series,
MSI phenotyping should be routinely performed to improve the
clinical management of patients with CRC.
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MSI AND IRINOTECAN RESPONSE IN COLORECTAL CANCER
ACKNOWLEDGMENTS
We thank Prof. Pierre Netter and Drs. Brigitte Bressac-de-Paillerets, Alain
Sarasin, and Anne Stary for stimulating discussions and critical reading of this
manuscript. We also thank Drs. Pierre Duvillard and Philippe Lasser for their
invaluable contribution and constant support, as well as Sophie de Oliveira,
Johny Bombled, Sylvanna Scolaro, and Valérie Velasco for their excellent
technical assistance. We also thank Rick Willett for correcting our English.
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Response to Irinotecan in Patients with Advanced Colorectal
Cancer
David Fallik, Francesco Borrini, Valérie Boige, et al.
Cancer Res 2003;63:5738-5744.
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