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Regular Articles |
From the Division of Gastrointestinal/Liver Pathology, Department
of Pathology*
and Oncology Center,
The Johns Hopkins University School of Medicine, Baltimore, Maryland;
Department of Medicine,
Hereditary Cancer
Center, Creighton University School of Medicine, Omaha, Nebraska;
Department of Pathology,§
School of Medicine,
University of Auckland, New Zealand; and Memorial University of
Newfoundland,¶
Health Sciences Centre, St.
John's, Newfoundland, Canada
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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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About 15% of colorectal cancers are characterized by microsatellite instability (MSI), also termed DNA replication errors or ubiquitous somatic mutations (reviewed in 7 to 16 ). Inactivation of one of a group of genes whose products participate in postreplicative repair of nucleotide mismatches leads to insertions and deletions of nucleotides in intrinsically unstable repeated sequences (ie, microsatellites) throughout the genome because of defective repair of the slippage mistakes made by DNA polymerases. MSI-positive tumors thus accumulate numerous frameshift mutations but also have a mutator phenotype that increases both base substitution mutations and frameshift mutations in expressed genes. In patients with hereditary nonpolyposis colorectal cancer syndrome (HNPCC, Warthin-Lynch syndrome; reviewed in 17 and 18 ), germline mutation of hMSH2 (human MutS homolog 2), hMLH1 (human MutL homolog 1), hPMS1 or hPMS2 (human postmeiotic segregation 1 and 2), or the GTBP (guanine/thymidine mismatch-binding protein)/hMSH6 gene predispose to tumorigenesis. In addition to germline and somatic alterations in these genes in HNPCC, somatic inactivation alone of mismatch repair genes have been identified as a cause of MSI in sporadic tumors. Loss of immunohistochemical expression of hMSH2 and hMLH1 gene products in MSI-positive tumors has been reported.19-23 MSI-positive colorectal carcinomas in both the inherited and sporadic settings have unusual pathological manifestations, including right-sided predominance and high frequency of large size; poorly differentiated, medullary, or mucinous histopathological type; and prominent lymphoid inflammatory response.24-31
Tumors with widespread MSI have extensive subtle alterations in repeated nucleotide sequences, including those within the coding regions of genes. Inactivation of the APC gene by subtle mutation is common in MSI-positive neoplasms,32 although some studies have reported low rates of APC mutation.33,34 In clear contrast to usual colorectal cancer, mutations are frequent in MSI-positive tumors among mononucleotide or other small repeats within the gene for the transforming growth factor ß type II receptor (TGFß RII),33-43 the E2F-4 transcription factor gene (reviewed in Ref. 44 ),45-47 the insulin-like growth factor II receptor gene,48,49 the hMSH3 and hMSH6 mismatch repair genes,47,50,51 and the Bax gene for a BCL-2-related protein50,52-54 that promotes apoptosis (reviewed in Ref. 55 ). Because of the high frequency of mutation in microsatellite sequences throughout the genome, it is not clear that the intragenic mutations are causally related to tumor progression, and many genes with repeat sequences do not show instability.54,56 In addition, allelic losses are infrequent, and total DNA content of tumor cells is typically in the normal range. Little is known, however, about the pattern in MSI-positive tumors of accumulated alterations in the oncogenes and suppressor genes that are important in the progression of usual colorectal neoplasia, because only small numbers of MSI-positive tumors have been studied, and conflicting results have been reported. For example, ras proto-oncogene mutations in MSI-positive colorectal carcinomas were reported to occur at frequencies similar to microsatellite-stable (MSS) cancers in some series28,34,40,57,58 but at low frequency in others.33,43,59-61 For p53 alterations, conflicting reports of similar27,42,57,58 or lower28,30,33,34,43,55,59-63 frequencies of abnormalities have also appeared. Furthermore, alterations such as loss of heterozygosity of chromosome 8p have been reported at different frequency in sporadic MSI-positive tumors than in HNPCC MSI-positive tumors,33 and frequency of mutation of some genes (eg, E2F-4) differed with severity of MSI.46
To address these uncertainties about genetic alterations in MSI-positive tumors, we studied the spectra of mutations in a large series of colorectal carcinomas from patients with HNPCC or sporadic colonic cancer. We evaluated MSI status; immunohistochemical expression of hMSH2 and hMLH1 gene products; mutation of the Kirsten ras proto-oncogene; allelic deletion of the long arm of chromosome 18q where the DCC, DPC4/Smad4, and JV-18/MADR2/Smad2 genes reside; overexpression of p53 gene product and mutation of the p53 gene; and mutations in nucleotide repeat sequences in the coding regions of the TGFß RII, E2F-4, and Bax genes. The results have implications for the diagnosis and treatment of MSI-positive tumors as well as understanding of the biology of colorectal neoplasia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied 39 colorectal cancers from patients in 20 families,
that met International Collaborative Group criteria for
HNPCC64
and/or had germline mutation of hMSH2 or
hMLH1,65,66
and 57 sporadic right-sided colonic
cancers located proximal to the splenic flexure (Table 1)
. The HNPCC cancers (HNPCCa) were
obtained for collaborative studies of the genetics of HNPCC from
registries at the Department of Preventive Medicine/Public Health,
Creighton University School of Medicine (Omaha, NE)
(n = 19); Department of Pathology, University of
Auckland School of Medicine (Auckland, NZ) (n =
5); Memorial University of Newfoundland (St. John's, Newfoundland,
Canada) (n = 5); and The Johns Hopkins
University School of Medicine (Baltimore, MD) (n
= 10). Germline mutation of the known mismatch repair genes in an
affected family member was evaluated previously by in vitro
synthetic protein assays and by sequencing of hMSH2,
hMLH1, hPMS1, hPMS2, and
GTBP in 17 of the families: 7 had hMSH2 mutation,
5 had hMLH1 mutation, and 5 had no mutation
found.67
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Three colon cancer cell lines with known MSI status and mismatch repair gene mutation status were used as controls in various analyses: LoVo is MSI positive with reported homozygous deletion of the hMSH2 gene,75 HCT116 is MSI positive with mutation of hMLH1, and SW480 is MSS with no known mutation of either mismatch repair gene.
Microdissection and DNA Extraction
For all tumors, areas of carcinoma and nonneoplastic control tissue were microdissected from numbered, routine buffered, formalin-fixed, paraffin-embedded tissue sections. Areas of cellular tumor with minimal stroma and inflammatory cells were selected, and multiple areas with high cellularity from each slide were combined, favoring detection of clonal abnormalities. DNA was extracted as in our previous studies.74,76 Frozen tissue was available for 11 MSI-positive SRSCCa; cryostat microdissection and DNA extraction for restriction fragment length polymorphism analysis were done as described previously.77,78 DNA was prepared from fresh aliquots and from histopathological sections of formalin-fixed, paraffin-embedded cell blocks of LoVo, HCT116, and SW480.
MSI Analysis
Five noncoding polymorphic dinucleotide repeat sequences on the
long arm of chromosome 18 (D18S69, D18S64, D18S55, D18S61, and D18S58
from centromere to telomere) and the noncoding nonpolymorphic
polyadenine (poly(A)) tract in the fifth intron of the
hMSH2 gene (BAT26) were chosen for determination of MSI
status by polymerase chain reaction (PCR) amplification, as in our
previous studies.36,74
The normal alleles were usually
represented by a major band accompanied by a few minor bands. Mobility
shift of PCR products from tumor DNA as compared with corresponding
nonneoplastic tissue was determined for each marker independently
(Figures 1 and 2)
. MSI-positive status of a tumor was
defined by mobility shift in at least two of five or six markers. The
MSI status of the SRSCCa based on the dinucleotide repeat sequences was
included in our previous publications.26,74
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Sections (6 µm) of formalin-fixed, paraffin-embedded cancers were deparaffinized with xylenes for 30 minutes and rehydrated using graded ethanols. Antigen retrieval was performed using a heat-induced epitope retrieval method.79 Immunoperoxidase staining using diaminobenzidine as chromogen was performed with the TechMate 1000 automatic staining system (Ventana/BioTek Solutions, Tucson, AZ). Immunoglobulin G mouse monoclonal antibody to hMSH2 gene product (Ab-2, Oncogene Science, Cambridge, MA) and two immunoglobulin G mouse monoclonal antibodies to hMLH1 gene product (Ab-1, Pharmingen, San Diego, CA, and NA28, Calbiochem, Cambridge, MA) were used. Counterstain was light hematoxylin.
To establish utility of antibodies and dilutions for use,
histopathological sections of buffered formalin-fixed,
paraffin-embedded cell blocks of the LoVo, HCT116, and SW480 colon
cancer cell lines were prepared. The sections were stained along
with eight MSI-positive HNPCCa from patients with known germline
mutation status of the hMSH2 and hMLH1 mismatch
repair genes (four with hMSH2 mutation and four with
hMLH1 mutation). Nuclear staining for hMSH2 gene product was
absent from the LoVo cell line (Figure 3)
with reported homozygous deletion of the hMSH2
gene75
and absence of hMSH2 gene product expression by
Western blot,80
and from the four HNPCCa of patients with
known germline hMSH2 mutation. By contrast, nuclear staining
for hMSH2 gene product was retained in the HCT116 cell line with
hMLH1 mutation, the SW480 cell line without MSI, and the
four HNPCCa from patients with germline hMLH1 mutation.
Dilution of 1:30 was selected for use. By contrast, nuclear staining
for hMLH1 gene product was retained unexpectedly over a range of
dilutions in the HCT116 cell line and the four HNPCCa from patients
with known germline hMLH1 mutation. Therefore, the hMLH1
antibodies were not used further in this study.
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The immunohistochemistry slides were interpreted on two occasions 3
months apart. The results were concordant in 89% (67/75) of all cases
(
statistic 0.61, good reproducibility) and in 81% (39/48) of
MSI-positive cancers ( statistic 0.53, good reproducibility). For
the discordant cases, all of which had intratumoral heterogeneity and
were found to be MSI positive when the classification was uncoded,
immunohistochemistry was repeated and the discrepancies were resolved
by re-review of the slide sets for final classification and data entry
before the MSI status and germline mutation status were known.
K-ras Proto-Oncogene Mutations
All possible sequence alterations of the K-ras proto-oncogene in codons 12 and 13 were determined. Exon 1 was amplified as in our previous studies,81,82 and the resulting PCR product from each tumor was sequenced using the SequiTherm EXCEL DNA sequencing kit (Perkin-Elmer Corp., Norwalk, CT).
Chromosome 18q Allelic Loss
Loss of the long arm of chromosome 18 in MSS tumors was defined by the complete or partial loss of the polymorphic alleles in dinucleotide repeats on the long arm of chromosome 18, as described previously.74 The results for the SRSCCa were included in our previous publication.74 Because 18q allelic loss cannot be accurately assessed by use of dinucleotide repeats in MSI-positive cancers, chromosome 18q loss was examined by loss of heterozygosity in restriction fragment length polymorphisms by Southern blot analysis of DNA isolated from cryostat sections of frozen tissue available in 11 MSI-positive SRSCCa. DCC 1.9, SAM 1.1, Josh 4.4, and p15–65 were used after digestion with HindIII, EcoRI, PstI, and EcoRI-SacI restriction endonucleases, respectively, as described previously.77,78
Immunostaining for p53 Gene Product
Detection of p53 gene product overexpression by immunohistochemistry serves as an indicator of p53 gene mutation with approximately 75% overall accuracy in colorectal neoplasms (reviewed in Ref. 83 ) because of the prolonged half-life of most mutated proteins in these tumors. Mouse monoclonal antibody DO7 against p53 (DAKO Corp., Carpinteria, CA) was used at 1:100 dilution in the TechMate 1000, and p53 labeling index was evaluated by computerized image analysis (CAS 200 system, Becton-Dickinson, Elmhurst, IL), as in our previous study.84
Sequencing of p53 Gene
A subset of p53 mutations is characterized by intragenic insertions and deletions that truncate the protein product and prevent accumulation of the mutated protein.85 Because insertions and deletions are common alterations in MSI-positive tumors, sequencing of exons 5 through 8 of the p53 gene was attempted in DNA extracted from 16 MSI-positive HNPCCa and 11 MSI-positive SRSCCa. A single 1.8-kb genomic fragment was generated by PCR using sense and antisense primers, as in our previous studies,86,87 and the PCR products were sequenced using the SequiTherm EXCEL DNA sequencing kit (Perkin-Elmer). Nucleotide sequence alterations could not be characterized because of the presence of only subtle abnormal bands that were difficult to interpret as mutations. Interpretation was complicated by the expected retention of the wild-type allele in MSI-positive cancers, which have very infrequent allelic loss, and by the expected contribution of wild-type DNA from contaminating intraepithelial lymphocytes, which were evident by histopathology of many of the microdissected specimens.
Mutation in the Gene for TGFß RII
Analysis of the poly(A) tract region in the fourth exon was done as reported previously.36 A mutated allele was represented by a major band shifted by 1 or 2 bp from the major wild-type band with intensity equal to or greater than that of the wild-type band.
E2F-4 Gene Mutation
The CAG trinucleotide repeat sequence region was analyzed in available HNPCCa and MSI-positive SRSCCa (no mutations have been reported in MSS tumors) using a previously reported method.46 A mutated allele was represented by a major band, which was shifted in position in comparison to the wild-type band. Amplification of the approximately 300-bp segment was obtained in only 66% (31/47) of DNA specimens from the formalin-fixed, paraffin-embedded cancers (P = 0.01 versus frequency of successful amplification of Bax and P < 0.000001 versus amplification of TGFß RII). Sequencing of the region containing the trinucleotide repeat was done by the previously reported method in one SRSCCa with an allelic shift and available frozen tissue.
Bax Gene Mutation
The polydeoxyguanosine tract region in the third exon was analyzed in available HNPCCa and MSI-positive SRSCCa (no mutations have been reported in MSS tumors) by the method reported previously.52 A mutated allele was represented by a major band that was shifted by 1 bp from the normal major band with intensity equal to or greater than that of the wild-type band.
Statistical Analysis
The sensitivity, specificity, predictive values of a positive and of a negative, and overall accuracy for MSI status of each of the noncoding dinucleotide and poly(A) tract markers were determined individually. Similar analysis was done for loss of nuclear staining for hMSH2 gene product in HNPCCa from kindreds previously tested for germline status of the hMSH2 and hMLH1 genes. Differences in frequencies were evaluated by Fisher's exact probability test. Differences in means were evaluated by Mann-Whitney U test. True Epistat (Richardson, TX) statistical software was used. All reported P values are two-tailed.
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Results |
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Of 39 HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa (P < 0.000001). The extent of instability, however, tended to be greater in MSI-positive SRSCCa than in HNPCCa: 94% (16/17) of MSI-positive SRSCCa had at least two-thirds of the dinucleotide markers shifted as contrasted with 69% (24/35) of MSI-positive HNPCCa (P = 0.08). HNPCCa from families with germline hMSH2 mutation, germline hMLH1 mutation, or no identified germline mutation had no statistically significant differences in the frequency of shifted markers. There was no evidence of subtle MSI in the two MSS HNPCCa: no shift was found in any of the markers tested.
The CA dinucleotide repeats on chromosome 18q (Figure 1)
and the poly
(A) tract in BAT26 (Figure 2)
showed no statistically significant
differences in their sensitivities (67 to 88%), specificities (95 to
100%), predictive values of a positive (96 to 100%), predictive
values of a negative (77 to 87%), and overall accuracies (84 to 92%)
for identifying MSI-positive carcinomas. D18S61 was least frequently
shifted in MSI-positive tumors, and BAT26 was the only marker shifted
in otherwise MSS tumors (n = 2, both SRSCCa).
Four MSI-positive HNPCCa and one MSI-positive SRSCCa (9% of all
MSI-positive cancers) had shifts only in dinucleotide repeats of
the noncoding markers.
Expression of hMSH2 Gene Product
Twenty-nine MSI-positive HNPCCa from families previously evaluated
for germline mutation status of hMSH2 and hMLH1
were studied by immunohistochemistry (Figure 3
, Table 2
). Absence of nuclear expression of
hMSH2 gene product, which was usually accompanied by retention of
cytoplasmic staining (Figure 3)
, was associated with germline
hMSH2 mutation: 12 of 14 cancers in patients with germline
hMSH2 mutation lacked nuclear expression as contrasted with
0 of 6 cancers in patients with germline hMLH1 mutation
(P = 0.0007) and 1 of 9 cancers in patients with
no identified germline mutation (P = 0.0007).
The two cancers that retained nuclear expression of hMSH2 gene product
in patients with germline hMSH2 mutation were from families
with other members whose cancers had lost expression, indicating that
the nature of the germline mutation was not responsible for the
differences in expression. Absence of nuclear staining for hMSH2 gene
product in MSI-positive cancers from patients evaluated for germline
status had sensitivity for germline mutation of hMSH2 of
86%, specificity of 93%, predictive value of a positive of 92%,
predictive value of a negative of 88%, and overall accuracy of 90%.
All MSI-positive SRSCCa and all MSS cancers had intense nuclear
expression of hMSH2 gene product accompanied by cytoplasmic staining.
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The prevalence of carcinomas with K-ras mutation in
codons 12 or 13 was similar in HNPCC and sporadic patients: 30%
(11/37) and 30% (16/54), respectively. MSI-positive SRSCCa had the
lowest frequency of K-ras mutations (18% (3/17); Table 3
), but the differences in frequencies
between MSI-positive and MSS carcinomas were not statistically
significant.
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), but no HNPCCa from patients with
germline hMSH2 mutation had a codon 13 K-ras
mutation (P = 0.02; Table 5
). Transition from G to A in the second
nucleotide of codon 12 was most common in SRSCCa (56%, 9/16 cancers
with K-ras mutation; Table 4
), but the differences in
frequency between HNPCCa and SRSCCa were not statistically significant.
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. The case
with three mutations had a histopathological section available for
microdissection to analyze the topography of the ras
mutations in samples of approximately 1 mm2. Regions with
two, one, and no ras mutations were identified (Figure 4)
, indicating the presence of multiple
subclones with different ras mutation status within the
cancer. Multiple ras mutations were not found in any SRSCCa
with ras mutation (P = 0.16
versus HNPCCa).
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In HNPCCa, loss could be evaluated by microsatellite analysis in
the two MSS cancers only; neither showed deletion (Table 3)
. Allelic
deletion was found in 48% (24/50) of SRSCCa, and loss was inversely
related to MSI; none of 11 MSI-positive SRSCCa evaluated by restriction
fragment length polymorphism analysis had deletion, as contrasted with
62% (24/39) of all MSS carcinomas evaluated with microsatellite
markers (P = 0.0004; Table 3
). Loss involved all
evaluable markers in 22 of the 24 carcinomas with deletion, and the
centromeric markers in an additional cancer. Thus, the entire
DCC/DPC4-Smad4/JV-18-MADR2-Smad2 region was lost in 96% (23/24) of
cancers with an 18q allelic deletion.
p53 Gene Product Overexpression
The labeling indices for p53 immunohistochemistry had a bimodal
distribution with all values either above 70% or below 45% (Figure 5)
. Extensive overexpression of the type
associated with p53 gene mutation was found in 8% (3/39) of
HNPCCa as contrasted with 23% (13/57) of SRSCCa
(P = 0.06; Table 3
). High p53 labeling index was
inversely related to MSI positivity: only 4% (2/55) of MSI-positive
HNPCCa and SRSCCa had extensive overexpression as contrasted with 34%
(14/41) of MSS cancers (P = 0.0001).
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Frameshift mutation resulting in loss or gain of 1 or 2 bp within
the 10-bp adenine repeat occurred in 79% (30/38) of HNPCCa and 28%
(16/57) of SRSCCa (P = 0.000001; Table 3
and
Figure 6
). All mutations were in
MSI-positive carcinomas, with high frequencies in both HNPCCa and
SRSCCa: 83% (30/36) of MSI-positive HNPCCa showed mutation, as did
89% (16/18) of MSI-positive SRSCCa.
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Mutation resulting in loss or gain of 3 bp occurred at high
frequency in both MSI-positive HNPCCa and MSI-positive SRSCCa (71%
(12/17) and 57% (8/14), respectively; Table 3
). Of note,
E2F-4 mutation was more frequent in HNPCC patients with
germline hMSH2 gene mutation than those with germline
hMLH1 mutation (100% (8/8) versus 40% (2/5),
P = 0.04; Table 5
). Sequencing of an SRSCCa specimen
with one altered allele of a size 3 bp smaller than wild type (fourth
lane in Figure 7
) and available frozen
tissue confirmed loss of one of the CAG trinucleotide repeat sequences
in codons 306 to 318 as compared with matched control DNA (Figure 8
; lowercase letters indicate nucleotides
with detectably altered mobility as compared with control): wild type,
(CAG)12 CAG CAA CAG TAA CAG CAG CAG TTC GTC; tumor,
(CAG)12 CAa CAg tAa cAg CAG CAG ttc gTC.
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Frameshift mutation within the homopolymeric region occurred with
higher frequency in MSI-positive HNPCCa as compared with MSI-positive
SRSCCa (55% (17/31) versus 13% (2/15), respectively,
P = 0.01; Table 3
and Figure 9
). No tumor had mutation of both
Bax alleles. The frequency of Bax mutation was
significantly lower than that of TGFß RII mutation in MSI-positive
carcinomas (Table 3)
.
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Our evaluation of a panel of genetic alterations permitted
assessment of MSI-positive carcinomas for mutational spectra relative
to clinical setting (HNPCC versus sporadic) and germline
mutation in families with HNPCC (Figure 10)
. Heterogeneity of the mutational
spectra was prominent. The most frequent pattern was characterized by
TGFß RII and E2F-4 mutation without Bax
mutation, ras mutation, or p53 gene product overexpression,
but this combination occurred in only 23% (8 of 35) of evaluable
cancers. This pattern was found in two HNPCCa from patients with
germline hMSH2 mutation representing two different families
(patients 11 and 12 in Figure 10
) and in six SRSCCa (patients 40 to 45
in Figure 10
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When all of the markers and gene mutations evaluated in our study were considered, there was no infallible single assay for MSI. As a consequence, our study supports the proposed use of a panel of assays but suggests use of markers in addition to those recommended by the consensus conference. The poly(A) tract in the coding region of the TGFß RII gene had high sensitivity, specificity, predictive values, and overall accuracy for MSI status in our study. This coding sequence and similar mononucleotide tracts have the practical advantage of showing no evident polymorphism in nonneoplastic tissue in our and previous studies.94,95 As a consequence, these assays can be used for microdissected tumors even if control DNA is not available, in contrast to assays that evaluate polymorphic markers and therefore require matched nonneoplastic control DNA for comparison. Mutations in the coding regions of E2F-4 and Bax occurred at lower frequency than the TGFß RII coding region mutation in our study, and the E2F-4 assay was often unsatisfactory in DNA from formalin-fixed, paraffin-embedded tissue, probably because of the relatively large size of the PCR product. The poly(A) tract in BAT26 showed shifts in two SRSCCa with no other evidence of MSI in our study, possibly because of somatic mutation in the GTBP/hMSH6 gene, which results in susceptibility to shifts in mononucleotide repeats but not in dinucleotide microsatellites,96-98 although contradictory results have been reported.99 Therefore, in screening for HNPCC, the BAT26 assay may have occasional false positives, and the importance for prognosis and therapeutic response of minor MSI confined to mononucleotide microsatellites is as yet unknown. Of note, shift in the poly(A) tract in the TGFß RII gene in our study was specific (100%) for MSI-positive cancers, although rare exceptions have been reported,54 and highly accurate (85%), so that assay of this gene is an excellent initial test for MSI. The 9% of MSI-positive cancers that lacked shifts in either poly(A) tract in our study did have shifts in various dinucleotide repeats. Thus, our data support the proposal that assay of dinucleotide tracts is needed to evaluate the MSI status of colorectal carcinomas that do not have a shift in the TGFß RII gene. Use of polymorphic dinucleotide markers for chromosomal regions of interest for allelic loss (eg 18q as in our study) provides useful information in MSS tumors as well as identifying MSI-positive tumors. Similar studies for methodological evaluation of MSI are needed in tumors of other organ systems.
Two HNPCCa in our study were MSS with no evidence of a shift in any
marker. Both of the cancers were from families with known germline
hMSH2 mutation. In the first patient, a 72-year-old woman,
nuclear expression of hMSH2 gene product was maintained in the cancer
available for immunohistochemistry, in contrast to absence of nuclear
staining in most MSI-positive HNPCCa in such families (Table 2)
. This
patient had not undergone germline testing, but the cancer was probably
a sporadic tumor occurring in an older patient in an HNPCC family, ie a
phenocopy. The second patient, a 42-year-old woman, had an identified
germline mutation of hMSH2, and her MSS cancer had
histopathological characteristics associated with MSI, including poor
differentiation and the presence of numerous signet ring cells and
prominent lymphoid response. No tissue was available for
immunohistochemistry for hMSH2 gene product, and the explanation of the
absence of identifiable MSI in repeated analyses of this cancer is
uncertain. Adenomas in HNPCC patients have a lower prevalence of MSI
than do HNPCCa,53,100-104
and this cancer may have been
early in the process of tumorigenesis, analogous to adenomas.
We found that hMSH2 gene product was usually absent from the nucleus of
MSI-positive cancers of patients in families with known germline
mutation in this mismatch repair gene, as reported
previously,19,20
and in a cell line (LoVo) with reported
homozygous deletion of the hMSH2 gene75
and
absence of hMSH2 gene product expression on Western blot.80
Intratumoral heterogeneity in nuclear staining was often evident in
these cancers (Figure 3)
. This unexplained heterogeneity adversely
impacts the diagnostic utility of immunohistochemistry by producing
difficulty in its interpretation in MSI-positive cancers. In addition,
there was retention of staining for hMSH2 gene product in the cytoplasm
of many tumors from patients with germline hMSH2 gene
mutation (Figure 3)
, raising the possibility of abnormal translocation
of the gene product to the nucleus rather than complete absence of the
protein. In previous studies, allelic loss of the wild-type allele of
hMSH2 was uncommon in HNPCCa,33,105
so that gene product
expression could be retained, but background staining could also
explain the cytoplasmic findings. The nature of the underlying germline
hMSH2 mutation did not explain the heterogeneity, because
patients from the same kindred with the same germline mutation had
different immunohistochemical results (Figure 10)
. No MSI-positive
cancers from patients with germline hMLH1 mutation,
MSI-positive sporadic cancers, or MSS cancers showed loss of hMSH2 gene
product expression in nuclei (Table 2)
. Thus, immunohistochemistry
appears to be useful in the identification of most patients who have
germline hMSH2 mutation, thereby indicating the gene to be
addressed by formal mutation analysis. Loss of hMSH2 gene product
expression in MSI-positive HNPCCa from patients with unknown germline
mutation of a mismatch repair gene and in sporadic MSI-positive colonic
carcinomas was infrequent in our study, suggesting infrequent
occurrence of germline hMSH2 mutation in these patient
subsets. In agreement with this interpretation, somatic mutation of
hMSH2 was rarely found in MSI-positive colonic cancers in
previous studies of patients without HNPCC.106-108
By
contrast with the close relationship between loss of nuclear hMSH2
expression and germline mutation of the hMSH2 gene, loss of
hMLH1 gene product expression can occur as a result of somatic
hypermethylation of the gene promoter, leading to transcriptional
silencing of expression in the absence of germline mutation of
hMLH1.109-111
The frequency of ras proto-oncogene mutation in MSI-positive
colorectal cancers is usually reported in the literature to be lower
than in MSS cancers.33,43,59-61
By contrast, and in
agreement with other studies,28,34,40,57,58
we found a
frequency in MSI-positive HNPCCa that was similar to MSS SRSCCa and a
lower frequency in MSI-positive SRSCCa that was not statistically
significantly different from MSS cancers (Table 3)
. Two aspects of the
ras mutations are of note. First, there was a suggestion
that the types of ras mutations differed between
MSI-positive cancers in the hereditary and sporadic settings: codon 12
mutations were predominant in MSI-positive SRSCCa whereas codon 13
mutations were somewhat more common in MSI-positive HNPCCa (Table 4)
but were absent in the subset of HNPCC patients with germline
hMSH2 gene mutation (Table 5)
. As a consequence, the
underlying mismatch repair gene defect appears to influence the
position of the G-to-A transition in the ras proto-oncogene.
This finding suggests complex relationships between defects in specific
components of the mismatch repair system and the sequence context or
DNA conformation of specific mispairs. Secondly, two MSI-positive
HNPCCa had multiple ras mutations (Figure 4
and Table 4
),
which appeared to result from the presence of subclones with different
mutation. Intratumoral heterogeneity has also been reported for loss of
heterozygosity in the hMSH2 and hMLH1 loci of MSI-positive sporadic
colon cancers.42
Our finding of heterogeneity for
ras mutations is similar to that reported in MSS colorectal
adenomas, but the MSS cancers associated with the adenomas had only one
of the mutations, indicating clonal dominance of the cancer by one of
the subclones from the adenoma.112
The presence of multiple
ras mutations in some HNPCCa may reflect the effects
of the mutator phenotype and also favors the concept that
ras mutations provide weaker clonal advantages in HNPCCa
with MSI as compared with MSS cancers.
Previous studies have shown low frequency of allelic losses in MSI-positive colorectal carcinomas.33,59 Our study shows that chromosome 18q, the site of the DCC, DPC4/Smad4, and JV-18/MADR2/Smad2 genes that are commonly lost in MSS tumors, was retained in MSI-positive SRSCCa. Frozen specimens to permit restriction fragment length polymorphism analysis of HNPCCa were not available, but similar retention of 18q is expected. Previous cytogenetic studies also showed retention of 18q in the karyotypes of MSI-positive colon cancer cell lines.113 These findings further emphasize the differing molecular pathogenesis between MSI-positive and MSS colonic cancers.
In agreement with previous studies of MSI-positive colonic carcinomas
in both the hereditary and sporadic settings,26,28,30,62
we
found a low rate of p53 gene product overexpression of the type seen
with p53 gene mutation (Figure 5)
. p53 mutations
were reported to be uncommon in MSI-positive colonic
cancers,33,34,53,59-61,63
although a recent study reported
p53 gene mutations in four of nine MSI-positive colon cancer
cell lines (two homozygous and two hemizygous).112
The
reason that p53 mutation status in MSI-positive tumors seems
to vary so much from study to study is unclear. MSI-positive tumors are
often heavily contaminated with nonneoplastic cells, especially
tumor-infiltrating lymphocytes.31
In addition, loss of
heterozygosity is uncommon so that retention of both alleles of p53
would be expected. These factors make sequence analysis difficult, as
occurred in our study, because of the dilution by wild-type DNA of any
DNA with p53 nucleotide sequence alteration. Subtle or unusual
mutations could also make it difficult to detect some mutations, such
as those that reduce expression. In addition, it has been reported that
there is a difference in p53 alteration status between
right-sided and left-sided colorectal cancers, regardless of MSI
status.27,69
Therefore, right-sided MSI-positive cancers
could have a low rate of p53 alteration because of their
location alone. Our study comparing sporadic MSS right-sided colonic
cancers with MSI-positive cancers favors genetic rather than anatomical
explanations of the inverse relationship between MSI and p53
alteration.
Genes with nucleotide repeats in the coding region are expected to show
high rates of mutation in those repeated sequences in MSI-positive
cancers because of disordered mismatch repair and the presence of
associated mutator phenotypes.7-9
Our study, however,
confirms the findings in previous studies that the frequency of
mutations in repeat sequences varies greatly among potential target
genes: the TGFß RII gene was usually mutated, but the proapoptotic
Bax gene had significantly lower rates of mutation and no
tumor showed mutation of both Bax alleles, in contrast to
TGFß RII. The differences in frequencies of mutations suggest
differences in the selective growth advantage for the tumor cells
conferred by the various mutations. Of particular note, the frequency
of mutation of the E2F-4 gene was higher in HNPCC patients
with germline hMSH2 mutation than those with germline
hMLH1 mutation, and the frequency of mutation in the
Bax gene was higher in MSI-positive HNPCCa than in
MSI-positive SRSCCa (Table 3)
. Recent studies showed that
E2F-4 mutations were common in a subset of colorectal
carcinomas with extensive MSI45-47
and were accompanied by
frameshift mutations in a poly(A) repeat within the seventh exon of the
hMSH3 gene.47
In contrast to our study, MSI in
Japanese HNPCCa was not associated with mutation of the Bax
gene.114
The importance of the molecular basis of mismatch
repair deficiency in affecting subsequent gene mutations is also
evident in cell lines that showed varying patterns of mutations
associated with mutations in different mismatch repair
genes.115,116
Similarly, the basis of mismatch repair
deficiency affects tumorigenesis in mouse models: germline mutation of
the human homologs of MLH1, PMS1, or PMS2 results in very different
types of murine tumors.117
Our evaluation of a panel of genetic alterations permitted identification of the striking heterogeneity in the combinations of mutations in individual MSI-positive colonic cancers, analogous to the intertumoral heterogeneity of oncogene mutations and suppressor gene alterations in MSS cancers.76,118 The heterogeneity of the gene mutational spectra in MSI-positive tumors may be explained in part by the underlying mechanisms of defective mismatch repair, because alterations of ras and Bax genes had evidence of differences between HNPCCa and SRSCCa, and E2F-4 mutation frequency differed in HNPCC patients with germline mutation of hMSH2 as compared with hMLH1. In turn, the heterogeneity in mutations may impact on the clinical behavior of MSI-positive cancers. For example, studies have shown improved prognosis in HNPCCa patients as compared to patients with sporadic colorectal cancer of equivalent stage.70-73 Similarly, patients with sporadic MSI-positive cancers are reported to have better outcome than MSS cases.28,89,90 Nonetheless, colorectal cancer is a major cause of death in HNPCC families, and many patients with sporadic MSI-positive cancers do die of their disease. The variability of the accumulated mutations and alterations in gene expression119,120 may help to explain the variability of the tumor phenotype. Additional studies are needed to define the crucial alterations that may then serve as prognostic markers. These genetic alterations may eventually define targets for unique therapies based on understanding of the specific pathways that are altered by mutations in the molecular subsets of colorectal cancer.
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Acknowledgements |
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Footnotes |
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Supported in part by the Clayton Fund and grants CA47527 and CA62924 from the National Cancer Institute, National Institutes of Health. HTL was supported by CA74684 and American Cancer Society grant EDT-84A.
Accepted for publication July 1, 1998.
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