Affiliations of authors: T. L. Chan, A. S. Y. Chan, J. C. Y. Ho, S. Y. Leung (Department of Pathology), S. T. Yuen, L. P. Chung (Department of Pathology and Hereditary Gastrointestinal Cancer Registry), J. W. C. Ho, K. Y. M. Kwan (Department of Surgery and Hereditary Gastrointestinal Cancer Registry), Queen Mary Hospital, The University of Hong Kong, Hong Kong; A. H. Wyllie, Department of Pathology, Cambridge University, U.K.
Correspondence to: Siu Tsan Yuen, M.B.B.S., Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Hong Kong (e-mail: styuen{at}hkucc.hku.hk).
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ABSTRACT |
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INTRODUCTION |
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We recently drew attention to the fact that there is an excess of patients with colorectal cancer, as high as fourfold, in the younger age groups (<46 years old) in Hong Kong compared with Scotland and other data from predominantly Caucasian countries (1). This high incidence in the young Hong Kong (Southern) Chinese was present 20 years ago and has not changed, despite the rapid rise in the overall incidence of colorectal cancer in recent years. This recent increase is entirely attributable to classical, late-onset (>50 years old) patients. Because of these data, we began to search for a genetic basis that would explain this high incidence of colorectal cancer in the young Hong Kong Chinese population.
Patients with constitutional (or germline) mismatch repair defects are prone to colorectal and certain other cancers at a young age (2,3). Mutations in the hMSH2 or hMLH1 gene are consistently associated with cancers that show microsatellite instability (MSI), characterized by the expansion or deletion of small repeat sequences during DNA replication (4-11). A small number of reports (12,13) document constitutional mutations of hMSH6: Tumors in these individuals may also show MSI. In contrast, tumors from mice genetically engineered with a homozygous null MSH6 gene mutation do not exhibit MSI (14). Patients with constitutional mutations in mismatch repair genes (hMSH2, hMLH1, and hMSH6) sometimes have strong family histories of colorectal cancer that fit the Amsterdam criteria (15) for hereditary nonpolyposis colon cancer (HNPCC) syndrome. However, this characteristic is not invariable (16). Analysis of the data in the Hereditary Gastrointestinal Cancer Registry in Hong Kong did not reveal a high proportion of families fitting the Amsterdam criteria.
To clarify the situation in Hong Kong, we analyzed the incidence of MSI and mutations in
hMSH2, hMLH1, and hMSH6 genes in a series of Hong Kong Chinese patients with colorectal
cancer. We sought to identify germline mutations in hMSH2, hMLH1, and hMSH6 in the young
patients with tumors showing instability in a high proportion (40%) of the
microsatellite sites investigated. We designated these tumors as highly unstable MSI (MSI-H).
We also searched for germline mutations in hMSH6 in young patients who had tumors in which
the microsatellites appeared completely stable (microsatellite stable) or revealed instability at less
than 40% of the sites investigated (low-level microsatellite instability
[MSI-L]). Moreover, we collected the family history of patients younger than 46
years of age. The data suggest that germline mutation in these three mismatch repair genes have a
substantial but perhaps not exclusive role in determining the susceptibility of young Chinese to
colorectal cancer.
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PATIENTS, MATERIALS, AND METHODS |
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We studied 117 colectomy specimens from patients with adenocarcinoma at Queen Mary Hospital, Hong Kong, from 1991 through early 1997. The following patients were selected: 1) all patients (total, 59 patients) for whom frozen tumor blocks were available and the patients' age at diagnosis was less than 46 years and 2) a similar number of patients (total, 58 patients) whose age at diagnosis was 46 years or older. In the latter group, the selection of patients was such that about equal numbers of patients were selected from each decade of life from 46 years onward. Overall, 72 specimens were from male Chinese patients and 45 specimens were from female Chinese patients. The patients ranged in age from 24 through 87 years. We received the specimens unfixed on ice from the operating theaters and took representative blocks of tissue from both the tumor and the normal mucosa. These blocks were snap-frozen in liquid nitrogen and stored at -70 °C. Other representative blocks from these specimens were fixed in buffered 10% formalin and embedded in paraffin for routine histologic examination.
The paraffin and frozen sections, prepared from stored blocks, were assessed by light microscopy. Only blocks with tumor tissue occupying more than 70% of the section area were used to prepare DNA. DNA was also extracted from the normal mucosa of each patient. DNA was extracted by standard protocols that included proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation.
A detailed family history was taken from all patients younger than 46 years old during interviews with patients and relatives. The interviewer (K. Y. M. Kwan) had undergone training at the Polyposis Registry (St. Mark's Hospital, London, U.K.). Venous blood was drawn, with informed consent, from the patients and from some of the available family members for extraction of DNA and RNA. This study was approved by the Ethics Committee of Queen Mary Hospital.
Analysis of MSI
The MSI analysis was performed as previously described (17,18). In
summary, segments of genomic DNA at 10 sites including known microsatellite sequences were
amplified by the polymerase chain reaction (PCR). Twenty-five to 30 cycles of PCR were
performed in 10 µL that contained 50-100 ng of DNA, 10 mM Tris-HCl (pH 8.3),
50 mM KCl, 1.5-3 mM MgCl2, 2-4 pmol of each primer, 0.1
µL of deoxycytidine 5'-[-32P]triphosphate (6000
Ci/mmol; 10 µCi/µL), all four deoxynucleoside triphosphates (each at 50 µM), and 0.5 U of Taq polymerase (Life Technologies, Inc. [GIBCO
BRL], Gaithersburg, MD). The annealing temperatures were 52 °C-60 °C.
Radiolabeled PCR products were subjected to electrophoresis in 6%
polyacrylamide-32% formamide-5.6 M urea gels at 70 W for 2-3
hours. The gels were then fixed, dried, and autoradiographed. The following 10 loci were used:
D2S123, D3S1067, D5S82, D5S346, TP53 (or p53), D18S58, DCC (deleted in colon cancer),
BAT-26, BAT-40, and TGFßRII (transforming growth factor-ß receptor II). For each
tumor, at least five loci were analyzed, including both dinucleotide and mononucleotide loci.
Tumors in which at least 40% of the microsatellite loci analyzed showed altered
electrophoretic mobility relative to the corresponding normal tissue (Fig. 1)
were designated MSI-H. Tumors with some (but <40%) loci showing
electrophoretic shift were designated MSI-L. All other tumors were designated microsatellite
stable.
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In vitro synthesized protein assays to screen for truncation mutations in hMSH2 and hMLH1, the two mismatch repair genes, were performed as previously described (18). These assays were performed for all young patients (<46 years) with MSI-H tumors and from whom blood leukocytes had been collected for RNA extraction. In brief, 3 µg of total RNA was reverse transcribed in a reaction mixture containing 20-200 ng of random hexamers or oligo(dT), 20 U of RNasin, all four deoxynucleoside triphosphates (each at 20 pmol), and 200 U of Superscript II reverse transcriptase (Life Technologies, Inc.), by use of the manufacturer's suggested reaction conditions. Forty cycles of PCR were performed in 50 µL containing 2-4 µL of the first-strand complementary DNA (cDNA) mixture, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3-5 mM MgCl2, 5 pmol of each primer, all four deoxynucleoside triphosphates (each at 200 µM), and 2.5 U of Taq polymerase (Life Technologies, Inc.). Both hMSH2 and hMLH1 were amplified in two overlapping segments that were between 1.2 and 2.0 kilobases long. The left-hand primers of each segment were tagged with a T7 promoter sequence and a translation initiation site. The products were then subjected to in vitro transcription and translation by use of the Linked T7 transcription-translation system (Amersham Corp., Little Chalfont, U.K).
DNA or cDNA Sequencing Analysis
When truncated protein products were detected by an in vitro synthesized protein assay, genomic sequencing of the regions of interest was performed to detect the site and nature of the underlying mutations. Because missense mutations are common in hMLH1 but are not detectable by an in vitro synthesized protein assay, we sequenced the whole hMLH1 cDNA from all young patients who had MSI-H tumors. Mutations found were confirmed by genomic sequencing. We designed primers for PCR amplification that spanned individual exons of hMSH2 and hMLH1 genes including intron-exon boundaries. Similarly, primers were designed to amplify overlapping regions of hMLH1 cDNA. Sequences of the primers are available from the authors on request. The PCR products were then purified by use of the High Pure PCR product purification kit (Boehringer Mannheim GmbH, Mannheim, Germany). Direct sequencing was performed by a manual sequencing method by use of Sequenase Version 2.0 (Amersham Corp.) or by automated sequencing by use of the dRhodamine terminator cycle sequencing kit (The Perkin-Elmer Corp., Norwalk, CT). Both forward and reverse primers were used in sequencing, following the manufacturer's protocols. For manual sequencing, the sequencing products were denatured at 80 °C for 5 minutes and subjected to electrophoresis through 6% polyacrylamide-urea gels at 70 W for 2-3 hours. The gels were then fixed, dried, and exposed to autoradiographic films. For automated sequencing, the sequencing products were analyzed by an automated sequencer (Applied Biosystems, Foster City, CA).
Single-Strand Conformation Polymorphism and Direct DNA Sequencing Analysis of hMSH6
Mutational analyses for hMSH6 were performed by use of blood leukocytes or normal colonic tissue from the following three groups of young patients: 1) all young patients (<46 years) who had MSI-H tumors but who did not have germline mutations in hMSH2 or hMLH1, 2) all young patients (<46 years) who had MSI-L tumors, and 3) all patients 40 years old or younger who had microsatellite stable tumors. In all patients, individual exons of hMSH6 including intron-exon boundaries were amplified by PCR. The sequences of the primers are available on request from the authors. The PCR products were denatured in the presence of NaOH and EDTA at 50 °C for 15 minutes. Six microliters of the denatured PCR product was snap-cooled, mixed with 3 µL of formamide dye, and loaded onto MDETM gels (FMC BioProducts, Rockland, ME). The gels were subjected to electrophoresis at 6-10 W overnight and silver stained, as described by the Promega Corp. (Madison, WI) protocol. Products with abnormal mobility were sequenced as described above.
Statistical Analysis
A binomial response model with age as the explanatory variable was used to assess the
prevalence of MSI with age. The statistical analysis was done by the statistical package GLIM (19). We also used the 2 test with the Yates
correction. All P values are two-sided.
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RESULTS |
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Of the 59 tumors from patients whose age at diagnosis was younger
than 46 years, 19 were MSI-H and four were MSI-L. Of those tumors from
patients whose age at diagnosis was 46 years old or older, eight were
MSI-H and seven were MSI-L. The profile of microsatellite instability
in all 27 MSI-H tumors is shown in Table 1. The
incidence of MSI-H had a striking association with the age of the
patients at diagnosis (Table 2)
. The model logit
(p) = 1.08-0.61 age provides a sufficient fit to the data in
Table 2
(
32 = 2.79). In other
words, the age
factor can be used to explain the differences in the incidence rate of
MSI-H tumors in the different age groups. The age effect is
statistically significant (P<.001), with a 95% confidence
interval of -0.25 to -0.96. The interpretation of the suggested
model is that each upward step in the age group decreases the logit by
-0.61 (i.e., multiplies the odds on the prevalence of MSI-H by
0.55). The corresponding Armitage trend test for age is statistically
significant (
12 = 11.24; P<.001).
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Germline Status of hMSH2 and hMLH1
Of the 19 patients with MSI-H tumors who were diagnosed before age
46 years, blood leukocytes from 15 patients were available for
analysis. In six of these patients, an in vitro synthesized
protein assay identified truncated hMSH2 proteins and direct DNA
sequencing of the corresponding regions identified the causative
mutations (Fig. 2; Table 3)
.
In vitro synthesized protein data did not reveal any truncated
hMLH1 protein products in these 15 patients.
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Patient 19 also developed a brain glioblastoma that exhibited MSI-H but did not have detectable truncated protein by an in vitro synthesized protein assay. However, he had been included in our earlier studies (18,21) and did have a germline mutation in hMSH2 that was detected by direct genomic sequencing. This missense mutation was also found in one sibling who had colorectal cancer.
Overall, eight patients had a mutation in either hMLH1 or hMSH2; seven patients had a mutation in hMSH2 and one patient had a mutation in hMLH1 in the germline.
Analysis of hMSH6
Single-strand conformation polymorphism and sequencing of individual
exons of hMSH6 revealed multiple nucleotide changes (Table
4). The following three changes were located within
the coding region: one change that resulted in no amino acid change,
one change that was a 4-base-pair (bp) insertion at the codon of the
7th amino acid from the C terminus of hMSH6, and the remaining change
that resulted in a single amino acid change (T1284M) in exon 9 (Fig.
3)
. We screened a number of normal individuals in
the Hong Kong Chinese population for the latter two changes. Eleven of
99 normal individuals (who had usable PCR results) had the same 4-bp
insertion in one allele, and we concluded, therefore, that this change
was a common polymorphism with a population allele frequency in the
Southern Chinese of 5.6%. However, none of 108 normal individuals had
the change T1284M in exon 9. T1284M was found in a 29-year-old male
patient whose colonic tumor was microsatellite stable.
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DISCUSSION |
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The data on hMSH6 are of special interest. To our knowledge, this is one of the first
systematic studies to reveal multiple nucleotide changes in authentic human tumors (Table 4). Most of these changes are unlikely to be responsible for tumor
development because they are intronic changes, conservative changes, or polymorphisms. Our
finding of a common polymorphism, the 4-bp insertion at the very end of the last exon of
hMSH6, in two patients is in agreement with a previous report of a similar 4-bp insertion at the
same location, although with different sequences, in a Japanese population (12). We did find, however, one missense mutation in a 29-year-old man whose tumor
did not exhibit MSI at any loci. Although the possibility of this mutation being a rare
polymorphism cannot be absolutely excluded, a search of 108 normal Chinese individuals did not
show a similar nucleotide change. Furthermore, this mutation alters an amino acid (threonine)
that is conserved in MSH6 and MSH2 in yeast, mouse, and human proteins (Fig 4)
, indicating its potential importance for the function of the protein. To our
knowledge, this is the first report of a germline hMSH6 mutation in a human tumor without MSI.
Tumors from mice with an engineered deficiency in MSH6 also do not exhibit MSI (14). On the other hand, there are two reports (12,13) of three
germline hMSH6 mutations in patients whose tumors exhibited MSI. It is possible that an MSH6
deficiency may have a less consistent association with MSI than a deficiency in other mismatch
repair genes.
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Although a family history of cancer is evident for some of our young colorectal cancer
patients, all but one of these family histories fell short of the Amsterdam criteria. Although the
Amsterdam criteria were necessary to define the core clinical HNPCC syndrome, it is now clear
that penetrance falls short of 100%. In particular, some patients with an unequivocal
germline deficiency in a mismatch repair gene develop colorectal cancer late in life. Table 1 documents the eight families with a positive family history of cancer
according to the Bethesda guidelines. There are five patients with germline mismatch repair gene
mutations whose family histories of cancer satisfy the more relaxed Bethesda guideline 3 but not
the Amsterdam criteria.
Patients younger than 36 years of age make up only 1%-2% of the total patients with colorectal cancers in most Caucasian populations (23-25). However, similar data in Hong Kong show that patients younger than 36 years of age account for up to 3%-5% of the total number of patients with colorectal cancer (26,27) and that the population incidence in this age group exceeds, in absolute terms, the incidence in Scotland by some fourfold (1). We have shown that these patients are a particularly important subgroup, with a high chance of harboring germline mutations in the mismatch repair genes. Indeed, 84% of our patients who were younger than 31 years of age carry a germline mutation in one of the mismatch repair genes. This observation has important implications for the management of these young patients. Total colectomy may be the surgical treatment of choice, and a subsequent surveillance program is essential for both the patients and their relatives.
It is of interest to note that the proportions of Hong Kong Chinese and Caucasian patients with young-onset cancer who have MSI-H tumors, germline mutation in mismatch repair genes, and a positive family history of cancer are very similar (16). Therefore, despite the fact that this study has emphasized the numeric importance of mismatch repair deficiency (involving hMSH2, hMLH1, and hMSH6) in the pathogenesis of young-onset cancer in Hong Kong Chinese, there is no evidence that these genes are uniquely responsible for the high susceptibility in Hong Kong Chinese. Other types of genomic instability are also proportionately more common, including those responsible for susceptibility to cancer with evidence of chromosome instability. The nature of this instability and susceptibility in the Hong Kong Chinese population needs further investigation.
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NOTES |
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We thank Dr. Paul Yip, Department of Statistics, The University of Hong Kong, for his advice and help in the statistical analyses of this article and Prof. L. C. Chan and Dr. R. J. Collins for their support through this project.
Presented at the 89th Annual Meeting of the American Association for Cancer Research, 1998, New Orleans, LA.
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Manuscript received November 30, 1998; revised May 19, 1999; accepted June 3, 1999.
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