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Carcinogenesis Advance Access originally published online on June 12, 2008
Carcinogenesis 2008 29(7):1351-1359; doi:10.1093/carcin/bgn133
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Differential cancer predisposition in Lynch syndrome: insights from molecular analysis of brain and urinary tract tumors

A.H.S. Gylling1, T.T. Nieminen1, W.M. Abdel-Rahman1,2, K. Nuorva3, M. Juhola3, E.I. Joensuu1, H.J. Järvinen4, J.-P. Mecklin5, M. Aarnio5 and P.T. Peltomäki1,*

1 Department of Medical Genetics, University of Helsinki, Helsinki, Finland
2 College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates
3 Department of Pathology, Jyväskylä Central Hospital, Jyväskylä, Finland
4 Second Department of Surgery, Helsinki University Hospital, Helsinki, Finland
5 Department of Surgery, Jyväskylä Central Hospital, Jyväskylä, Finland

* To whom correspondence should be addressed. Department of Medical Genetics, Biomedicum Helsinki, PO. Box 63 (Haartmaninkatu 8), University of Helsinki, Finland, FIN-00014. Tel: +358 9 19125092; Fax: +358 9 19125105; Email: paivi.peltomaki{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Hereditary non-polyposis colorectal carcinoma (Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). Lynch syndrome patients are predisposed to different cancers in a non-random fashion, the basis of which is poorly understood. We addressed this issue by determining the molecular profiles for different tumors from a nationwide cohort of Lynch syndrome families (~150 tumors in total). We focused on some less prevalent cancers, affecting the brain (n = 7) and urinary tract (five bladder and five ureter uroepithelial cancers and four kidney adenocarcinomas), and compared their molecular characteristics to those of the most common cancers, colorectal, gastric and endometrial adenocarcinomas, from the same families. Despite origin from verified MMR gene mutation carriers, the frequency of high-level microsatellite instability in tumors varied between high (100–96% for ureter, stomach and colon), intermediate (63–60% for endometrium and bladder) and low (25–0% for kidney and brain). In contrast to gastrointestinal and endometrial carcinomas, active (nuclear) β-catenin was rare and KRAS mutations were absent in brain and urological tumors. Compared with other tumors, frequent stabilization of p53 protein characterized urinary tract cancers. Promoter methylation of tumor suppressor genes discriminated the tumors in an organ-specific manner. Our findings suggest that different Lynch syndrome tumors develop along different routes. Uroepithelial cancers of the ureter (and bladder to lesser extent) share many characteristics of MMR deficiency-driven tumorigenesis, whereas brain tumors and kidney adenocarcinomas follow separate pathways.

Abbreviations: ACVR2, activin A type II receptor; APC, adenomatous polyposis coli; LOH, loss of heterozygosity; MLPA, multiplex ligation-dependent probe amplification; MMR, mismatch repair; MRE11A, meiotic recombination 11 homolog; MS, methylation specific; MSI, microsatellite instability; MSS, microsatellite stable; PCR, polymerase chain reaction; PTEN, phosphatase and tensin homolog


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Hereditary non-polyposis colorectal cancer (Lynch syndrome) is a multiorgan cancer syndrome, which is caused by germ line mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 (1). Whereas the definition of the Lynch syndrome tumor spectrum remains controversial, epidemiological studies suggest that at least cancers of the colon and rectum, endometrium, small intestine, ureter and renal pelvis are associated with sufficiently high relative risk compared with the average population to justify their inclusion in the clinical consensus criteria for the syndrome [Amsterdam criteria II, (2)]. Microsatellite instability (MSI) is a hallmark of tumors in Lynch syndrome and is present in 15–25% of corresponding sporadic tumors as well (3).

For sporadic gliomas, MSI frequencies of 0–30% have been reported (4–9), and MSI is absent or rare in gangliogliomas [0%, (4)] and meningiomas [1–2%, (4,10)]. A few studies have detected loss of MMR protein expression in MSI brain tumors at low frequencies (8,9). An increased (~4-fold) incidence of brain tumors, mainly gliomas, has been reported in Lynch syndrome families (11,12). Turcot syndrome is a rare disorder clinically defined by the concurrence of brain and colorectal tumors (13). In addition to dominantly inherited Lynch syndrome, recessively inherited forms of Turcot syndrome have been described that are associated with biallelic MMR gene mutations (14). With sensitive techniques, MSI can be demonstrated even in normal tissues from biallelic mutation carriers (14).

MMR defects have been found at a low frequency (up to 10–18%) in sporadic primary renal cell carcinoma (15–18). The frequency of such defects is low (<10%) in sporadic urinary bladder carcinomas (19), but higher (4–27%) in upper urothelial cancers (20–25). Urothelial cancers of the renal pelvis and ureter occur in excess in Lynch syndrome, with a lifetime risk of 4% compared with <1% in the general population, and possibly urothelial carcinoma of the bladder as well (11,12,26,27).

It is poorly understood why certain organs are more susceptible than others to develop cancer in the context of deficient MMR. Lynch syndrome offers a useful model to address this issue; however, the available literature is mainly limited to colorectal cancer. We therefore set out to study the molecular pathogenesis of Lynch syndrome by determining the tumor profiles for all available brain and urological carcinomas from Finnish families with Lynch syndrome and compared these with more prevalent Lynch syndrome-associated cancers, colorectal, endometrial and gastric cancer, from the same families.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Patients and specimens
This study was based on ~150 tumors from Lynch syndrome mutation carriers from a nationwide Hereditary Colorectal Cancer Registry of Finland. Specifically for this investigation, all available specimens of brain tumor (n = 7, Table I) and urological tumors (n = 14, Table II) were collected. The urinary tract tumors included 4 renal cell adenocarcinomas and 10 uroepithelial cancers (five ureter and five urinary bladder). All patients were from families fulfilling the Amsterdam criteria (2,28) or revised Bethesda guidelines (29). The findings in brain and urological tumors were compared with those in Lynch syndrome gastric cancer, colorectal cancer and endometrial cancer, in part published previously (30–35).


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  		Table I. Histology and MMR features of brain tumors from Lynch syndrome patients

 


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  		Table II. Histology and MMR features of urological cancers from Lynch syndrome patients

 
Formalin-fixed paraffin-embedded specimens of tumor and matching normal tissues were collected. Based on histological verification, areas with pure normal or high tumor percentages were selected and dissected out and used for DNA extraction according to the method of Isola et al. (36). In addition, 5 µm tissue sections were cut, mounted on glass slides (Dako, Erembodegem, Belgium) and air-dried overnight at 37°C for immunohistochemistry analysis. The appropriate institutional review boards of the Helsinki University Central Hospital and Jyväskylä Central Hospital approved this study.

Immunohistochemical analysis
The following mouse primary antibodies were used: anti-MLH1 (clone G168-15, PharMingen, Erembodegem, Belgium), anti-MSH2 (clone FE-11, Calbiochem/Oncogene Research, Darmstadt, Germany), anti-MSH6 (clone 44, Transduction Laboratories, Erembodegem, Belgium), anti-β-catenin antibody (clone 14, BD Transduction Laboratories, Erembodegem, Belgium) and anti-p53 (clone DO7, DakoCytomation, Glostrup, Denmark) (32). The Dako EnVision+ System (DakoCytomation, Glostrup, Denmark) was applied according to the manufacturer’s instructions with antigen retrieval step by microwave boiling for 15 min in citrate buffer pH 6.0 (MSH6) or ethylenediaminetetraacetic acid buffer pH 8.0 (MLH1, MSH2, β-catenin and p53). Immunohistochemical results for the DNA MMR proteins were interpreted as described (37). β-Catenin expression was considered aberrant if there was nuclear staining in >10% of tumor cells (not observed in the matching normal tissue). In reporting p53 protein stabilization, a cutoff level of >10% positive tumor cells was used.

MSI analysis
MSI status was determined using mononucleotide repeat markers (BAT25 and BAT26) and dinucleotide repeat markers (D5S346, D2S123 and D17S250) from the Bethesda panel (38). Tumors with two or more unstable markers were considered to have high-degree MSI, whereas those with one or no unstable markers were microsatellite stable (MSS).

Small-pool polymerase chain reaction
Fluorescently labeled dinucleotide markers D5S346 and D2S123 were used (38). Initial genotyping was done by standard polymerase chain reaction (PCR). The small-pool PCRs were essentially performed as described earlier (39), in a 25 µl reaction volume and 39 parallel aliquots per sample, using Expand High-Fidelity PCR System enzyme mix (Roche, Mannheim, Germany) and 50 pg of DNA template. The annealing temperature was 62°C. From each aliquot (clone), 0–4 were alleles amplified, of which those that deviated >1 bp from the constitutional allele size (determined by standard PCR) were considered unstable. The average total number of alleles analyzed per tumor specimen was 15 and 34 for D2S123 and D5S346, respectively, and 40 and 64 per blood specimen. Mutation frequency was calculated by dividing the number of alleles with MSI by the total number of alleles in each sample. For reference, four pairs of normal colorectal mucosa and sporadic MSS colorectal carcinoma (all representing paraffin-derived DNA) were studied for D2S123. The mutant allele frequencies were 2–7% (mean 5%) for normal tissues and 2–4% (mean 3%) for tumor tissues based on an average of 54 alleles examined per specimen.

Loss of heterozygosity analysis
In MLH1-associated cases, loss of heterozygosity (LOH) was determined by single-nucleotide primer extension analysis using the I219V polymorphism (37). In carriers of a genomic deletion of MLH1 exon 16, multiplex ligation-dependent probe amplification (MLPA) was also applied (40). In MSH2 or MSH6 mutation carriers, LOH was analyzed using flanking microsatellite markers, D2S2378, CA7 and D2S123 (41). LOH at adenomatous polyposis coli (APC) was examined by primer extensions utilizing a polymorphism in exon 11, combined with standard LOH analysis with flanking microsatellite markers, D5S1965 and D5S346 (42). Ratios of allelic peak areas in normal relative to tumor tissue were calculated, and values ≤0.6 or >1.67 (indicating that the transcript of one allele had decreased 40% or more) were considered to indicate strict LOH (41) and ratios between 0.61–0.75 and 1.66–1.33 putative LOH (43).

Mutation analysis
To examine if the patients carried germ line mutations previously identified in their families (41, 44–46), the respective exons of MLH1, MSH2 and MSH6 were screened by direct sequencing of genomic PCR products using primers given in Chadwick et al. (47). For the genomic deletion affecting MLH1 exon 16, a direct assay described in Nyström-Lahti et al. (45) was used.

KRAS (exon 2) and CTNNB1, gene for β-catenin (exon 3), were screened by single-strand conformation polymorphism analysis of genomic DNA, followed by sequencing with primers from Deng et al. (48) and Gylling et al. (49), respectively. Previously published primers were used to examine mononucleotide repeats in phosphatase and tensin homolog (PTEN) exons 7 and 8 (35), activin A type II receptor (ACVR2) exons 3 and 10 (48), tumor growth factor β type II receptor exon 3 and meiotic recombination 11 homolog (MRE11A) intron 4 (49).

Methylation-specific MLPA
The methylation-specific (MS) MLPA method detects methylation of DNA using probes containing a digestion site for the methylation-sensitive HhaI enzyme. The SALSA MS MLPA ME001B (MRC Holland, Amsterdam, the Netherlands) probe mix was used to study aberrant methylation of CpG islands in promoter regions of 24 tumor suppressor genes, and the SALSA MMR MS MLPA ME011 (MRC Holland, Amsterdam, the Netherlands) to study promoter methylation in MLH1, MSH2 and MSH6. All reactions were carried out and results analyzed according to the manufacturer’s instructions (http://www.mrc-holland.com). For each MLPA reaction, 100–150 ng of paraffin-extracted DNA was used. Normal DNA specimens derived from lymphocytes from healthy controls and tumor cell lines (HCT116, HCT15, RKO, HEC59, LoVo and SW48), with verified methylation status, were included in every assay. A dosage ratio of 0.15 or higher (corresponding to 15% of methylated DNA) was regarded to indicate promoter methylation. This threshold value also provided the best discrimination of tumor DNA relative to paired normal DNA where no methylation was generally observed (49).

Statistical analysis
Statistical significance for differences between groups (P-value) was determined using Fisher’s or t-test as appropriate. All reported P-values were two tailed, and values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
For a better understanding of carcinogenesis associated with Lynch syndrome and deficient MMR in general, brain (Table I) and urological carcinomas (Table II), which represent rare Lynch syndrome tumors, were molecularly characterized and compared with common tumors, colorectal, endometrial and gastric cancer, from the same families (Table III). The existing data for gastric, colorectal and endometrial cancers in Table III were supplemented in the present study as necessary.


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  		Table III. Age at onset and summary of molecular data on MMR gene germ line mutation positive cancers

 
Brain tumors
The average age at diagnosis of brain tumors was 38 years, which is lower compared with other tumors occurring in Lynch syndrome (Table III). Glioma was the commonest histological subtype (Table I). Three patients with brain tumor (118:1, 132:1 and 153:1) had developed metachronous colorectal cancer and thus represented Turcot syndrome.

The seven individuals with brain tumor were all found to have the predisposing MMR gene germ line mutation of their families, three in MLH1, three in MSH2 and one in MSH6. Loss of the corresponding MMR protein was found in three out of four available cases, including one with MSH2 mutation that additionally lacked MSH6 as expected (30) (Table I, Figure 1A). One case with MLH1 germ line mutation (83:27) showed normal staining for MLH1, which is presently unexplained. A possibility remains that this particular meningioma arose sporadicly since the same mutation was associated with loss of MLH1 protein in colorectal, endometrial and gastric cancers from other patients (30,49). No MLH1, MSH2 or MSH6 promoter methylation was found in brain tumors, whereas LOH at MLH1 appeared to be an important mechanism for somatic inactivation of the wild-type allele.


Figure 1
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  		Fig. 1. (A) Immunohistochemical staining with anti-MLH1 antibody and anti-MSH2 antibody for kidney carcinoma from patient 19:45 (upper panels) and glioblastoma from patient 130:1 (lower panels). In patient 19:45 with a germ line mutation in MLH1, the tumor cell nuclei (red arrow) are negative for MLH1, whereas the brown color in normal tubular cells (green arrows) indicates nuclear staining (upper left). MSH2 staining for the same tissue area is positive (upper right). In patient 130:1 with a germ line mutation in MSH2, tumor cells (red arrow) do not express MSH2, whereas normal endothelial cells (green arrows) do (lower right). MLH1 protein staining is positive (lower left). Original magnification x200. (B) MSI tracings of mononucleotide markers BAT25 (left) and BAT26 (right) for the tumors shown in Figure 1A as well as for a normal reference sample. The tumors from 19:45 and 130:1 are MSS despite the lack of MMR protein corresponding to the germ line mutation.

 
Surprisingly, all brain tumors were MSS by standard PCR (Table I). One family had three cases of brain tumors, a grandfather with glioblastoma (130:1), a grandchild with astrocytoma (130:3) and another grandchild with ganglioglioma (130:2). All three brain tumors were MSS (Figure 1B), whereas at least two other tumors, a gastric and colorectal carcinoma, from close relatives carrying the same predisposing mutation had high-degree MSI [(49) and this study]. None of the brain tumors displayed any truncations or expansions in the mononucleotide repeat tracts of growth-regulatory genes PTEN, ACVR2, tumor growth factor β type II receptor gene or MRE11A, either. We therefore applied a more sensitive method, small-pool PCR, which can be used to study single genome amounts of DNA and found significantly higher frequencies of alleles with MSI at two dinucleotide repeat loci tested from the Bethesda panel (D2S123 and D5S346) in brain tumors compared with matching normal tissue (blood) (Table I). The results suggest that the brain tumors had MSI with a pattern different from that occurring in, e.g., colorectal or gastric carcinomas and which is easily masked by the normal alleles present (see Discussion below).

Overall, brain tumors were characterized by the rare occurrence of molecular alterations tested (Table III). For example, p53 was stabilized in one tumor only (glioblastoma multiforme from patient 118:1), and the present frequency of 17% (1/6) is lower than p53 stabilization rates generally reported for sporadic gliomas [13–60%, (51,52)].

Urological tumors
The average age at diagnosis for urological cancer was 56 years, being the lowest for bladder (55 years) and highest for kidney cancer (64 years) (Table III). Cancers of the bladder and ureter were of transitiocellular type, whereas kidney tumors were adenocarcinomas (Table II). Urological cancer was seldom the first or only cancer in the respective individuals.

Of the 14 patients examined, all were found to have the predisposing MMR gene germ line mutation of their families, 11 in MLH1 and 3 in MSH2. All lacked the MMR protein corresponding to the germ line mutation, and all three cases with MSH2 protein loss additionally lacked MSH6 (Table II). As for brain tumors, MLH1, MSH2 and MSH6 promoter methylation was absent and LOH mainly responsible for the inactivation of the wild-type allele in urological tumors (7/11, 64%).

High-degree MSI was found in 100% (5/5) of ureter cancers, 60% (3/5) of bladder cancers and in 25% (1/4) of kidney cancers. Figure 1 gives an example of kidney carcinoma (from 19:45) that was MSS despite lack of MLH1 protein expression. Since bladder cancer has been described to show a higher frequency of alterations in tetranucleotide than mononucleotide repeats (‘elevated microsatellite alterations at selected tetranucleotides’; (22)], we also tested three tri- or tetranucleotide repeats (CAGR1, D9S242 and D20S85; (53,54). Their combined instability frequencies were similar to MSI frequencies determined by the Bethesda panel (5/5 for ureter, 2/5 for bladder and 2/4 for kidney cancer).

The urological tumors showed mutation frequencies of 29% (4/14) for both ACVR2 and tumor growth factor β type II receptor gene and 46% for MRE11A, whereas PTEN frameshift mutations were absent (Table III). These genes have previously been implicated mainly in the development of sporadic gastrointestinal cancer (50,55–57) and PTEN also in endometrial cancer (58). Nuclear β-catenin and mutations in KRAS were absent and the overall rates of tumor suppressor promoter methylation generally low with some variation according to the type of urological tumor. In contrast, p53 protein stabilization played a major role in all urological tumors (33–75%), especially those of uroepithelial origin (Table III), which is in line with observations from corresponding sporadic tumors (59–61).

Comparison of Lynch syndrome tumors representing different tissues
The tumors included in Table III originate from a well-characterized series of Lynch syndrome families from a nationwide registry with the predominant involvement of MLH1 (>90% of the families) and a high rate of shared mutations (with MLH1 exon 16 deletion, ‘Mutation 1’, accounting for half of the families) (44). Despite the predominance of MLH1 in the entire series, the relative share of MSH2 was increased among uroepithelial cancers of the bladder and ureter and in brain tumors, which is compatible with previous observations of elevated risk of these cancers in MSH2 versus MLH1 mutation carriers (62). All tumors lacked the MMR protein corresponding to the germ line mutation, and with the exception of the known instability of MSH6 in the absence of the MSH2 protein, endometrial cancer was the only type of tumor lacking additional MMR proteins [21/39, 54% of tumors from MLH1 mutation carriers additionally lacked MSH2 and/or MSH6; Table III and (30)]. Although all tumors originated from verified MMR gene mutation carriers, the frequency of high-level MSI in tumors displayed a wide range over high (100–96% for ureter, stomach and colon), intermediate (63–60% for endometrium and bladder) and low values (25–0% for kidney and brain), which probably reflected tissue-specific patterns of malignant growth [(35) and Discussion below].

While the occurrence of frameshift mutations in mononucleotide repeats within growth regulatory genes in part depended on the length of the repeat and the general MSI frequency in the tumors, differences specific to tumor type were also seen, in line with observations from sporadic tumors (see above). For example, among tumors with similar MSI frequencies, frameshift mutations leading to the inactivation of the tumor suppressor gene ACVR2 were significantly more common in gastric and colorectal carcinomas than ureter carcinomas (32/33 versus 1/5, P = 0.00033). PTEN involvement was characteristic of, and practically limited to, endometrial carcinoma [(35) and Table III]. MRE11A, which encodes a repair protein interacting with MLH1 (63), was mutated in all tumors except brain with frequencies closely following the general MSI frequencies in the same tumors.

The Wnt-signaling pathway is known to be activated, usually due to inactivating mutations in APC or activating mutations in the β-catenin (CTNNB1) gene, in 90% of sporadic colorectal cancers and less frequently in other cancers, leading to the stabilization and accumulation of β-catenin in the nucleus (64). In the present series, brain and urological tumors seldom displayed nuclear β-catenin in contrast to gastrointestinal and endometrial carcinomas; the difference between urological (0%) and gastrointestinal (stomach and colon, 61%) cancers and between urological (0%) and endometrial carcinomas (53%) was statistically significant (P-values 0.000066 and 0.0016, respectively). In the urological samples, strict or putative LOH at APC occurred in seven out of 11 informative tumors (64%), which is significantly higher compared with colon cancers (P = 0.016) or endometrial cancers (P = 0.0032). Since APC–LOH, however, showed a generally poor correlation with nuclear β-catenin in the present series, APC–LOH may reflect general chromosomal instability instead of providing a targeted mechanism to inactivate the wild-type copy of APC.

In contrast to the gastrointestinal and endometrial carcinomas, KRAS mutations were absent in the brain and urological tumors, the difference between urological cancers (0%) and colon cancer (31%) being statistically significant (P = 0.028). Compared with gastrointestinal cancers (15%), p53 protein stabilization occurred at a similar rate in brain tumors (17%) but significantly more often in the various urinary tract cancers (33–75%, P = 0.0047).

Among 24 tumor suppressor genes examined for promoter methylation, the highest average methylation frequencies were observed in the colorectal (4.0) and gastric carcinomas (4.1) and the lowest in the urinary tract (1.0–2.3; P = 0.010 relative to gastrointestinal tumors) and brain tumors (1.4; P = 0.039 relative to gastrointestinal tumors) (Table III). Figure 2 shows genes with promoter methylation in a minimum of 30% of tumors in at least one tumor type. RASSF1 turned out to be one of the most commonly methylated genes in the analyzed tumors, with the highest methylation frequencies in kidney (75%) and endometrial carcinoma (71%). Except for kidney, APC promoter 1A methylation was found in all carcinomas (especially in stomach, colorectal and endometrial carcinoma, 48–62%), whereas no APC methylation was detected in brain tumors. Some genes were methylated in a tissue-specific manner, for instance significant CD44 promoter methylation was detected in kidney cancer only (75%). ESR1 methylation was limited to adenocarcinomas (especially those of the gastrointestinal tract) with no methylation in urothelial and brain tumors. The highest frequency of GSTP1 promoter methylation was found in brain tumors (57%).


Figure 2
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  		Fig. 2. Promoter methylation in tumors from Lynch syndrome patients. Genes with promoter methylation in ≥30% of tumors in at least one tumor type are included. Y-axis shows percentage of tumors with methylation at a given gene promoter.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Tissue specificity of cancers that arise from defects in ubiquitously expressed genes, such as those responsible for human MMR, remains unexplained. Lynch syndrome with a characteristic non-random distribution of different cancers provides an excellent model to examine this question. Among tumors we studied, uroepithelial cancer of the ureter is included in the Amsterdam criteria II because of its clearly increased incidence in Lynch syndrome versus average population, whereas the relative risks for uroepithelial cancer of the bladder, adenocarcinoma of the kidney and tumors of the brain are not considered high enough to warrant their inclusion (2). To gain additional insights into the issue, we investigated if the tumors, all of which were from carriers of MMR gene mutations, were probably to arise through deficient MMR. All brain (except one, Table I) and urological tumors (Table II) lacked the MMR protein corresponding to the germ line mutation suggesting that in keeping with the classical two-hit hypothesis, the wild-type copy, too, of the MMR gene in question had become inactive (supported by our LOH findings). The consequences of inactivation in terms of MSI, however, drastically differed between the different tumors, the MSI frequencies being 100% for ureter, 60% for bladder, 25% for kidney and 0% for brain tumors. Previous observations in colorectal tumors from Lynch syndrome patients as well as sporadic MSI colorectal tumors suggest the frequent occurrence of multiple subclones within the tumors (65–67). Such clonal heterogeneity, which could be of different degree for different tumor types, may offer one possible explanation for the present findings in brain and kidney tumors. In fact, small-pool PCR experiments of brain tumors suggested that the tumors did have MSI but it was diluted by the presence of multiple minor clones with unstable alleles (with mutant allele frequency <30%) and the high proportion of clones with normal alleles, resulting in a MSS pattern by conventional PCR.

One patient with a heterozygous MSH6 mutation and meningioma (132:1) had relatively high frequencies of unstable alleles in blood DNA as well (Table I), which is exceptional since previous reports of MSI in constitutional tissue mainly concern carriers of biallelic MMR gene mutations (14). This individual turned out to have an additional germ line mutation in another DNA repair gene, CHEK2 [1100delC, (68)], which could theoretically play a role although genomic instability possibly caused by the 1100delC mutation may manifest itself differently [in polyploidy and DNA double-strand breaks; (69)]. Alternatively, the phenomenon could represent the accumulation of somatic mutations induced by lost MMR function or haploinsufficiency in analogy to low-level MSI detected by a sensitive PCR–cloning technique in blood lymphocytes from heterozygous carriers of MLH1 and MSH2 mutations even before tumor diagnosis (70).

The present ureter and bladder cancers showed high MSI frequencies that clearly exceed frequencies reported for the corresponding sporadic tumors (see Introduction). In addition, our findings are compatible with the available literature reports of four urothelial tumors of the ureter or bladder from verified or putative carriers of MSH2 mutations (21,71–73) and five urothelial tumors of the ureter and renal pelvis from carriers of a single MSH6 mutation (74) that show high-degree MSI and/or MMR protein loss in these tumors as a rule. Our observations on kidney carcinomas agree with Mongiat-Artus et al. (73), who described a Lynch syndrome case with papillary kidney carcinoma which was MSS, had normal MMR protein expression and no frameshift mutation in target genes. Together with the infrequent occurrence of MSI in sporadic renal cell carcinomas (see Introduction), the findings suggest that renal cell carcinomas are unlikely to be part of Lynch syndrome, contrary to ureter and possibly bladder carcinoma.

DNA methylation differences constituted perhaps the most distinguishing feature between the different tumor types (Table III, Figure 2). To our knowledge, no previous data exist comparing the methylation patterns in the various tumors from Lynch syndrome patients. Whereas some genes showed frequent methylation in virtually all tumor types (RASSF1), other genes displayed patterns specific to the main histological category of the malignancy (ESR1 in adenocarcinomas) or the organ in question (CD44 in kidney cancer). In regard to the different tumors of the urinary tract, methylation patterns were more often concordant than discordant between bladder and ureter cancers (both uroepithelial) but more often discordant than concordant between the uroepithelial cancers and kidney adenocarcinoma (Figure 2), suggesting patterns specific to the cell type of origin. Finally, the methylation pattern in brain tumors differed from all other tumor types. Therefore, the methylation findings further support the differences observed in other molecular characteristics between these tumors.

In conclusion, our findings combined with few available reports from literature (75,76) suggest that the events that follow the first ‘hit’ (the predisposing MMR gene germ line mutation) and ultimately lead to tumor formation in Lynch syndrome are different in different tissues. Uroepithelial cancers of the ureter (and bladder to lesser extent) share many features of tumorigenesis driven by profuse MMR deficiency, whereas brain tumors and kidney adenocarcinomas develop along separate routes.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Academy of Finland (121185); Finnish Cancer Foundation; Sigrid Juselius Foundation; K. Albin Johansson Foundation; Victoria Foundation; Middle-Finland Healthcare District Research Grant; Jyväskylä Central Hospital Science Foundation; Cancer Society of Middle-Finland.


   Acknowledgments
 
We thank Kirsi Pylvänäinen, Tuula Lehtinen and Katja Kuosa for sample collection and Kaija Koivula for procurement and Saila Saarinen for laboratory analyses.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received January 3, 2008; revised May 21, 2008; accepted May 25, 2008.


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