Accuracy of the routine detection of mutation in mismatch repair genes in patients with susceptibility to hereditary upper urinary tract transitional cell carcinoma


Morgan Rouprêt, 23 quai dí Anjou, 75 004 Paris, France.



To establish the clinical benefits of systematic testing for hMSH6 and hMLH1 mutations in the very rare patients with upper urinary tract transitional cell carcinomas (UUT-TCCs), a clinical predisposition for hereditary tumour and no mutation detected in hMSH2 gene.


In all, 164 UUT-TCC specimen blocks were screened for microsatellite instability (MSI); 27 (16%) had high MSI levels. Eight patients (30%) had clinical criteria suspicious of hereditary tumour; in three a mutation in hMSH2 was detected. For the other patients, clinical data were collated, and DNA gene sequences analysed to detect mutations in hMLH1 and in hMSH6 genes.


Five patients were assessed (mean age at the diagnosis of UUT-TCC 65.2 years, sd 8, range 54–71; two aged < 60 years). Three patients had a personal history of hereditary nonpolyposis colorectal related-cancer (three colorectal). There were only mutations in hMSH2 gene detected, with none in hMSH6 and hMLH1.


For the rare patients with UUT-TCC who are suspected of carrying mismatch repair gene mutations if no hMSH2 mutation is found by genetic testing, complementary DNA sequencing for hMLH1 and hMSH6 mutation does not seem to contribute and should not be recommended in daily practice.


upper urinary tract


hereditary nonpolyposis colorectal carcinoma


microsatellite instability.


Upper urinary tract (UUT) TCCs are rare tumours which account for < 5% of all urothelial carcinomas [1]. UUT-TCCs belong to the spectrum of hereditary nonpolyposis colorectal carcinomas (HNPCC) [2,3], an autosomal dominant syndrome predisposing to colorectal carcinoma but sometimes also to extracolonic tumours such as UUT-TCCs (5% of cases) [4]. HNPCC is caused by germ-line mutations affecting one or several mismatch repair genes, i.e. hMSH2 (60% of the time), hMHL1 (30%) and hMSH6 (5–8%) [4–6]. More recently, genes such as hMLH3, hPMS1, hPMS2, TGFβRII and EX01 have also been implicated [7,8]. Together, deleterious mutations of these other genes, account for < 5% of cases. Tumour microsatellite instability (MSI) indicates probable mutations or epigenetic alterations in these mismatch repair genes [4,9]. High MSI levels are detected in nearly 15% of patients with UUT-TCC [10,11]. We already established that high MSI status is useful to indicate a hMSH2 mutation in these patients [12]. To avoid overlooking a hereditary cancer, we showed that patients with a high MSI level and a history of HNPCC-associated cancer or aged < 60 years should be tested for hMSH2 mutation [12]. The aim of the present study was to establish the clinical benefits of systematic testing for hMSH6 and hMLH1 mutations in the very rare patients with UUT-TCC, a clinical predisposition for hereditary tumour and no mutation detected in hMSH2 gene.


The files of 164 patients treated for sporadic UUT-TCC over 12 years were reviewed; all tumour blocks retrieved were screened for MSI. Paired DNA from tumours and normal tissues were amplified by PCR using five microsatellite markers from the Bethesda panel [13]: BAT25 (4q12), BAT26 (2p16), D2S123 (2p16), d5s346 (5q21–22) and D17S250 (17q) (for primer sequences, sense/antisense, see In accordance with National Cancer Institute consensus [13], any pair of samples of normal DNA and tumour DNA that had instability at two or more of these five loci was scored as having high-frequency MSI, whereas a sample pair with no instability at these five loci was scored as having MSI. Any sample pair having instability at one of the five loci was tested again at that locus to exclude artefact. If MSI was confirmed additional loci were tested to determine whether the phenotype of the sample was low-frequency (1–4 loci) or high-frequency MSI (five or more loci). For additional loci, we used markers that we had already tested in UUT-TCC [10,14]: MFD15 (1q23), APC (5q22), BAT40 (1p13.1), d18s58 (18q22), D18S69 (18q21), d10s197 (10p12), MYC1L (1p34), UT5320 (8q24), ACTBP2 (6q13), CFS1R (5q33-q35), D20S82 (20p12), d11s488 (11q24) and D9S242 (9q33). PCR amplification was carried out with ≈ 10 ng of DNA in a 20-µL final volume of reaction mixture (0.25 mmol/L dNTP in 1 mol/L Tris, 0.9 mol/L boric acid, 0.01 mol/L EDTA, 20 pmol of each primer (MWG Biotech, Ebersberg, Germany), 0.75 µL of DMSO, and 1 U Taq Polymerase (Qbiogen, Illkirch, France). Cycling parameters were described previously [10]; 1 µL of PCR product was added to 1 µL blue Dextran and 3 µL formamide. After a 2-min denaturation step at 94°C, the mixture was immediately immersed in an ice bath. The amplified fragments were separated by denaturing gel electrophoresis in Tris-borate-edetic acid buffer/4% polyacrylamide (acryl-to-bisacryl 29 : 1), 6 mol/L urea (gel) using an PRISM 377 Genetic Analyser (Applied Biosystems, Palo Alto, California); GeneScan 3.1 Fragment Analysis software (Applied Biosystems) was used to analyse the data.

Twenty-seven patients (16%) had high MSI levels; the following data were collated: age, personal or family history of a HNPCC-associated tumour, history of other cancers, tumour stage (TNM 1997) and grade. None of these 27 patients met the Amsterdam clinical criteria for HNPCC [15]. These 27 patients had their DNA sequenced to detect hMSH2 mutation, which were found in three (11%). Consequently, clinical criteria were defined to suspect a predisposition for hereditary UUT-TCC, i.e. a personal or familial history of HNPCC-associated cancer and/or aged < 60 years [12]. Only those patients who met these criteria and had no hMSH2 mutation were included in the present study.

For genetic testing, blood samples were obtained from all participating subjects and kept frozen at − 30 °C until DNA extraction. DNA was isolated from peripheral blood lymphocytes using a purification kit (QIAamp blood kit, Qiagen, Courtaboeuf, France). All coding exons of the hMSH6 and hMLH1 were sequenced by PCR amplification with intronic flanking primers, as described elsewhere [16]. Briefly, primers were designed using the human genome sequence (GenBank NM_000179). Sequencing reactions were conducted with the ABI Prism Big Dye Terminator Cycle sequencing kit and analysed on an ABI310 sequence analyser (both Applied Biosystems).


Of the 27 patients with high MSI levels, eight (30%) met the clinical criteria for hereditary UUT-TCC. Their gender, age, personal and family history, and tumour characteristics are given in Table 1. Of these eight patients five were included in the present study (mean age at the diagnosis of UUT-TCC 65.2 years, sd 8, range 54–71; two aged < 60 years). Three patients had a personal history of cancer related to the HNPCC spectrum; UUT-TCC was never the first cancer in their personal history. No patient had a family history of HNPCC-associated cancer. Of the five patients, two developed UUT-TCC in the renal pelvis and three in the ureter. No patient had metastases when the UUT-TCC was diagnosed. Of the five tumours, two were superficial (pT1) and three were invasive (pT2, pT3). All patients had a radical nephroureterectomy; two had a recurrence (one bladder cancer, one cancer of the contralateral upper urinary tract). As reported in Table 1, only mutation in hMSH2 was detected, with no mutation on MLH1 and MSH6 after DNA sequencing in these patients.

Table 1.  The characteristics of patients predisposed to hereditary UUT-TCC
N/sex/age, yearsHistory of cancerUUT-TCC siteGradeStagehMSH2*hMSH6hMLH1
  • *

    results from previous study [12].

1/F/58Colon, breastNoRenal pelvis21T1N0M0811del4*
3/M/57ColonNoRenal pelvis22T2N0M0R711X*
4/M/59NoNoRenal pelvis32T2N0M0
5/M/73ColonNoRenal pelvis33T3N1M0
6/M/69ColonMother (breast)Ureter21T1N0M0


This study of patients suspected of having hereditary UUT-TCC caused by germline mutation of mismatch repair genes and previously explored for hMSH2 gene [12] provides no support for the hypothesis that DNA sequencing of hMLH1 and hMSH6 might be useful to detect hereditary disease among these rare cases of UUT-TCCs. Undoubtedly some hereditary cancers, whether of the colon or UUT-TCC, are misclassified as sporadic and their incidence is underestimated [3,4,12]. In addition, the incidence of de novo mutations is not negligible, especially in hMSH2[17,18]. Moreover, in half of patients, UUT-TCC shows the presence of HNPCC and, conversely, the relative risk of UUT-TCC in HNPCC patients is 14 [3]. The daily practice of diagnosis in hereditary cancers must be improved; in such cases, when gene mutations are detected, the patient and his family benefit from multidisciplinary management [5,19]. The presence of other HNPCC-associated cancers is sought and patients closely monitored. Genetic counselling is provided to the patient's family. So that a hereditary cancer is not overlooked, we already suggested changing screening strategies in UUT-TCCs based on the above tests [12]. Screening for MSI is now warranted as routine in all patients with UUT-TCC, as for colorectal cancers, irrespective of age at diagnosis. A panel of five microsatellite markers is usually used to determine MSI; when the discrimination is poor, a further 10 markers or more are used [13]. In our choice of markers, we applied the criteria of the 1998 consensus [13] and used markers that were relevant in our earlier studies on UUT-TCC [10,14]. In future, the more precise 2004 criteria need to be implemented [20]. MSI screening identifies a further 5% of hereditary cancers than when applying the stringent clinical criteria for diagnosing HNPCC (Amsterdam criteria) [4,12]. Immunohistochemistry can be a useful additional test to indicate which mismatch repair gene might be involved [12]; some authors even consider that the results are sufficiently well correlated with MSI phenotype to act as a surrogate for MSI determination, especially as it is quicker [21,22]. However, tumour MSI phenotype determined by PCR is more specific for changes in DNA repair genes and is still the standard method [12,20,23].

DNA sequencing, as the last step for a positive diagnosis, is long, complex and expensive, and needs to be restricted to few patients [12,16]. DNA sequencing requires specialized equipment and is time-consuming. Given that a significant relationship was shown between the presence of hMSH2 mutation, a history of HNPCC-associated cancer and UUT-TCC occurrence when aged < 60 years [12], in the present study we selected patients for further investigation. Mutations in hMLH1 and hMSH6 are involved in hereditary tumours in only 30% and < 8% of cases, respectively [4,5,8]. Furthermore, detecting mutations in these two genes is not currently always available [24]. As sporadic UUT-TCCs are very rare tumours, only five of the present 164 patients were included in this study, and the final result was of little practical value. The reported risk for all HNPCC-related tumours is significantly lower in MSH6 or in MLH1 than in MSH2 mutation carriers [5,8]. Consequently, there is no doubt that searching systematically for hMLH1 and hMSH6 mutations by genetic testing is not cost-effective and is unwarranted in daily practice for managing UUT-TCC.


None declared.