Infrequent Microsatellite Instability in Urothelial Cell Carcinoma of the Bladder in Young Patients

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1 european urology 49 (2006) available at journal homepage: Bladder Cancer Infrequent Microsatellite Instability in Urothelial Cell Carcinoma of the Bladder in Young Patients Pierre Mongiat-Artus a, *, Catherine Miquel b,c, Madelon van der Aa d, Olivier Buhard e, Richard Hamelin e, Chris Bangma f, Pierre Teillac a, Theodorus van der Kwast d,1, Françoise Praz b,1 a Department of Urology, Saint-Louis Hospital and University Paris VII, Paris, France b Centre National de la Recherche Scientifique-Unité Propre de Recherche 2169, Genetic Instability and Cancer, Institut Gustave Roussy, Villejuif, France c Department of Pathology, Sainte-Anne Hospital and University Paris V, Paris, France d Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands e INSERM U434, Centre d Etude du Polymorphisme Humain, Paris, France f Department of Urology, Erasmus Medical Center, Rotterdam, The Netherlands Article info Article history: Accepted November 30, 2005 Published online ahead of print on January 4, 2006 Keywords: Bladder Urothelial cell carcinoma Microsatellite instability Abstract Objective: Defects in the DNA mismatch repair result in microsatellite instability (MSI), which characterise most tumours related to the hereditary nonpolyposis colorectal cancer syndrome and some sporadic tumours. Several studies have reported the occurrence of MSI in urothelial cell carcinoma (UCC) of the bladder with a particularly high incidence in tumours from young patients. In this study, we have evaluated the occurrence of MSI in primary bladder UCC arising in seventeen young patients selected for being below 45 years of age at diagnosis. Methods: Microsatellite analysis has been performed using the panel of five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27) recently recommended to detect MSI tumours. The original Bethesda panel including BAT-25, BAT-26 and three dinucleotide repeats (D2S123, D5S346, D17S250) has further been studied in 10 UCC samples. Results: MSI has been observed in only one of the 17 bladder UCC studied. Using the original Bethesda panel, identical results were obtained, indicating that the panel of five mononucleotide markers adequately detected MSI in UCC tumours. Conclusions: Our data indicate that classical MSI affecting mono- or di-nucleotides are rarely involved in bladder UCC developing in young patients. Further studies using gold standard criteria would help clarifying the involvement of MSI in the pathogenesis of bladder UCC. # 2005 Elsevier B.V. All rights reserved. * Corresponding author. Department of Urology, Saint-Louis Hospital, 1 avenue Claude Vellefaux, Paris, France. Tel ; Fax: address: pierre.mongiat-artus@sls.aphp.fr (P. Mongiat-Artus). 1 Theodorus van der Kwast and Françoise Praz contributed equally to the work /$ see front matter # 2005 Elsevier B.V. All rights reserved. doi: /j.eururo

2 686 european urology 49 (2006) Introduction Urothelial cell carcinoma (UCC) of the bladder is the fourth most common cancer with an annual incidence of approximately 120,000 cases per year in Europe [1]. Though newly diagnosed bladder UCC are superficial tumours in 70 to 80% of the cases and can be completely resected, half will recur and 10 to 20% will progress in stage or grade [1,2]. Two mechanisms of genetic instability have been documented in cancer, characterized by high levels of chromosome abnormalities or mutations at the nucleotidic level. The latter generally results from defects in the post-replicative DNA mismatch repair system (MMR), and is characterized by the accumulation of insertion/deletion mutations in simple repeated sequences called microsatellites. Microsatellite instability (MSI) has been observed in a variety of sporadic cancers and in the hereditary non polyposis colorectal cancer syndrome (HNPCC), which predisposes to the development of cancers at different sites including colon, stomach, endometrium and upper urinary tract. In contrast, UCC of the bladder occurs in HNPCC patients at a frequency comparable to that of the general population [3]. Several studies have evaluated the frequency of MSI in bladder UCC using various panels of mono-, di- tri- or tetra-nucleotides, leading to considerable variations in the MSI frequency (0 to 100%) observed. These variations are likely to reflect differences in the sensitivity, the number and nature of the microsatellite markers analysed, the techniques, the criteria for MSI classification, and the patient populations [4]. In particular, a subset of urothelial bladder carcinomas that display MSI at tetra- or trinucleotide markers rather than at mono- and dinucleotides has been described. This uncommon phenotype termed elevated microsatellite instability at selected tetra-nucleotide repeats (EMAST) is not associated with reduced MMR protein expression suggesting that the two forms of instability are unrelated [2,4]. This situation is in sharp contrast with that of colorectal cancer for which strict criteria for MSI diagnosis have been defined by the National Cancer Institute (NCI) workshop group held in Bethesda [5]. This international consensus meeting proposed a panel of five markers including 3 dinucleotide repeats (D2S123, D5S346 and D17S250) and 2 mononucleotide repeats (BAT-25 and BAT-26). Two previous studies investigated MSI in bladder UCC from a non-age-selected population with the NCI criteria and reported frequencies varying from 0 to 16% [6,7]. In another recent study in which a large series of tumours were studied with another panel of monoand di-nucleotides, MSI was reported in 73% of the cases and was proposed to be considered as a prognosis factor [8]. Finally, the detection of MSI has been proposed as a potential non-invasive diagnosis tool when performed on voided urines [9]. Interestingly, the highest MSI frequency ever reported (100%) was obtained using dinucleotide markers in a series of bladder tumours arising in young patients (35 55 years old) [10]. The use of simple and accurate markers that improves the current gold standard for MSI detection is needed to establish whether classical MSI is involved in UCC carcinogenesis. In our study, we have used a panel of 5 mononucleotidic markers recommended by the recent NCI consensus workshop, because they are more accurate and sensitive than the aforementioned classical Bethesda panel to detect tumour MSI status [5,11 13]. In addition, because UCC arising at a young age had been reported to be exclusively of the MSI phenotype, we have investigated the MSI status of bladder UCC occurring in 17 adults younger than 45 years of age [10]. 2. Materials and methods 2.1. Patients Seventeen cases of primary bladder UCC selected for being younger than 45 years-old at diagnosis were retrieved from the files of the Department of Pathology from Erasmus Hospital, Rotterdam, the Netherlands, between 1992 and Tumour specimens were archival formalin-fixed paraffin-embedded tissues and consisted of transurethral resection material in 16 cases and a resected radical cystectomy in one case. Clinical information including sex, age, histological grade, pathological stage, time to recurrence, patient status at last follow-up was retrieved by review of pertinent records. The histological type, tumour staging and grading were determined according to the criteria defined in 1998 by the World Health Organization and the International Union Against Cancer. The TNM classification of all patients was based on intravenous urography and/or computerized tomography. Follow-up consisted in urine cytology, cystoscopy designed on a regular 3 months basis during the first year, and 6 months during the 4 following years and in annual intravenous urography DNA extraction For molecular analyses, tumoral DNA were extracted from microdissected paraffin-embedded blocks using the DNeasy TM Tissue Kit (Qiagen, Germany). Sections from each paraffin block were stained to select the tumour site containing the largest amount of tumour tissue (>80% of tumour tissue) that was further microdissected with a scalpel under an inverted

3 european urology 49 (2006) microscope (40 magnification). For 10 patients, germline DNA was also extracted from frozen blood MSI Analyses The MSI status of the tumours was established using the new pentaplex PCR recommended by the National Cancer Institute including 5 quasimonomorphic mononucleotide repeats: BAT-25, BAT-26, NR-21, NR-24, NR-27; tumours were classified as MSI if three or more markers displayed instability [11 13]. The 5 mononucleotide repeats were coamplified in a multiplex PCR containing 0.2 mmol/l of BAT-26, NR-21, and NR-27 primers, 0.3 mmol/l NR-24 primers, 0.4 mmol/l BAT-25 primers 100 mmol/l dntp, 3 mmol/l MgCl 2, and 0.75 U Taq DNA polymerase. One primer in each pair was labelled with fluorescent dye FAM, HEX, or NED. The pentaplex PCR was performed under the following conditions: denaturation at 95 8C for 3 min, cycles of denaturation at 95 8C for 30 s, annealing at 55 8C for 30 s, and extension at 72 8C for 30 s, followed by an extension step at 72 8C for 5 min. The original Bethesda panel including 2 mononucleotide repeats (BAT-25 and BAT-26) and 3 dinucleotide repeats (D5S346, D2S123, and D17S250) has been further performed in 10 tumours for which normal counterpart DNA was available; using this panel, tumours areclassifiedasmsi-high(msiattwoormoremarkers), MSI-Low (MSI at one locus) or MSS (microsatellite stable, no MSI) [5]. PCR products were run in Performance Optimized Polymer 6 on a 36-cm capillary array on the ABI PRISM 3100 Genetic Analyzer. Thefragmentsizes wereanalyzedusingthegenescan 3.1 software (Applied Biosystems). 3. Results 3.1. Patients Thirteen males and four females (sex ratio 3.25) with primary bladder UCC diagnosed before 45 years of age were studied (Table 1). The mean age was 35 years (range 20 45). Thirteen patients presented with a non-invasive tumour (pta) with 8 being grade 1 and 5 grade 2, and 3 patients presented with tumours invading the lamina propria (pt1), with 1 being G1, 1 G2 and 1 G3 (Table 1). The last patient presented with a pt4 tumour, with a prostate involvement. The median follow up was 35.9 months (4 to months). Ten patients (8 males and 2 females) developed a recurrence; the recurrence rate was defined as the number of recurrences per year. The median global recurrence rate was 0.18 (0 to 1.96) and the median recurrence rate for the patients presenting a recurrence was 0.33 (0.17 to 1.96). Six patients received intravesical instillation of BCG or chemotherapy. None of the patients developed recurrence in the upper urinary tract. None of them had familial history of colorectal, gastric or endometrial carcinoma, the cancer locations most often encountered in the context of HNPCC syndrome, or had developed any other primary tumour MSI assessment We have analyzed the MSI status of bladder UCC using the PCR pentaplex of five quasimonomorphic mononucleotide repeats according to the 2004 revised Bethesda Guidelines [13]. Only one patient (case #14) had a tumour that exhibited widespread MSI. In this tumour, all five mononucleotide markers showed deletions varying from 1 to 3 bp (Fig. 1). Because the corresponding normal DNA was available for this patient, we could exclude the possibility that these alleles corresponded to rare Table 1 Clinical and pathological data of the 17 young patients with bladder carcinomas Case Gender Age (*) Stage/grade (TNM) Number of recurrence Recurrence rate per year Time to first recurrence (months) Follow-up Time (months) 1 M 36 pt4g3 0 ( ) ( ) M 24 ptag1 0 ( ) ( ) M 30 ptag M 32 ptag M 33 ptag1 0 ( ) ( ) M 35 pt1g F 35 ptag M 39 pt1g F 40 ptag F 41 ptag2 0 ( ) ( ) M 41 ptag M 41 ptag2 0 ( ) ( ) M 20 ptag1 0 ( ) ( ) M 45 pt1g F 28 ptag M 35 ptag1 0 ( ) ( ) M 40 ptag M: male; F: female; (*) age in years at diagnosis of the first bladder carcinoma.

4 688 european urology 49 (2006) Fig. 1 Allelic profiles of the five mononucleotide markers used for the determination of MSI status. Germline DNA (top panel) and tumour DNA (lower panel) obtained from patient #14 were co-amplified by PCR using fluorescent primers and migrated on an ABI3100 genetic analyzer (Applied Biosystems). The profiles were analyzed using the GeneScan 3.1 software. Shorter alleles are indicated by a star. The size of the deletion is indicated for each marker. polymorphisms. The sizes of the deletions observed in this tumour were significantly shorter than those generally observed in gastro-intestinal tumour, but comparable to those of endometrial tumours. Sixteen patients had microsatellite stable tumours. The ten tumours for which normal DNA was available were further analyzed using the original National Cancer Institute microsatellite panel which, in addition to BAT-25 and BAT-26, comprises three dinucleotides D2S123, D5S346, and D17S250. The classification was identical for all tumours; the only MSI tumour (case #14) displayed instability at both the D2S123 and the D5S346 loci and was stable at the D17S250 locus. 4. Discussion The MSI phenotype has been reported in a subset proportion of upper urinary tract UCC, and in bladder UCC with variable frequencies [2,4,6 8,10,14 16]. Both sporadic and HNPCC-related MSI gastro-intestinal tumours display specific molecular, pathological and clinical features, notably a favourable prognosis and different clinical response rates to various chemotherapeutic regimens [13,17]. Based on these observations, we aimed at investigating the involvement of MSI in the UCC carcinogenesis. Considering the extremely high MSI tumour rate reported in sporadic UCC arising at a young age, we focused our study on young patients with a cutoff at 45 years of age. MSI is defined as the presence of alternate-sized repetitive microsatellite sequences in tumour DNA that are not found in the corresponding normal germline DNA. Such variations have been described using various types of microsatellite markers, classically mono- or di-nucleotides. The number and type of microsatellites analysed vary significantly among studies, as does the MSI incidence reported in these tumours [4,6 8,10]. Moreover, the populations studied differ in ethnic, stage and grade representation. Such an heterogeneity in the frequency of MSI also emerged from the first studies carried out on colorectal carcinomas, which prompted the community to hold an International Consensus Meeting at the National Cancer Institute in Bethesda in 1997 [5]. During this meeting, the use of a panel of five markers, two mononucleotides and three dinucleotides was recommended for MSI testing [5]. It was decided that only tumours displaying instability at two or more microsatellites should be defined as MSI-High (MSI-H), while tumours with instability at a single microsatellite should be classified as MSI-Low (MSI-L). Interestingly, it was later shown that MSI-H but not MSI-L phenotype identifies tumours with defective MMR and distinctive favourable features [18]. Owing to the clinical and pathological similarities between the MSI-L and the MSS colorectal tumours, the NCI recommended to group these tumours in a single entity. Their conclusions pointed out the need to strictly follow the consensus guidelines for microsatellite typing and interpretation of profiles. In our study, we chose to use for the first time in this tumour location the panel of five quasi-monomorphic mononucleotides that has recently been recommended by the National Cancer Institute as an alternative to the original Bethesda panel for the detection of MSI in gastrointestinal HNPCC tumours [13]. This panel has been shown to be highly sensitive and specific to detect MSI-H in gastrointestinal tumours with no need to match counterpart germline DNA [12]. Interestingly, we recently further established that this method was also adequate for the diagnosis of MSI in urothelial cell carcinomas of upper urinary tract [19]. In our series of bladder UCC, MSI could be detected in only one tumour, which displayed widespread instability. Because of the small size of allelic shifts in this tumour, we recommend the concomitant analysis of the normal counterpart DNA for MSI screening of UCC, as in the case of endometrial tumours [12]. Our results are in close agreement with a previous study based on 30 non-selected bladder UCC and reporting a single MSI tumour, using the original Bethesda panel and the NCI criteria to determine the MSI status [7]. Furthermore, no MSI tumour could be detected in a study performed on a non-selected population including 21 tumours and using the NCI diagnostic criteria, even if the microsatellite panel

5 european urology 49 (2006) differed from the Bethesda panel by one mononucleotide marker [6]. Nevertheless, such a low MSI frequency is in sharp contrast with the high frequencies claimed by other groups who used significantly different methods [8,10]. The use of a large number of dinucleotide markers could explain the surprisingly high MSI incidence (100%) reported by the group of Christensen who used a panel of 22 dinucleotide markers in young patients more or less age-matched with ours [10]. Firstly, most dinucleotide repeats are highly polymorphic in contrast to mononucleotide markers and their amplification profiles are sometimes difficult to interpret leading to discrepancies in scoring and misclassification of MSS tumours into MSI tumours [20]. On the other hand, the use of dinucleotide markers not always leads to a high proportion of MSI tumours, as illustrated by a study in which instability in six dinucleotide repeats was detected in only four of 200 UCC bladder tumours [14]. Secondly, the use of extended panels of dinucleotide repeats increases the chance to discover incidental instabilities in tumours which are not deficient in mismatch repair, and to over-classify tumours as MSI [21]. Moreover, the recommendations of the NCI stipulate that, when instability is restricted to the dinucleotide markers of the Bethesda panel, instability must be confirmed with a secondary panel of mononucleotides [13]. Yet, microsatellite instability restricted to dinucleotides rather than mononucleotides would correspond to MSI-L tumours, while MMR-associated MSI destabilizes both the mononucleotide and the dinucleotide microsatellites. Whether MSI-L UCC share clinical, pathological and molecular characteristics of MSS tumours as in the case of colorectal cancers remains to be established using standardized criteria. Many new microsatellite markers have been proposed for MSI testing in different studies with often an arbitrary definition for MSI phenotype. Two studies reported 21% (8/38) and 17% (12/72) of patients with MSI UCC but alterations involving more than one microsatellite marker was present in only 8 and 3% of the cases [15,16]. In another recent study reporting 73% of MSI bladder UCC and proposing MSI as a predictive factor of UCC recurrence, the authors used ten markers of which only two were shared with the Bethesda panel and defined MSI at 30% of unstable loci [8]. To date, few tumours with EMAST have been described and the molecular defect underlying this variety of instability remains unknown. Owing to the natural instability of tri- and tetra-nucleotide repeats, caution should be given to the MSI phenotype diagnosed with these markers, emphasizing the need for more studies to elucidate the role of such genetic instability, notably in UCC tumorigenesis. The use of various MSI testing methods that have not yet been validated most likely explains the heterogeneity in the results and hampers the comparison between studies. These conflicting data prompted us to investigate MSI using methods that have been validated in tumours of various locations [11,13]. 5. Conclusion In conclusion, classical MSI reflecting a defect in MMR gene expression is detected in a minority of bladder UCC from young patients. Even if this observation favours the existence of distinct classes of UCC, the role of MSI is most probably marginal, explaining why the incidence of these tumours is not significantly increased in HNPCC patients. The MSI analysis using the five mononucleotide markers recommended for MSI testing by the NCI that are more sensitive and easy to interpret compared to other molecular markers may help clarifying the role of MSI in bladder UCC in terms of clinical behaviour or oncogenic pathway. Acknowledgments Pierre Mongiat-Artus received support from the Association Française d Urologie. Catherine Miquel was supported by the Fondation pour la Recherche Médicale. This research was supported by a grant from the Association pour la Recherche sur le Cancer (#4683 to FP). References [1] Chopin DK, Gattegno B. Superficial bladder tumors. Eur Urol 2002;42: [2] Catto JW, Meuth M, Hamdy FC. Genetic instability and transitional cell carcinoma of the bladder. BJU Int 2004;93: [3] Aarnio M, Sankila R, Pukkala E, Salovaara R, Aaltonen LA, de la Chapelle A, et al. Cancer risk in mutation carriers of DNA-mismatch-repair genes. Int J Cancer 1999;81: [4] Catto JW, Azzouzi AR, Amira N, Rehman I, Feeley KM, Cross SS, et al. Distinct patterns of microsatellite instability are seen in tumours of the urinary tract. Oncogene 2003;22: [5] Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998;58:

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