Low Frequency of HNPCC-Associated Microsatellite Instability ...

AJCP / Original Article

634 Am J Clin Pathol 2014;142:634-639 DOI: 10.1309/AJCPVTCJ4VU5HKVZ

© American Society for Clinical Pathology

Low Frequency of HNPCC-Associated Microsatellite Instability and Aberrant MMR Protein Expression in Early-Onset Bladder Cancer

Johannes Giedl, MD,1 Roland Schneckenpointner, MD,2 Thomas Filbeck, MD,3 Petra Ruemmele, MD,4 Ferdinand Hofstaedter, MD,4 Maximilian Burger, MD,5 Arndt Hartmann, MD,1 and Robert Stoehr, PhD1

From the 1Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-University, Erlangen-Nuremberg, Germany; 2Center of Pneumology, Psychosomatic Medicine and Psychotherapy, Clinical Center Donaustauf, Donaustauf, Germany; 3Department of Urology, University Hospital Erlangen, Friedrich-Alexander-University, Erlangen-Nuremberg, Germany; and 4Institute of Pathology and 5St Josef Medical Centre, Department of Urology, University of Regensburg, Regensburg, Germany.

Key Words: Bladder cancer; Early onset; Microsatellite instability; Lynch syndrome

Am J Clin Pathol November 2014;142:634-639

DOI: 10.1309/AJCPVTCJ4VU5HKVZ

ABSTRACT

Objectives: Recently, it was shown that patients with Lynch syndrome due to an MSH2 mutation are at increased risk for the development of bladder cancer. To further this discussion, we screened the largest investigated cohort of patients with early-onset bladder cancer for microsatellite instability (MSI) and mismatch repair (MMR) deficiency to determine a possible role of Lynch syndrome in young patients with bladder cancer.

Methods: A total of 109 cases of bladder tumors from young patients (aged <45 years) were examined for MSI (Bethesda consensus panel). Expression of MMR proteins (hMLH1, hMSH2, and hMSH6) was evaluated by immunohistochemistry using a tissue microarray. Results were compared with a series of unselected consecutive bladder tumors (n = 95).

Results: Regarding the frequency of MSI high (1% vs 0%) or abnormal expression of MMR proteins (2% vs 6.5%), no significant difference between the early-onset and unselected patient group was found.

Conclusions: In young patients with bladder tumors, MSI and defects in MMR protein expression were not more frequent than in a series of consecutive bladder tumors. Most bladder tumors in young patients are not to be attributed to Lynch syndrome.

Urothelial bladder cancer is the seventh most common cancer worldwide in men and the 17th in women. The main risk factors for urothelial bladder cancer are cigarette smoking and occupational exposure to aromatic amines. Around half of the cases in men and more than one-third of the cases in women can be attributed to cigarette smoking. Certain medical conditions such as chronic urinary retention and chronic inflammation (eg, due to schistosomiasis), medical drugs (eg, phenacetin, chlornaphazine, and cyclophosphamide), radiotherapy (eg, for prostate carcinoma), and a certain genetic susceptibility (eg, NAT2 slow acetylator and GSTM1 null genotypes) are known to increase the risk of urothelial bladder cancer, while consumption of vegetables and fresh fruits lowers this risk.1,2

Bladder cancer is mainly found in elderly people, with 90% of the patients older than 55 years and the mean age of diagnosis being 73 years. Very few patients (1%−2.4%) with bladder tumors are younger than 40 years at the time of diagnosis.3

The close examination of young patients with a malignant disease led to the discovery of predisposing changes such as in the case of hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome. Cancers known to be associated with Lynch syndrome or HNPCC are colorectal cancer, endometrial and ovarian cancer (with gynecologic cancers posing the largest manifestation of extra-colonic Lynch syndrome–spectrum malignancies), gastric cancer, urothelial cancer of the upper urinary tract, small bowel cancer, central nervous system tumors, and tumors of the pancreaticobiliary system.4

van der Post and colleagues5 recently showed that patients with Lynch syndrome due to an MSH2 mutation were also at an increased risk for developing urothelial bladder cancer.

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Am J Clin Pathol 2014;142:634-639 635 DOI: 10.1309/AJCPVTCJ4VU5HKVZ


The authors of this study proposed adding urothelial cancer of the bladder to the spectrum of Lynch syndrome–associated cancers. Furthermore, recommendations for surveillance of urothelial carcinomas in Lynch syndrome were issued, proposing surveillance (using a combination of ultrasound of the bladder and upper urinary tract, urinary cytology, and sediment) in every MSH2 mutation carrier aged 40 years and older, performed every 1 to 2 years.

These risk data were strengthened by a second study analyzing proven Dutch and German mismatch repair (MMR) mutation carriers. Engel and colleagues6 also found an increased lifetime risk for bladder cancer in hMLH2 mutation carriers compared with the general population.

These epidemiologic data suggest that it might be reasonable to introduce young patients with bladder cancer to routine HNPCC testing since young age seems to be an indicator for HNPCC in patients with bladder cancer. To further evaluate this hypothesis, we investigated the frequency of microsatellite instability (MSI) and MMR deficiency (and thus the frequency of Lynch syndrome) in the largest cohort of bladder tumors from young patients (aged <45 years) investigated so far compared with a consecutive, unselected bladder tumor series using microsatellite analysis and immunohistochemistry.

Materials and Methods

Bladder Cancer Tissue SamplesIn total, 109 patients with early-onset bladder cancer

were identified from the files of the contributing institutes of pathology. Formalin-fixed, paraffin-embedded (FFPE) primary tumors and corresponding normal tissues were obtained from the contributing institutions. All cases were reclassified according to the 2004 World Health Organization classification.7 The control group consisted of 95 consecutive patients with urothelial bladder tumors. ❚Table 1❚ shows the characteristics regarding age, sex, and tumor grade and stage of the young collective and the control groups. Prior institutional review board (University Hospital Erlangen) approval was obtained for molecular analysis on archival material.

Microdissection and DNA IsolationTumor and normal DNA were extracted from paraffin-

embedded tumor tissues as described previously.8 In brief, 5-µm histologic tissue sections were deparaffinized and stained with 0.1% methylene blue for 15 seconds. Manual microdissection was used to obtain tumor cells with a purity of at least 80%. Prior to microdissection, the areas with the highest tumor cell density were marked on a representative

H&E-stained section by an experienced surgical pathologist (A.H. or J.G.). Nonmalignant tissue was removed from the section by scraping off with a sterile needle. Tumor tissue was then collected with a separate sterile needle and transferred into a sterile tube. DNA isolation was performed using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.

Microsatellite AnalysisThe microsatellite panel recommended by the National

Cancer Institute for the detection of MSI (Bethesda consensus panel), consisting of two mononucleotide repeats (BAT25 and BAT26) and three dinucleotide repeats (D2S123, D5S346, and D17S250), was used for analysis of MSI.9 Polymerase chain reaction (PCR) amplification was performed in an MJ Research Thermocycler (PTC100; MJ Research, Watertown, MA) as previously described using 100 ng of purified DNA in a final volume of 20 µL.10 Analysis of PCR products was performed by 6.7% polyacrylamide/50% urea gel electrophoresis (1 hour, 1,500 V, 55°C) in a SequiGen sequencing gel chamber (Bio-Rad, Hercules, CA), followed by silver nitrate staining as described previously.11 MSI was defined as the presence of new bands after PCR amplification of tumor DNA not present in PCR products of the corresponding normal DNA. All gels were evaluated independently by three authors (R. Schneckenpointner, J.G., and R. Stoehr). A tumor was

❚Table 1❚Characteristics of the Study Cohorts

Characteristic

Patients With Early-Onset Bladder Cancer (n = 109)

Unselected Consecutive Cohort (n = 95)

Age distribution, y Minimum/maximum 17/45 50/87 Mean ± SD 37.6 ± 6.6 70.1 ± 7.8 Median age 40 70Sex, No. Male 83 74 Female 24 21 Not available 2 0Stage distribution, No. Urothelial papilloma 2 0 Inverted urothelial papilloma 5 1 PUNLMP 0 9 pTa low grade 53 55 pTa high grade 19 7 pT1 high grade 13 17 pT2 high grade 8 6 pT3 high grade 6 0 pT4 high grade 3 0

PUNLMP, papillary urothelial neoplasm of low malignant potential.

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Giedl et al / MSI and MMR Defects in Early-Onset Bladder Cancer



considered as having a high-level instability (MSI-H) if at least two of the five tested markers were unstable. If only one marker was unstable, the tumor was classified as having a low-level instability (MSI-L). Instable markers were verified by a second PCR amplification. A loss of heterozygosity was not counted as MSI. A case was considered interpretable if at least two markers yielded interpretable results.

Immunohistochemistry (hMLH1, hMSH2, and hMSH6)To achieve a high level of standardization for

immunohistochemical analysis, we constructed a tumor tissue microarray from tissue cores (1.2 mm in diameter) of each paraffin block as described previously.12

Expression of the mismatch repair proteins hMLH1, hMSH2, and hMSH6 was analyzed by immunohistochemistry as described previously.13 The slides were evaluated by one surgical pathologist (A.H.) without knowledge of the clinical data. Immunohistochemical expression of MMR was scored as negative or positive. If the tumor showed complete absence or nuclear expression in less than 5% of tumor cells in at least one MMR (showing regular MMR expression in the associated normal tissue), the case was scored as negative. Cases with both strong (>30%) and weak (<30% but >5%) nuclear positive reaction were scored as positive.

Statistical AnalysisA two-sided Fisher exact test was used to evaluate

differences in the distribution of MSI status and MMR

Mfd15 Mfd15

NO TU

D5S346

NO TU

D5S346

NO TU

NO TU

MSI

MSI

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❚Figure 1❚ A, Representative examples for microsatellite analysis. Arrows indicate single alleles of the markers. Microsatellite instability (MSI) (bandshift) is clearly visible on the right side of the figure. NO, DNA from nonmalignant tissue; TU, DNA from bladder tumor. B, Distribution of microsatellite-stable and microsatellite-instable tumors between the study cohorts. MSI-H, high-level MSI; MSI-L, low-level MSI; MSS, microsatellite stable. P = .683.

expression of the tumors between both cohorts. Statistical analysis was done using SPSS version 13.0 (SPSS, Chicago, IL). P values less than .05 were interpreted as statistically significant.

Results

MSI AnalysisUsing the Bethesda consensus panel, we evaluated the

cases for MSI. Only cases with more than two interpretable markers (of the five tested markers) were used for statistical analysis.

In the group of young patients (n = 109), DNA was available for every case, and interpretable results could be obtained for all cases. In the control group (n = 95), DNA was available for only 54 (56.8%) cases, which were conducted for statistical analysis.

In the group of young patients, one (0.9%) of 109 tumors showed an MSI-H status (patient age of 35 years, papillary bladder tumor, pT3 high grade). Six (5.5%) of 109 patients were MSI-L. All other cases (102 [93.6%] of 109) were microsatellite stable (MSS). In the consecutive group (n = 54), no case of MSI-H was found. Two (3.7%) of 54 cases were MSI-L. All other cases (52 [96.3%] of 54) in this group were MSS. There was no statistical significance in the frequency of MSI between both cohorts (P = .683) ❚Figure 1❚.

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Immunohistochemical Analysis of MMR Protein Expression

The expression of MMR proteins hMLH1, hMSH2, and hMSH6 was evaluated and compared between the young and consecutive groups.

In 13 (11.9%) of the 109 cases, the FFPE material was insufficient for further immunohistochemical analysis. In two (2.1%) of the remaining 96 cases, loss of MMR expression was found. Both cases showed a loss of MSH6 expression. The other 94 cases did not show loss of MMR expression. Of the 95 cases in the consecutive group, three (3.2%) cases did not have sufficient material for

immunohistochemical studies. Of the remaining 92 cases, six (6.5%) showed a loss of MMR protein expression (two cases with loss of MSH6, two cases with loss of MLH1, one case with loss of MSH2, and one case with loss of MSH2 and MLH1). Using the Fisher exact test, no significant differences regarding loss of expression of the MMRs hMLH1, hMSH2, and hMSH6 were found between the young and consecutive groups (P = .162) ❚Image 1❚ and ❚Figure 2❚. In addition, there was no correlation between MSI and loss of MMR protein expression. The single MSI-H tumor from a young patient showed a strong expression of all three analyzed MMR proteins.

A B

C D

❚Image 1❚ Representative examples for mismatch repair protein immunohistochemical staining. A, hHSH6-negative tumor (×400). B, hMSH6-positive tumor (×400). C, hMSH2-positive tumor (×200). D, Weak hMSH6 expression in a bladder tumor (×200).

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Giedl et al / MSI and MMR Defects in Early-Onset Bladder Cancer



Discussion

Pronounced MSI due to defects in DNA MMR is a hallmark of Lynch syndrome (HNPCC) and its associated malignancies. In Lynch syndrome, tumors of the gastrointestinal tract and urogenital organs (upper urinary tract) can arise early in life. Recently, several studies on affected families suggested that bladder cancer should be added to the spectrum of HNPCC-associated tumors, especially in hMSH2 mutation carriers.5,6

In our cohorts, bladder tumors from young patients did not show an MSI phenotype or aberrant expression of MMR proteins more frequently than did tumors from an unselected, consecutive group of patients with bladder cancer. The results of our study are in concordance with a previously published study, which did not find pronounced MSI in young patients with bladder tumors using the original Bethesda panel or an advanced marker panel, including quasimonomorphic mononucleotide repeats (NR21, NR24, and NR27).14 In addition, analyses of microsatellite status (also using the Bethesda microsatellite panel) and expression of MMR proteins of bladder neoplasms of adolescent patients (aged <19 years) also showed no MSI in these very young patients.10

In contrast, high frequencies of MSI in bladder tumors from young patients were reported in some studies. Christensen and coworkers15 examined 22 different microsatellite loci and found allelic imbalances in all (n = 14) of the analyzed tumors. Migaldi and colleagues16 used 19 different microsatellite loci for their analyses and found length variations in 48 of 51 tumors. Nevertheless, the results of these studies could not be compared with our study as the microsatellite markers used did not include the classic Bethesda panel optimized for detection of HNPCC-associated MSI. Both studies concentrated their marker selection on chromosomal regions known to be frequently affected by allelic imbalances in bladder cancer generally, so

a higher frequency in detecting chromosomal alteration not related to HNPCC might have been expected.

MMR defects and MSI have already been analyzed in bladder cancer. It was shown that loss of MMR protein expression could be found in approximately 20% of cases. MSI in bladder tumors detected by the Bethesda consensus panel was described as a very rare event (MSI-H, ~1%-3%; MSI-L, 7%).17-19 These data are in line with our findings and strengthen the validity of our results and the consistence of our cohorts.

An interesting observation of our study was the lack of correlation between MSI and MMR deficiency. This phenomenon was also found in both bladder cancer and other tumor entities (eg, colon cancer, breast cancer, Wilms tumor) before.17,20-22 There might be various reasons for this finding. Only three MMR proteins were analyzed in our study. In microsatellite-instable tumors, the expression of other MMR proteins (eg, hMLH3 or hMSH5) could have been decreased or lost, but we focused our study on the most frequently affected MMR gene in Lynch syndrome. The cellular loss of one MMR protein (eg, hMLH1) could not be sufficient to cause detectable microsatellite defects. In vitro studies in human colon cancer cell lines revealed that an almost complete knockdown of hMLH1 expression using antisense RNA did not cause loss of MMR function.23 In addition, a dramatically reduced expression of both hMLH1 and hMSH2 without mutations in both genes did not cause MSI in a human gastric cancer cell line. Only cell lines with mutated hMSH2 or hypermethylated hMLH1 promoter showed an MSI phenotype. These data suggested that an MSI phenotype is associated only with genetic alterations in MMR genes, and extremely low levels of MMR protein expression are sufficient for microsatellite stability.23,24 In combination with our data, it could be suggested that even in patients with early-onset disease, most microsatellite-instable or MMR-deficient bladder tumors have no HNPCC-associated background. Young age of onset is not a convincing or reliable factor for Lynch syndrome in patients with bladder cancer.

In conclusion, MSI and MMR deficiency are rare events in bladder cancer independent from age of disease onset. Occurrence of bladder tumors at a younger age than usual (here <45 years) is not an indicator of MSI and thus is not sufficient to justify an HNPCC screening procedure. Nevertheless, in patients with proven Lynch syndrome, screening for bladder cancer appears reasonable since the reported association between the hMSH2 mutation carrier and an increased bladder cancer risk suggests a close surveillance.

Address reprint requests to Dr Stoehr: Institute of Pathology, University Hospital Erlangen, Krankenhausstr. 8-10, 91054 Erlangen, Germany; [email protected]. Parts of this study were presented at the 90th annual meeting of the German Association of Pathology; June 19-21, 2006; Berlin, Germany.

❚Figure 2❚ Distribution of regular and abnormal mismatch repair (MMR) protein expression between the study cohorts. P = .162.

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De�cientMMRRegularMMR

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