Laboratory-Bladder cancer
MMR deficiency in urothelial carcinoma of the bladder presents with temporal and spatial homogeneity throughout the tumor mass

https://doi.org/10.1016/j.urolonc.2019.12.012Get rights and content

Highlights

Abstract

Background

Microsatellite instability (MSI), a hypermutator phenotype described in many cancers, has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in bladder cancer, but data on the possible extent of intratumoral heterogeneity are lacking.

Methods

To study MSI heterogeneity in bladder cancer, a tissue microarray (TMA) comprising 598 muscle-invasive urothelial carcinomas of the bladder was utilized to screen for MSI by immunhistochemistry with antibodies for MLH1, PMS2, MSH2, and MSH6.

Results

In 9 cases suspicious for MSI, MMR status was further evaluated by large section examination and polymerase chain reaction (PCR)-based analysis of microsatellites (“Bethesda panel”) resulting in the identification of 5 validated MSI cases from 448 interpretable cancers (prevalence 1.1%). MMR deficiency always involved PMS2 loss, in 3 cases with additional loss or reduction of MLH1 expression. Four cancers were MSI-high and 1 was MSI-low in the PCR analysis. Parallel sequencing revealed an inactivating MLH1 mutation in 1 tumor but no further known pathogenic MMR gene mutations were found. Immunostaining of all available 72 cancer-containing tissue blocks of the 5 confirmed bladder cancer with MSI including prior and subsequent biopsies showed complete homogeneity of the MMR protein defects and the status of the 4 MMR proteins did not markedly change in sequential resections. In all 4 cases with noninvasive precursor lesions, MSI was also detectable.

Conclusion

These data suggest that MSI occurs early in invasive bladder cancer and immunohistochemical MMR analysis on limited biopsy material is sufficient to estimate MMR status of the entire cancer mass.

Introduction

Urinary bladder cancer is the tenth most common cancer globally and the sixth leading cause of cancer death in males [1]. About 80% of patients present with low-grade noninvasive (pTa) or minimally invasive (pT1) cancers, which can be removed by transurethral resection (TUR-B) and exhibit a good prognosis. However, 20% of patients present with muscle-invasive disease (pT2 and higher), a condition with an overall mortality of almost 50%. In the absence of detectable metastases, these patients are treated by cystectomy. Chemotherapy is additionally applied in high-risk situations and in case of subsequent metastasis. The systemic treatment options include immune checkpoint inhibitors, which have shown striking efficiency in a fraction of patients [2,3]. Microsatellite instability (MSI) predicts response to immune checkpoint inhibition, which led to the site-agnostic approval of the PD-1 inhibitor pembrolizumab for advanced cancers with MSI irrespective of tumor origin [4].

MSI reflects a deficient mismatch repair (MMR) system that occurs in about 21% of endometrial cancers [5], 17% of colorectal cancers [6], 5.6% to 22% of gastric cancers [7], [8], [9], and in a wide variety of other cancer types typically within a range of 0.5% to 3% [10], [11], [12]. MMR deficient cancers accumulate mutations in monomorphic microsatellites (short tandem repeats) that are particularly prone to DNA mismatch errors. MSI is mostly caused by inactivation of one of the MMR genes MLH1, PMS2, MSH2, or MSH6, which may result from inherited mutations (Lynch Syndrome, estimated to cause 16% of all MSI-high cancers [11]) but more commonly occurs sporadically. MSI can be detected by molecular tests, including classic polymerase chain reaction (PCR)-based testing of a predefined panel of microsatellites or indirectly by immunohistochemical evaluation of MMR protein expression. Recent evidence demonstrated that MSI is a predictive biomarker for a favorable response towards immune checkpoint inhibitor therapy, and in 2017, the FDA approved the PD-1 inhibitor Pembrolizumab for therapy against advanced cancers with MSI-high or MMR deficiency irrespective of tumor origin.

Reports on MSI in urinary bladder cancer have described variable results. MSI has been reported to occur in 0% to 10% of cases when immunohistochemistry (IHC) was used to assess MMR status [13,14] and in 1% to 45% in reports describing PCR based methods [15,16]. Given the pivotal impact on the selection of treatment, precise assessment of MSI is important. Therapeutically relevant molecular information is generally obtained from a small fraction of the primary tumor, which may not relate to the molecular status of the entire cancer mass. Tumor heterogeneity can confound molecular diagnostics and diminish the success of targeted therapies. Intratumoral heterogeneity of MMR status has been described in some cases of colorectal [17], [18], [19] and endometrial cancer [20,21], but has so far not been analyzed in urothelial carcinoma.

To learn more on MSI heterogeneity in bladder cancer, a cohort of 598 muscle-invasive urothelial carcinomas of the bladder was screened by IHC on a tissue microarray (TMA) format. Cases with suspected MSI were further analyzed with PCR and repeated MMR immunohistochemistry on whole sections followed by a thorough analysis of all available cancer-containing tissue blocks including all previous and subsequent resections when MSI was confirmed.

Section snippets

Tissue microarray

A tissue microarray (TMA) was constructed from a consecutive series of 598 transurethral resection specimen from patients diagnosed with muscle-invasive urothelial carcinomas (pT ≥ 2) from transurethral resection specimen (TUR-B) at the Institute of Pathology of the University Medical Center Hamburg-Eppendorf between 2009 and 2017. All tumors were grade 2 or grade 3. Further clinical and pathological information was not available. TMA construction was done as described [22]. In brief, tissue

TMA screening

About 448 out of 598 (75%) bladder cancers on the TMA were considered interpretable for determination of the MMR protein status. Tumors were considered interpretable if either an unequivocal loss of staining of at least 1 of the 4 examined MMR proteins MLH1, PMS2, MSH2, or MSH6 was seen (MMR deficiency) or unequivocally retained expression of at least 3 MMR proteins was present without concomitant loss (intact MMR status). Noninterpretable cancers (25%) were due to lack of unequivocal tumor on

Discussion

A TMA containing 598 muscle-invasive bladder cancers was used for a rapid screening for tumors with MMR deficiency in this study. Detection of rare events is an ideal application for TMAs. For example, in earlier studies, 42 tumors with IDH1 mutations were identified in a screening of 15,531 prostate cancers (0.3%) or 43 tumors with CD117 overexpression were found in a cohort of 1,654 breast carcinomas (2.6%) [23,24]. It is of note that only 5 of 9 cancers that were initially suspected to have

Disclosure statement

The authors declare no conflicts of interest.

Ethical permissions

The usage of archived diagnostic left-over tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12,1) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

Acknowledgment

We are grateful to Melanie Witt, Maren Eisenberg, Gabi Rieck, Sina Dietrich, and Jana Hagemann for excellent technical assistance.

Authors’ contribution

CF, RS, GS: contributed to conception, design, data collection, data analysis and manuscript writing.

DH, FB, DD, AL, EB, TC: conception and design, collection of samples.

CF, CHM, GMF, CK, MK: collection and data analysis.

WW, GS, SS: study supervision.

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