Development of Multiplex Polymerase Chain Reaction (PCR)-Based MSA Assay for Bladder Cancer Detection

Sep 24, 2026

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Microsatellite analysis (MSA) can profile urine microsatellite changes to detect bladder cancer, but conventional workflows often require analyzing many individual markers, up to 15–20, making them technically complex. To enable fast, efficient, standardized, and lower-cost testing, three multiplex PCR-based MSA assays were developed and run on a genetic analyzer. Six cancer and fourteen healthy samples showed complete correct classification with a 15-marker Triplet assay. Overall results indicate time- and cost-effective genetic testing for patient care.

LanguageEnglish

CategoryHealthcare

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Pages13

Reading Time33 min

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Abstract

1. Introduction

Microsatellite instability (MSI)

Bladder cancer and diagnostic challenges

Article Development of Multiplex Polymerase Chain Reaction (PCR)-Based MSA Assay for Bladder Cancer Detection Thomas Reynolds 1, Maxie Gordon 2,3, Gabriela Vanessa Flores Monar 2, David Moon 2 and Chulso Moon 2,3,4, * Citation: Reynolds, T.; Gordon, M.; Monar, G.V.F.; Moon, D.; Moon, C. Development of Multiplex Polymerase Chain Reaction (PCR)-Based MSA Assay for Bladder Cancer Detection. Int. J. Mol. Sci. 2023, 24, 13651. [https://doi.org/](https://doi.org/)[ ](https://doi.org/)[10.3390/ijms241713651](10.3390/ijms241713651) Academic Editor: Stephen Bustin Received: 23 July 2023 Revised: 18 August 2023 Accepted: 28 August 2023 Published: 4 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ([https://](https://)[ ](https://)[creativecommons.org/licenses/by/](creativecommons.org/licenses/by/)[ ](creativecommons.org/licenses/by/)[4.0/](4.0/)) . 1 NEXT Bio-Research Services, LLC, 11601 Ironbridge Road, Suite 101, Chester, VA 23831, USA 2 HJM Cancer Research Foundation Corporation, 10606 Candlewick Road, Lutherville, MD 21093, USA 3 BCD Innovations USA, 10606 Candlewick Road, Lutherville, MD 21093, USA 4 Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins Medical Institution, Cancer Research Building II, 5M3, 1550 Orleans Street, Baltimore, MD 21205, USA * Correspondence: [csmoon6@gmail.com](csmoon6@gmail.com); Tel.: +1-(443)-370-5056 Abstract: Several studies have shown that microsatellite changes can be proﬁled in the urine to detect bladder cancer. Microsatellite analysis (MSA) of bladder cancer detection requires a comprehensive analysis of up to 15–20 markers based on amplifying and interpreting many individual MSA markers, which can be technically challenging. To develop fast, efﬁcient, standardized, and less costly MSA to detect bladder cancer, we developed three multiplex polymerase chain reaction (PCR) based MSA assays, all of which were analyzed by a genetic analyzer. First, we selected 16 MSA markers based on nine publications. We developed MSA assays based on triplet or three-tube-based multiplex PCR (Triplet MSA assay) using samples from Johns Hopkins University (JHU Sample, ﬁrst set of samples) . In the second set of samples (samples from six cancer patients and fourteen healthy individuals), our Triplet Assay with 15 MSA markers correctly predicted all 6/6 cancer samples to be cancerous and 14/14 healthy samples to be healthy. Although we could improve our report with more clinical information from patient samples and an increased number of cancer patients, our overall results suggest that our Triplet MSA Assay combined with a genetic analyzer is a potentially time-and cost-effective genetic assay for bladder cancer detection and has potential use as a dependable assay in patient care. Keywords: bladder cancer; loss of heterozygosity; microsatellite instability; triplet MSA assay 1. Introduction Microsatellite instability (MSI) is a molecular tumor phenotype resulting from genomic hypermutability and manifested by genome-wide variations in the length of microsatellite sequences. As a familiar part of colon cancer syndrome, MSI is common in individuals with Lynch syndrome [1–3] . However, since its discovery, MSI has been a generalized phenomenon in a wide spectrum of sporadic cancers [4–8] . The underlying mechanisms of sporadic cancers may be based on epigenetic mechanisms, namely MLH1 methylation. The accumulation of frameshift mutations in tumor-associated genes is the most detrimental outcome of MSI, either in inherited or sporadic tumors. These mutations can activate crucial pathways promoting human carcinogenesis. Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal

FAQs

Why is microsatellite analysis useful for bladder cancer detection?

Microsatellite changes can be detected in urine and used to profile molecular alterations associated with bladder cancer.

What challenge limits current microsatellite analysis workflows?

Comprehensive MSA often requires analyzing many individual microsatellite markers, typically 15–20, which is technically challenging.

How did the authors validate their multiplex PCR-based Triplet MSA assay?

They tested a triplet multiplex assay with 15 markers on two sets of samples and found correct prediction for all cancer and healthy samples in the second set.

Sep 24, 2026

Uploaded by Cipher