The role of Tropomyosins and their Isoforms in Urothelial Carcinoma of Bladder ('TropICana-B')

Humayun-Zakaria, Nada ORCID: 0000-0001-5106-5689 (2024). The role of Tropomyosins and their Isoforms in Urothelial Carcinoma of Bladder ('TropICana-B'). University of Birmingham. Ph.D.

HumayunZakaria2024PhD.pdf
Text - Accepted Version
Restricted to Repository staff only until 31 December 2025.
Available under License All rights reserved.
Download (6MB) | Request a copy

Abstract

High throughput sequencing has informed molecular pathology for classification of urothelial bladder cancer (BC). Noting the prominence of ‘biomarkers’, they may be a key tool to avoid impaired quality of life (QoL) for patients undergoing investigation and surveillance for BC. This is predicated on the practicality of implementing such tests as a point-of-care (POC) urine-based test. It can potentially aid in early detection and diagnosis and facilitate screening pathways of patients prior to the development of T1 disease. Altered transcriptional events generate tropomyosin (TPM) isoforms that appear to have selective upregulation in the presence of BC. They are stable coiled-coil dimers, resistant to degradation and can be found in urine. We created a multistep verification model to assess the biomarker potential of TPM isoforms for detection of BC, incorporating sequencing techniques and wet lab experiments.
IsoTPM, a bespoke bioinformatics pipeline was created to identify upregulated TPM isoforms that contributed to overall gene expression by exploring short-read RNA sequences from 6 datasets: Cancer Cell Line Encyclopaedia (CCLE n=26, BC cell lines), West Midlands Bladder Cancer Prognosis Program (BCPP n=85, non-muscle-invasive), The Cancer Genome Atlas (TCGA n=414, muscle-invasive), a paracancerous dataset (GEO Accession GSE133624 n=29), University of York (n=8, bladder stroma and urothelium) and Genotype Tissue Expression (GTEx n=7, benign). Coupled with targeted long-read sequencing, we evaluated the expression of these target TPM isoforms in BCPP archived tissues and BC cell lines (n=96). Experimental validation of in silico analyses was performed on the top candidates (Tpm1.4, Tpm1.7, Tpm4.2 and Tpm 4.1). Isoform specific primer-pairs with probe-based multiplex qPCR assay was created to measure ‘relative’ isoform expression. Preliminary assay testing was performed on urine supernatants from cancer patients and those with benign urological conditions to define its potential as a urine-based test.
We found 2 pairs of isoforms with strong disease diagnostic value (p-value: 0.031, 9.18e-06). Furthermore, our qPCR assay showed a 1.41-fold increase in relative isoform expression in cancer vs benign conditions (p-value: 0.00312, n=28). Our assay can effectively predict the presence of BC when compared to other benign bladder conditions (AUC= 0.82).
We have pioneered the use of relative isoform usage as a diagnostic urine-based-assay for detection of BC. Currently available detection kits utilize mutation/ methylation-based techniques incorporating molecular adaptations at the gene-level but cannot evaluate isoform expression which constitutes an additional layer of information indicative of gene expression to understand the mechanisms of carcinogenesis.
Translational Relevance:
With the use of isoform evaluation and differences found in expression levels, a urine-based-diagnostic test can be utilized for creation of a point-of-care test to triage patients for further investigations, if required, via referral to the haematuria pathway, decreasing the unnecessary need for undergoing flexible cystoscopy as a first-line investigation for identifying bladder cancer.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):