Unravelling bladder cancer in Uganda: insights from the discrepancies in TP53 assessment between immunohistochemistry and whole exome sequencing

African Urology, Journal Year: 2025, Volume and Issue: 5(1), P. 5 - 9

Published: March 1, 2025

Open AccessUnravelling bladder cancer in Uganda: insights from the discrepancies TP53 assessment between immunohistochemistry and whole exome sequencing B Ssekitooleko, H Nalwoga, S Kalungi, N Kiwanuka, M Galukande, D Namuguzi, I Kajja, Ssuna, F Asiimwe, J Kuteesa, JB Masaba Muwonge Ssekitooleko12 https://orcid.org/0000-0002-3561-055X , Nalwoga1 https://orcid.org/0000-0002-2965-9445 Kalungi3 https://orcid.org/0009-0003-4877-3184 Kiwanuka1 https://orcid.org/0000-0003-2471-9290 Galukande1 https://orcid.org/0000-0001-9086-968X Namuguzi1 https://orcid.org/0009-0002-1674-7347 Kajja1 https://orcid.org/0009-0007-1311-7979 Ssuna4 https://orcid.org/0000-0002-2031-7377 Asiimwe3 https://orcid.org/0009-0000-2767-9250 Kuteesa3 https://orcid.org/0009-0000-0410-2336 Masaba3 https://orcid.org/0009-0008-4114-1148 Muwonge1 https://orcid.org/0000-0003-3964-2169 Affiliations 1Makerere University College of Health Sciences, Uganda 2Islamic Uganda, 3Mulago National Referral Hospital, 4Uganda Tuberculosis Implementation Research Consortium, Published Online:1 Mar 2025https://doi.org/10.36303/AUJ.0204https://hdl.handle.net/10520/ejc-mp_afurol_v5_n1_a2SectionsPDFAbstract ToolsAdd to favouritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditGMailOutlookCopy LinkMendeley AboutAbstractBackgroundBladder (urothelial carcinoma) is highly heterogeneous. Despite several cutting-edge treatment practices, prognosis muscle-invasive urothelial carcinoma (MIUC) remains very poor. Immunohistochemistry (IHC) (WES) have been used understand heterogeneous nature this aggressive cancer. Unfortunately, TP53, guardian genome, most mutated gene MIUC. Overexpression its products a predictor poor carcinoma. This study assessed mutations their protein expression using IHC WES techniques.MethodsWe conducted cross-sectional where 50 formalin-fixed paraffin-embedded (FFPE) tissue blocks, processed biopsy specimens patients admitted Mulago Hospital (MNRH) with MIUC, were consecutively selected re-examined. done according standard protocols.ResultsAll presented haematuria mass. The female-to-male ratio was 1:1.5. mean age participants 59.3 years (standard deviation [SD] 12.9). Using IHC, 26 (52%) samples stained positive for yet only one had missense mutation at WES.ConclusionIn sample Ugandan patients, do not drive MIUC as they various parts world. overexpressed proteins seen are possibly wild-type gene. Therefore, regarding clinical practice, high discordance immunopositivity makes (a cheaper readily available option our setting) surrogate WES.IntroductionBladder ninth leading cause globally second common malignancy urinary system, presenting either or non-muscle-invasive disease.1,2 About 81 400 new diagnosed 2020, which 17 980 died. These deaths primarily attributable (MIUC).2 Moreover, even good settings, five-year overall survival rate ranges 10% 20%.2,3Immunohistochemistry determines cancer-associated biomarkers involved biological pathways.4 Several markers investigated proposed predicting cancer; however, none routinely current practice.5-8 Nonetheless, does undermine value signatures supporting diagnosis management, prognosis.TP53, tumour suppressor (guardian genome) located on chromosome 17p, regulates cell cycle processes maintains genomic stability by averting genotoxic stimuli.9,10 Its overexpression known be associated life-threatening outcomes.11 protein-coding play significant roles growth progression.12Next-generation reliable technique detecting mutations; it costly many centres sub-Saharan Africa. Hence, analysis mutant commonly marker these settings. However, some studies reported reasons that can stabilise protein, rendering marker.13-17Because carcinoma's heterogeneity, immunobiology always similar different human populations.8,18 aimed determine if could gene.MethodsA descriptive, complex. After size calculation, we archived blocks confirmed biorepository MNRH 2019 2022. Only FFPE aged 18 above selected. Damaged those extensive necrosis, missing vital demographic data excluded. Two laboratories used. ran histopathology studies, while Unipath Specialty Laboratory Ltd. (Ahmedabad, India) carried out all studies.The histopathologic staging performed per 2004 World Organization (WHO) classification.19 retrieved trimmed cut into 4 μm thickness microtome machine. sections spread surface water (5–10 °C) below melting point wax remove wrinkles, then mounted onto labelled, silanised glass slides, fixed dry heat an oven 55–65 °C 30–60 minutes melt wax. Later, dewaxed xylene about five rehydrated series graded alcohol follows: absolute 100%, 90% alcohol, 70% 1–3 each. They washed distilled minimum 30 seconds. Haematoxylin Eosin (H&E) staining operating procedures.For extra thick taken Poly-L-lysine-coated slides subjected antigen retrieval microwave method. anti-TP53 monoclonal antibody, BP53-11 (GSI: 760-2542J28654-0043), recognises both proteins. Sections incubated secondary biotinylated antibody avidin-biotin-peroxidase complexes minutes. A Tris-buffered saline solution wash off primary antibodies.Reaction revealed diaminobenzidine chromogen, counterstained Harris haematoxylin enhance nuclear detection. colonic controls. Nuclear positivity dark brown colour bluish background. percentage immunopositive cells calculated counting least 1 000 areas maximum positivity. cutoff 15% considered positive.19,20 grouped three categories: > 50%, 25–49%, 15–24%, intensity strong, moderate, mild.Two consultant pathologists independently examined tissues without knowledge data. In case disagreement, consensus reached re-examining multi-head microscope.Isolation quantitative DNA analysisDeoxyribonucleic acid (DNA) isolated Alexgen extraction kit (CAT# AGFF50). quantity measured Qubit® 4.0 fluorometer, quality analysed gel electrophoresis. Importantly, same sample.Library preparationThe libraries prepared Twist Bioscience 2.0 104207). TapeStation checks.Cluster generation sequencingAfter obtaining library's Qubit concentration peak profile, library loaded Illumina NovaSeq 6000 cluster sequencing. Raw reads obtained after run demultiplexed.Bioinformatics analysisRaw quality-checked filtered adapters low-quality bases Fastp v0.20.0. high-quality mapped hg37 reference genome. Each sample's Sequence Alignment/Map files generated later converted Binary files. Variant annotation Franklin (Genoox) tool. RefSeq database identify characterise gene-associated variants. disease association variants derived COSMIC ClinVar databases. For specific study, discussed.Statistical analysisData collected questionnaire, entered REDCap (Research Electronic Data Capture), exported comma-separated values (CSV) file RStudio analysis. Patients' ages categorised groups (i.e. < 40, 40–49, 50–59, 60–69, 70–79, 80) summarised SD. Categorical frequencies proportions table format. Crude associations immunoreactivity independent variables (age groups, sex, lymphovascular invasion [LVI]) Pearson's chi-square test. p-value less than 0.05 statistically significant.ResultsOverall, participant included study. All participants' SD ± 12.9 years. majority (60%) males. Of samples, analysis, strong immuno-intensity WES. There no relationship participant's age, LVI. Most exhibited moderate intensity.Immunohistopathological characteristics examinedImages Figure (A – H&E X60, X100) show dysplastic urothelium forming fragmented papillae nests, detrusor muscle invasion, ≥ pT2, LVI; hence, lower likelihood regional nodal metastasis. images (C X40, negative immunoreactivity.Images 2 extending muscle, prominent possible Variable-sized cysts lined flattened cuboidal intraluminal necrotic material. immunopositivity.In 3, image (H&E X60) shows pT2. Image solid papillary nests infiltrating inciting desmoplastic stromal reaction. intense immunopositivity.DiscussionOf when 52% tested (Table I, Figures 1–3). Firstly, just infrequent. Secondly, having nearly corresponding uncommon. According recent Cancer Genome Atlas (TCGA), frequently driver across types.16,21 finding consistent few published papers area, notably 49% 60.7%.22,23The findings because, traditionally, there immuno-expression sample, expected.17 has long half-life quickly accumulates cell, creating stable target detection.10,17 contrast, normal protein) short half-life, making almost undetectable IHC.17However, necessarily related explain scenarios lead stabilisation protein. instance, now mechanisms also protein.13,16 heterogenous functional structural defects theTP53 but rather agents promote exogenous stress.16,24 Foods such soybeans, eggs, sweet potatoes, rice, among others, contain ceramides bind disrupt MDM2-TP53 interactions, stabilisation.24 Likewise, levels β-catenin increase nucleus, proteolytic degradation reduces, transcriptional activity increases, further accumulation proteins.25It should noted cancers large numbers epigenetic changes affecting chromatin proteins.21 other words, possibility led products. Similarly, results comprehensive molecular characterisation showed RB1 mutations. tumours luminal-papillary messenger ribonucleic (mRNA) subtype, displayed histology, predominantly node-negative, tended occur younger patients.22Therefore, although rare, likely overly expressed 1–3) (non-mutant) and, Alternatively, might effect dysregulation, including alterations methylation, histone modification, remodelling, essential role controlling expression.Study limitationsThe portion mutational differed immunostained part. conceivable heterogeneity account discordant findings. Furthermore, inability re-sequence newer AVITI, provided additional comparative insights. note system's accuracy reliable, over 85–90% typically exceeding Q30 score (indicating 99.9% accuracy) mapping specificity consistently 99%.26 robust performance ensures credibility despite lack cross-platform validation.This single-institution small selection bias (consecutive sampling). given national referral hospital, manages cases country, provide insight patient population.ConclusionOur contradict report correspond cancers, underscoring may indicate presence tissue.16,27,28Figures TableFigure 1: Muscle-invasive carcinoma; nested variantDownload FigureFigure 2: carcinoma, Microcystic 3: squamous differentiationDownload FigureTable I: immunohistochemical patterns TP53Cell (%)IntensityTotalStrongModerateWeakZero≥ 50830011Positive 26/50 (52%)25–4974101215–2402103Negative 24/50 (48%)< 150132024Total151052050Conflict interestThe authors declare conflict interest.Funding sourceMakerere Innovations Fund (REF: MAKRIF/CH/02/21). university influence protocol, management interpretation, manuscript writing.Ethical approvalEthical approval Makerere School Medicine research ethics committee under protocol number: Mak-SOMREC-2021-257.1. 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Medeyi Vicent, Namugga Martha, Nassaka Victo, Tinka Ann, nurses working ward MNRH. thank staff pathology department.PDF download Disclosure confirm read approved named persons who satisfied criteria authorship listed. due consideration protection intellectual impediments publication, timing respect property. Ethical conduct state appropriate institutional review board outlined Declaration Helsinki animal experimental investigations. signed informed consent document

Language: Английский

Bladder cancer biomarkers

Exploration of Targeted Anti-tumor Therapy, Journal Year: 2025, Volume and Issue: 6

Published: March 25, 2025

Bladder cancer (BCa) is among the most frequently diagnosed urinary tract cancers, characterized by a high recurrence rate and significant clinical heterogeneity. Effective diagnosis treatment of BCa demand continuous advancements in medical technologies, particularly given limitations classical methods such as cystoscopy urine cytology. A comprehensive search PubMed Web Science was conducted using relevant keywords to structure this narrative review. Additionally, specialist journals were reviewed. Only articles English included, with selection based on titles, abstracts, availability full texts. In recent years, biomarkers have emerged crucial tools complementing traditional techniques, providing more precise, sensitive, non-invasive for early detection, prognosis, monitoring response BCa. Molecular, genetic, protein enable deeper understanding biology, creating opportunities personalized therapy tailored individual patient needs. However, despite their potential, certain challenges remain, including standardization, validation, integration into routine practice. This review highlights transformative potential oncological care. It underscores importance incorporating these innovations refine diagnostic therapeutic approaches, ultimately improving outcomes. Modern prognostic can enhance outcomes enabling disease detection reducing risks. progress promises improve patients’ quality life minimizing burden fostering effective, care strategies.

Language: Английский