Somatic mutations in protein-coding genes and noncoding regulatory regions are the major drivers of cancer. Only a relatively small number of somatic noncoding mutations that are likely drivers have been described to date, including those in the promoters of the TERT,FOXA1, andTP53 genes. The impact of these alterations can be profound by initiating, increasing, or abolishing gene expression. Promoter mutations in particular have been difficult to identify even from whole tumor genomes due to their high content of G and C nucleotides, which leads to loss of sequencing coverage in these regions. Therefore, the landscape of somatic drivers in gene promoters remains incomplete. Here, we present a hybrid capture assay optimized for >3000 promoters of cancer genes. We show that this assay allows for deep sequencing of challenging GC-rich promoter regions, enabling discovery of reliable point mutations, short insertions and deletions, copy number variants, and mutational signatures in cell line models as well as formalin-fixed, paraffin-embedded archival tissue samples. Our assay nominated candidate noncoding driver mutations in CDK4, SMAD3, andGATA3 in breast cancer for future functional follow-up.

Osteosarcoma (OS), the most frequent primary malignant bone tumor in children and adolescents, is characterized by substantial inter- and intra-tumoral heterogeneity and an immunosuppressive tumor microenvironment (TME), constraining the efficacy of both standard chemotherapy and emerging immunotherapies. Recent advances in single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) have enabled high-resolution profiling of OS tumors, revealing diverse malignant, immune, and stromal cell populations. These studies have identified proliferative, inflammatory, and angiogenic tumor states, immunosuppressive myeloid subsets, exhausted T cells, and complex cell-cell communication networks that contribute to tumor progression and immune evasion. However, several challenges constrain the broader application of single-cell approaches in OS. The mineralized structure of bone tissue complicates dissociation into viable single cells, and the rarity of OS limits access to fresh specimens. Most existing datasets are based on small, heterogeneous cohorts and are generated using diverse protocols, which complicate data integration and comparison. Emerging methods are beginning to overcome these barriers. These include snRNA-seq for frozen and archival tissue, improved dissociation protocols for mineralized tumors, and integration with spatial transcriptomics to retain spatial context. Moving forward, combining single-cell transcriptomics with complementary modalities, such as immune repertoire analysis, chromatin accessibility profiling, and spatial proteo-genomics, combined with functional validation, will provide deeper insights into immune dynamics, regulatory mechanisms, and the cellular architecture of the OS TME. This review summarizes the current landscape of single-cell transcriptomics in OS and highlights methodological challenges in single-cell studies in OS tumors, recent biological insights, and their implication for immunotherapies.

Human nephrogenesis is complete at 34-36weeks gestation, with 60% of nephrons forming during the third trimester through lateral branch nephrogenesis (LBN). Currently, no mechanism exists for LBN as there are no late gestation human kidney transcriptional datasets. We hypothesized that an induced but dividing population of nephron progenitor cells (NPCs) would contribute to the amplification of nephrons in late gestation. We used the rhesus macaque, an established model of LBN, to help identify potential mechanisms. Single-cell RNA-sequencing (scRNA-Seq) was performed on cortically-enriched fetal rhesus kidneys (n = 9) from late second trimester and third trimester during LBN. This data was integrated with publicly available human scRNA-seq datasets from 8-18weeks gestation kidneys (n = 8) using Harmony package. Differentially expressed genes and ligand-receptor interactions were assessed and validated using RNAScope™ on human and rhesus archival tissue. Label transfer of previously defined kidney cell populations was performed on scRNA-seq data from 64,782 rhesus cells, including 7,879 nephron progenitor cells (NPCs) identified based on marker gene expression. Pseudotime analyses identified a late gestation-specific lineage branch of induced NPC in rhesus that was not observed in mid-gestation humans. Differential expression analyses identified increasedSFRP1,FZD4,andTLE2and decreasedFZD7,SHISA2,SHISA3, andTLE4within the late-gestation rhesus NPC compared to mid-gestation human NPC and increased SEMA3D within the rhesus ureteric bud (UB) tip, suggesting a compositional shift in WNT and SEMA signaling components within the naive NPC population during LBN. The rhesus macaque uniquely enables molecular studies of late-gestation primate nephrogenesis. Our study suggests the hypothesis that a transitional state of self-renewing NPCs supported by compositional shifts in key pathways may underlie the switch from branching phase nephrogenesis to lateral branch nephrogenesis and support ongoing nephron formation in late gestation. However, it remains to be determined if these changes within the late gestation NPC are time-dependent or species-dependent.

Oral squamous cell carcinoma (OSCC) is a life-threatening disease that ranks sixth amongst the most common human cancers worldwide. In most cases, OSCC development is preceded by oral premalignant disorders (OPMDs). There is an urgent need for more biomarker studies that will aid in early detection, diagnosis, and treatment of OPMD to prevent their transformation to OSCC. Leucine rich repeats and immunoglobulin like domains 1 (LRIG1) are known to negatively regulate EGFR expression. Little is known about the significance of LRIG1 in oral cancer pathogenesis. In a retrospective study, immunohistochemical analysis of EGFR and LRIG1 was carried out in 212 archival oral tissues (100 OSCC, 87 OPMD, and 25 normal), correlated with clinicopathological parameters and disease outcome over 62 months for OSCC patients. Further, gene expression for EGFR and LRIG1 was also evaluated in 160 tissue samples. Significant increase in EGFR in OSCC tissue was observed compared to controls, while LRIG1 levels were found to be downregulated in OSCC and OPMD cases compared to normal tissues (p < 0.05). Kaplan-Meier analysis of patients stratified to high and low LRIG1 expression showed significantly different overall survival (p < 0.05, median survival 1397 and 561 days, respectively). A unique translational impact of our study is that it provides clinical evidence for the potential of LRIG1 downregulation as predictor of poor prognosis in OSCC. These preliminary findings support further research to validate LRIG1 based prognostic tools for oral cancers.

Small partial deletion of a highly GC-rich FOXF1 exon 1 in two deceased siblings with alveolar capillary dysplasia.

BackgroundMultiplex immunostaining combined with digital image analysis has become a central tool in hepatology research because it allows for the simultaneous visualisation and spatial mapping of multiple cellular markers within a single tissue section, providing critical insights into the complex, heterogeneous microenvironments of liver diseases like cholangiopathies, hepatocellular carcinoma, metabolic dysfunction-associated steatohepatitis or hepatic fibrosis. Nevertheless, significant barriers prevent the widespread adoption of most multiplexing platforms, including the technological complexity that necessitates specialised equipment and personnel. Additionally, several technologies remain incompatible with standard laboratory workflows and archival formalin-fixed paraffin-embedded (FFPE) tissue or in vitro culture samples.MethodsWe developed a customised immunofluorescence workflow based on sequential cycles of antibody stripping to generate multiplexed digital images from FFPE liver sections, intrahepatic cholangiocyte organoids and primary mouse liver cells cultured either in conventional two-dimensional systems or within a biliary niche-on-a-chip platform. Subsequent image processing and channel alignment were performed using an in-house and open-source, Python-based software tool.ResultsWe demonstrate the functionality of a multiplex immunofluorescence protocol that can be readily adapted to a broad range of archival tissues and primary cell-based samples. This workflow allows the visualisation of antigen-expressing cells and protein–protein interactions using proximity ligation assays. This study also details the technical considerations necessary for its rapid integration into routine workflows in virtually any laboratory equipped for conventional immunohistochemistry.ConclusionsThis methodology integrates multiplex immunofluorescence into standard laboratory workflows, thereby enabling researchers to overcome challenges such as technological complexity and cost for transitioning from conventional to multiplexed microscopy.

By 2050, one-fifth of the world is expected to be over 60, and the prevalence of age-related neurological conditions is predicted to increase dramatically. Aged animals are currently underutilised in neurological research, leading to a gap in knowledge about the contribution of biological age to the pathophysiology of age-related neurological conditions. Additionally, it is unclear whether age-related changes differ across species used in preclinical models, and how these differences may compare to the aged human brain. Understanding these points is critical for successful translation of findings from preclinical studies to the human context. The current study presents a cross-species characterisation of microglia, the key regulator of the brain's immune response, during ageing. Microglial number, proliferation and morphology were assessed in archival tissue from Sprague Dawley rats (males; 3 to 18 months old) and Merino sheep (males and females; 1 to 6 years old), with these two species selected for their relevance to preclinical modelling of neurological disease. Increased numbers of proliferating microglia were observed in the cortex, hippocampus and portions of the striatum in both species. This proliferation declined at the oldest timepoint assessed (i.e. 18months old) in rats, a pattern not seen in the sheep. Total microglial number was largely unchanged with age in the rat brain; however, in sheep, the number of microglia decreased significantly in the dentate gyrus in older animals. Notably, microglia in 18-month-old rats were larger in all regions, but changes in branching were observed exclusively the striatum. Similarly, in sheep, morphological changes were localised to the striatum, with increased cell and soma size in the caudate nucleus, and increased cell size and process length in the putamen. These changes suggest a shift away from homeostasis in the cortex and hippocampus and towards a semi-ramified morphology in the striatum in late middle adulthood that is largely conserved across these two species. Nevertheless, the age of the oldest animals here equates to only ~ 60years old in humans, rather than reflecting an aged human population. Thus, future work is needed to understand how species-specific differences continue to evolve in older age.

Refining spatial proteomics by mass spectrometry: an efficient workflow tailored for archival tissue

Primary TB and post-primary TB (PPTB) are different disease entities. PPTB occurs only in humans, and no animal model mimics the actual pathology of PPTB. Information on the immune pathogenesis of PPTB is lacking due to the scarcity of untreated human material. In the early stages of PPTB, lesions can either progress or regress. Mycobacterial proteins present in these early lesions are vital to identifying targets for future development of better vaccines and therapeutics. Using proteomics, we aimed to establish a methodology to identify mycobacterial proteins expressed in lesions of human PPTB from the archival formalin-fixed and paraffin-embedded lung tissue from 1937−1941. Five untreated TB patients with a total of eleven samples were used. Micro-dissected tissue from six early and five necrotic lesions were processed for proteomics. Using a mass spectrometry and multiplexing tandem-mass-tag (TMT) approach, a total of 3531 Homo sapiens and 110 bacterial proteins were identified and quantified. Four M. tuberculosis proteins; mIHF (accession p71658), groEL2 (accession P9WPE7), RV2971 (accession P9WQA5), cycA (accession O33203), and 1 Mycobacterium avium protein hup (accession A0A0H3A054) were identified. Comparison of early lesions with necrotic lesions showed significantly more RV2971 proteins expressed in early lesions, and mIHF expressed in necrotic lesions (Log2 fold change necrotic – early; 1.401 & −0.581 respectively). In conclusion, we established the methodology and proof of principle for detecting M. tuberculosis proteins in PPTB lesions at different stages of the disease. We identified four M. tuberculosis proteins, two showing significant differential expression in early and necrotic lesions.

Concordance and prognostic value of antibody drug conjugate and checkpoint inhibition targets in matched bladder cancer specimens from transurethral resection of the bladder, radical cystectomy and lymphadenectomy.

Recently, oncology practice has undergone a paradigm shift, emphasizing precision-driven and patient-centered treatment models. Droplet digital polymerase chain reaction (ddPCR) is considered a top-tier technique for detecting cancer-associated genetic changes due to its high precision and reliability. Today, ddPCR is widely used for absolute quantification of alleles, identification of rare mutations, assessment of copy number variations, evaluation of DNA methylation, and detection of gene rearrangements in diverse clinical samples. It has proven especially valuable in the analysis of archival tumor tissues, where degraded DNA and limited material often hinder traditional methods, offering objective and automated quantification even underchallenging conditions. Additionally, new avenues are emerging that leverage other body fluids such as cerebrospinal fluid and urine, further expanding the potential of ddPCR-based molecular testing. It can also be considered a tool for noninvasive prenatal diagnosis and neonatal screening. This article provides a comprehensive overview of the current and evolving applications of ddPCR in oncologic molecular screening and pediatric-onset genetic disease research.

A reliable isolation of the dentate gyrus (DG) is a critical pre-analytical step for region-specific neurobiological assays, yet DG microdissection practices vary widely and are rarely compared quantitatively under standardized conditions. In addition, long-term paraformaldehyde-fixed archival brain tissue is commonly regarded as unsuitable for microdissection because of reduced pliability and poor anatomical contrast, limiting its use for training and protocol development. Here, we quantitatively compare two commonly used DG microdissection strategies, a medial (intact-block) approach and a coronal (slice-guided) approach across fresh, fixed, and softened-fixed rat brain hemispheres under matched conditions. To enable the use of archival material, fixed hemispheres were subjected to a simple 15-day slow-running tap water softening protocol to improve tissue handling and landmark visibility. Dissection duration and anatomical specificity were evaluated, the latter quantified by measuring residual cornu ammonis (CA)1-3 area on hematoxylin-eosin-stained coronal sections following DG removal. In fresh tissue, the medial approach enabled significantly faster DG isolation than the coronal approach, while both strategies achieved comparable anatomical specificity. In softened-fixed tissue, dissection times increased for both approaches, but the same relative performance ranking was preserved. Softening markedly improved tissue pliability and boundary visualization, particularly benefiting the coronal, stepwise dissection strategy. Residual CA1-3 areas did not differ significantly between approaches or tissue states. This study provides a validated, training-oriented DG microdissection workflow that supports methodological standardization, reproducibility, and 3R-aligned use of archival tissue, strengthening the pre-analytical foundation for downstream region-specific neuroscience assays.

Objective: Brain branks preserve extensive material relevant toneurodegenerative disease research. As these collections age, tissue becomesarchival, raising the question of whether long-term fixed and stored human braintissue remains suitable for contemporary immunohistochemical analyses.Materials and Methods: Forty-one autopsy brains collected between1946 to 1980 were examined. For each case, midbrain and hippocampus wereavailable both as original paraffin-embedded blocks and as tissue stored longterm in fixative. New paraffin blocks were prepared from the long-term fixatedtissue. Sections from original and newly prepared blocks wereimmunohistochemically stained for α-synuclein, hyperphosphorylated tau andamyloid-β. Immunoreactivity was assessed using semi-quantitativescoring.Results: Original blocks consistently showed good staining intensityand morphological preservation for each protein pathology. Newly prepared blocksshowed slightly lower semi-quantitative scores for Lewy-related pathology,without statistically significant differences, except for astrocyticα-synuclein in the substantia nigra in cases from the 1960s. Tau pathologydisplayed modestly reduced labelling, particularly of the neuropil threads andneurofibrillary tangles, most evident in cases from the 1950s.Amyloid-β-positive senile plaques showed similar or slightly higher scoresin newly prepared blocks, with no significant differences across regions.Conclusion: Human brain tissue preserved as paraffin-embedded blocksor stored in fixative for up to 78 years remains suitable forimmunohistochemical analyses. Adequate-to-good detection of aggregatedα-synuclein, hyperphosphorylated tau and amyloid-β is achievable,indicating preserved pathological hallmarks of Lewy Body Disease and Alzheimer’sDisease in archival tissue.

Kaposi sarcoma (KS) is an angioproliferative tumor caused by Kaposi sarcoma herpesvirus (KSHV) that occurs in people with HIV. Concurrent KSHV-associated diseases (KAD), including multicentric Castleman disease, primary effusion lymphoma, and KSHV-associated inflammatory cytokine syndrome may modify KS biology and impact clinical outcomes. Transcriptomic profiling of archival KS tissue enables investigation of molecular heterogeneity associated with these overlapping disease states. Archival formalin-fixed paraffin-embedded (FFPE) KS skin biopsies from 42 patients with HIV-associated KS between 2017 and 2022 were analyzed based on confirmed histopathologic diagnosis, tissue adequacy for RNA profiling, and availability of linked clinical data. Bulk transcriptomic analyses were conducted using Nanostring nCounter PanCancer ImmunoOncology panel supplemented with KSHV-specific probes. Spatial RNA profiling was performed on four tissues from participants with KS and concurrent KAD (KS+KAD) using GeoMx digital spatial profiling (DSP) platform. Regions of interest were selected using LANA-1, CD45 and CD31 staining to characterize tumor (LANA-1+, CD31+), vessel (LANA-1-negative, CD31+) and immune cells (CD45+) areas. For bulk transcriptomic analyses and spatial transcriptomic analyses, p-values were adjusted for multiple comparisons using the Benjamin-Hochberg FDR approach, and adjusted p-values (padj) are reported. KS samples were obtained from 42 men with HIV (median age 40 years). Median HIV viral load of 27 copies/mL and median CD4+ T-cell count was 211 cells/µL. Forty-eight percent had KS alone and 52% had KS+KAD. Patients with KS+KAD had worse survival compared to those with KS alone. Transcriptomic analyses identified increased expression of STC1 (log2FC = 2.02, padj = 0.001), a secreted glycoprotein, and MKI67 (log2FC = 1.11, padj = 0.02), a common proliferation marker, in KS+KAD lesions, along with lower expression of cytokine-associated pathways. Spatial RNA profiling from 4 KS samples from patients with KS+KAD identified increased abundance of lymphatic endothelial cells, elevated LYVE1 expression in LANA-1+ tumor areas as compared to LANA-negative areas. Bulk and spatial transcriptomic profiling of archival HIV-associated KS lesions revealed disease-specific molecular programs associated with concurrent KAD that altered tumor and microenvironment features. These findings demonstrate the heterogeneity of HIV-associated KS lesions that may guide future studies on KS pathogenesis and potential therapeutic targets.

Aromatase inhibitors (AI) are used to treat estrogen receptor (ER)-positive low-grade endometrioid endometrial cancer. In breast cancer, ESR1 mutations are rare at diagnosis (<5%) but are frequently acquired in AI-resistant cases and are considered one of the major resistance mechanisms to endocrine therapy. This study aimed to assess the prevalence of ESR1 mutations in hormonotherapy-naïve endometrial cancer samples and correlate them with molecular profiles, ER expression, and clinical outcomes. A total of 147 patients with advanced endometrial cancer who had responded to first-line chemotherapy were recruited into the UTOLA trial. Archival endometrial cancer tumor tissues underwent sequencing of 127 genes, including ESR1. Only hotspot mutations in the ligand-binding domain were evaluated. ESR1 mutation prevalence was validated in the Genomics England dataset. In UTOLA, tumors were classified as POLE, MMR deficient, TP53abn, or no specific molecular profiles (NSMP) based on the Proactive Molecular Risk Classifier for Endometrial Cancer (PROMISE) classification. Of 147 patients, 137 had sufficient tumor material for sequencing. ESR1 mutations were identified in eight tumors (6%), including Y537S/C/N (n = 4), L536H/P (n = 2), and E380Q (n = 2). A similar prevalence (3.5%) was found among 1,311 tumors in the Genomics England dataset. All ESR1 mutation cases were low-grade endometrioid endometrial cancer, ER-positive, and PR-positive, and classified as NSMP. Among patients with metastatic NSMP low-grade endometrioid endometrial cancer, 22% (8/37) harbored ESR1 mutations. Survival outcomes after platinum chemotherapy were similar between patients with ESR1 mutation endometrial cancer and ESR1 wild type (median, not reached vs. 25.3 months; P = 0.114). ESR1 mutations, while rare overall in treatment-naïve endometrial cancer, are more prevalent in patients with NSMP low-grade endometrioid endometrial cancer, potentially affecting AI efficacy. ESR1 status should be considered in selecting hormonotherapy and as a stratification factor in AI trials.

LBA639 Background: PPARG is a master regulator of luminal lineage in UC with two-thirds of adv tumors classified as luminal. FX-909 is a first-in-class oral small molecule that potently and selectively inhibits both ligand-mediated and basal PPARG activity, offering a novel approach to targeting key cancer cell-intrinsic biology. Preliminary phase 1A data have demonstrated objective responses with FX-909 monotherapy (Gao, AACR Targets 2025). Here we report updated safety, tolerability, antitumor activity and predictive biomarker discovery to identify pts most likely to benefit from FX-909. Methods: 56 pts enrolled in a phase 1A open-label dose escalation study (30-100 mg PO QD, 28-day cycles), including additional 10 adv UC patients in 30 mg PO and 50 mg PO QD backfill cohorts. Baseline archival tissue (&lt;30 months old) or a fresh biopsy collected for PPARG IHC (SP500 clone) and NGS correlative biomarker analysis. A provisional PPARG TPS cutoff was determined using linear regression modeling leveraging molecular real-world data (RWD) (N=2609 adv UC pts, Tempus xT). Serial blood samples collected for ctDNA analysis. Results: As of Nov 10, 2025, 46 pts with adv UC have been treated across four dose levels; 30 mg (17); 50 mg (16); 70 mg (11); and 100 mg (2). Median age was 71 (range 44-86), 71.7% ECOG PS 1, and 100% had mUC. Median lines of prior therapy was 3 (range, 1-8), including prior EV and anti-PD(L)1 treatment in 69.6%. Concordance between PPARG mRNA and TPS (r = 0.88, p&lt;0.001) supported linear regression modeling, which inferred a provisional TPS cutoff of ≥60% (PPARG high ). Among 40 efficacy-evaluable adv UC pts, 35 had PPARG IHC results. 18/26 PPARG high pts showed tumor regressions, including 5 confirmed and 1 unconfirmed partial response. Decreases in ctDNA VAF occurred in 7/8 PPARG high pts with available results at cycle 2 (3 PR, 3 SD). Additional data demonstrating the relationship between PPARG high status, luminal subtype, and specific genomic alterations (PPARG(CN≥3), RXRA S427F and FGFR3) will be presented. At 30mg and 50mg doses, the most common ≥Gr3 TRAEs were anemia (18.9%), thrombocytopenia (16.2%) and fatigue (10.8%). Other common TEAEs were diarrhea (32.4%), hypertriglyceridemia (27%) and hyperglycemia (24.3%). The 30 mg dose was associated with fewer Gr3 TRAEs (35.3%), longer time to TRAE onset (52 days, range 44, 85), and fewer dose interruptions (29.4%) and dose reductions (11.8%). Conclusions: FX-909, a first-in-mechanism orally bioavailable PPARG inhibitor, demonstrates clinical proof-of-concept for targeting the master regulator of luminal lineage in heavily pretreated adv UC pts and may offer a novel therapeutic strategy for PPARG high adv UC. A randomized phase 1B expansion study is ongoing (NCT05929235). A combination study of FX-909/anti-PD1 is planned for early 2026. Clinical trial information: NCT05929235 .

FAS/FAS-ligand (FAS-L) complex is implicated in critical cell functions including programmed cell death (apoptosis) and immune response regulation. FAS and FAS-ligand are members of the tumor necrosis factor (TNF) superfamily. Their activation triggers the caspase-mediated apoptotic cataract. The aim of this study was to investigate the role of their altered co-expression in a series of laryngeal squamous cell carcinomas (LSCCs). A set of fifty (n=50) LSCC archival tissue sections was analyzed by performing a combination of immunohistochemistry (IHC) and digital image analysis (DIA) assays for determining the expression of FAS/FAS-L expression. A broad spectrum of FAS/FAS-L expression values were detected across the examined cases. A significant negative correlation was found between FAS and FAS-L overall expression (p<0.001) indicating that higher FAS expression is associated with lower FAS-L expression. Furthermore, the expression of FAS was highest in stage IV carcinomas, whereas FAS-L was higher in stage III tumors, however, the differences were not statistically significant (p=0.260, p=0.101, respectively). Concerning the size of the examined malignancies (max diameter), the FAS/FAS-L expression showed a statistically significant difference in both (p=0.001, p=0.011, respectively). According to their expression status, larger tumors tend to demonstrate higher levels of FAS protein, whereas smaller tumors exhibit overexpression of FAS-L. FAS/FAS-L apoptotic system deregulation is a relatively frequent event in LSCCs. Dysregulation of the system - due to altered FAS and FAS-L co-expression levels - negatively affects the normal apoptotic mechanism. Additionally, this abnormality is clearly observed in aggressive phenotypes (advanced stage, enlarged tumor volume).

Background: Cervical cancer is the second most prevalent malignancy among South African women, with high-risk human papillomavirus (HPV) infection as a critical risk factor. HPV plays a central role in cervical carcinogenesis, particularly in high-grade squamous intraepithelial lesions (HSIL). Increased programmed death ligand 1 (PD-L1) expression has been implicated in cervical carcinoma tumorigenesis. Using immunohistochemistry, this study investigated the correlation between high-risk HPV-driven cervical intraepithelial neoplasia (CIN) and PD-L1 expression. Methods: An analytical cross-sectional study was conducted on archival tissue from the Department of Anatomical Pathology, University of Pretoria (2018–2021). Formalin-fixed paraffin-embedded tissues from loop electrosurgical excisions, cone biopsies, punch biopsies, and polypectomies were analysed. PD-L1 expression was assessed using the combined proportion score (CPS). Three pathologists independently evaluated histological grade, p16 immunohistochemistry, and PD-L1 expression. Results: Among 108 cases (mean age: 37.36 years), 89.8% were CIN 3, 9.3% CIN 2, and 0.9% CIN 2–3. p16 was positive in 97.2% of cases. PD-L1 expression (CPS ≥ 1) was observed in 9.3% of cases, with a mean CPS of 1.57%. No significant association was found between PD-L1 expression and CIN grade (p = 0.6433, Cramer’s V = 0.1191) or between PD-L1 and p16 positivity (p = 1, Cramer’s V = 0.05976). Conclusions: This study demonstrates no correlation between PD-L1 expression and high-risk HPV-driven high-grade CIN. These findings suggest that immune checkpoint inhibition targeting PD-L1 may have limited therapeutic relevance in HSIL among South African women.

Aberrant microRNA expression has been implicated in cervical carcinogenesis, making miRNAs promising biomarkers for early detection and risk stratification of cervical precancer and cancer. This study aimed to identify and validate miRNA expression profiles and signatures that distinguish benign, precancer, and cancer cervical tissues, providing insights into their potential utility in cervical cancer screening and diagnosis. We analyzed miRNA expression from archival FFPE cervical tissues of 272 women (100 benign, 89 precancer, 83 invasive cancer) using next-generation sequencing. Differential expression was assessed using DESeq2 with significant miRNAs defined by an absolute log2 fold change ≥ 1 and an adjusted p-value < 0.05. Functional enrichment of miRNA targets was performed using multiMiR and DAVID, and protein-protein interaction (PPI) networks were constructed using STRING and Cytoscape. Machine learning classifiers were developed with recursive feature elimination (RFE) and XGBoost and evaluated in internal and external cohorts. miRNA signatures were further assessed for association with overall survival in the TCGA cervical cancer cohort using multivariable Cox regression models. Notably, hsa-miR-1246 was the most significant miRNA in cancer vs. benign (log₂FC = 5.58, p = 3.20 × 10⁻41) and cancer vs. precancer (log₂FC = 4.89, p = 5.23 × 10⁻33). hsa-miR-149-5p was most significant in benign vs. precancer (log₂FC = −1.48, p = 1.53 × 10⁻¹⁴). Enrichment analyses highlighted cancer-related pathways with hub genes including TP53, BCL2, STAT3, ESR1, and MYC. RFE with XGBoost identified a 26-miRNA panel for multiclass classification and a 4-miRNA panel for binary cancer vs. benign classification. The binary model achieved strong performance with an AUC of 0.97 and 0.91 in internal and external validation sets respectively. Both the 26-miRNA (p < 0.001) and 4-miRNA (p = 0.01) signatures were associated with poorer overall survival. Our findings identify distinct miRNAs and miRNA-based signatures that differentiate benign, precancer, and cancer cervical tissues, highlighting their potential clinical utility as diagnostic and prognostic biomarkers. Furthermore, the development of compact, biologically interpretable miRNA signatures demonstrates strong promise as robust classifiers for early cancer detection and tissue characterization, with possible therapeutic relevance.

Abstract Background: HER2 targeted therapy (tx) has transformed the trajectory of HER2+ metastatic breast cancer (MBC), with a subgroup of pts remaining on first line (1L) tx for many years (yrs). Detection of minimal residual disease (MRD) via ctDNA to guide tx is promising but data supporting its use in HER2+ MBC are virtually absent. Methods: We identified 70 pts with HER2+ MBC treated with 1L HER2 targeted tx consented to the EMBRACE cohort study with archival tissue, buffy coat and plasma at ≥1 key timepoints while on 1L tx (baseline, first-on-tx, year [Y] 1 landmark [L], Y2L, Y3L), or at time of disease progression (PD). We employed MAESTRO, an ultrasensitive whole-genome, tumor-informed, mutation enrichment sequencing assay, in a pooled testing format (MAESTRO-Pool). Exceptional responders were pts without PD or death for ≥3 yrs from 1L start (real-world progression-free survival [rwPFS] ≥3 yrs). Conventional responders were pts with PD or death within 3 yrs of 1L start (rwPFS&amp;lt;3 yrs). Local- or brain-only PD that did not lead to tx switch were not considered events. The primary objective was the association between Y1L MRD status (plasma samples collected 1 yr from 1L start +/- 3 months) and exceptional response. Sixty-three pts (90%) (39 exceptional and 24 conventional responders) had sufficient samples and successful assay design; MAESTRO-Pool was run on 147 samples. Results: More exceptional than conventional responders had de novo MBC (69.2% vs 41.7%, p=0.03). High (3+ by IHC) HER2 expression (87.2% vs 75.0%), estrogen receptor (ER)+ status (38.5% vs 33.3%) and visceral disease (61.5% vs 62.5%) were similar between exceptional and conventional responders. Most pts in both groups had 1L chemotherapy plus trastuzumab/pertuzumab (82.1% vs 70.8%) and maintenance endocrine tx if ER+ (86.7% vs 62.5%). With 284.4 person-yrs of follow up, 6/39 exceptional responders had late PD. In a landmark analysis at Y3, the 2-yr rwPFS was 97.4% (95%CI 82.8%-99.6%) (5 yrs from 1L start); 5-yr overall survival (OS) was 91.5% (95%CI 68.7%-97.9%) (8 yrs from 1L start). For conventional responders, total follow up was 110.5 person-yrs, median rwPFS was 1.65 yrs (95%CI 1.1-2.51), median OS was 4.58 yrs (95%CI 3.78-6.95). A median of 1,370 (range 136-4,635) tumor-specific mutations were tracked per patient. MRD was detected in 50 (34%) samples (median tumor fraction (TFx) 1352.8 ppm; range 4-206,399 ppm), including 21 (42%) samples with TFx &amp;lt;500 ppm, the limit of detection (LOD) of tumor-agnostic MBC assays, and 14 (28%) with TFx &amp;lt;100 ppm, the LOD of 1st generation tumor-informed MRD tests. After excluding baseline and PD samples, MRD was detected in 7 (7.4%) and 28 (80%) samples from exceptional and conventional responders, respectively. All exceptional responders who remained progression-free were always MRD- (n=30) or cleared MRD by Y1L (n=3). Indeed, Y1L MRD status was associated with exceptional vs conventional response (0/26 [0%] vs 10/13 [76.9%], p&amp;lt;0.001). Four of the 6 pts who had late PD had a Y3L sample, which was MRD+ in 3 cases (lead time range 2.77-13.47 yrs) and MRD- in one case of breast-only PD. For 20/24 conventional responders, the last sample collected before PD was MRD+. Of the 4 MRD- cases, one pt had 2 primary breast cancers, one had brain-only PD. Conclusions: Here we report, to our knowledge, the first data showing a significant association between MRD detection and duration of response to 1L tx for HER2+ MBC. MRD status at Y1L was highly predictive of exceptional response. Only 2 conventional responders had distant PD without a prior MRD+ sample. All exceptional responders with late distant PD were MRD+ at Y3L. Fourteen (28%) MRD+ samples had TFx&amp;lt;100ppm and would have been misclassified by 1st generation MRD tests, supporting the value of ultrasensitive MRD tracking to predict outcomes and its potential to guide tx de-escalation in prospective clinical trials. Citation Format: S. Morganti, C. Song, N. Zhou, K. Santos, P. Jain, L. Walsh, R. Li, J. Rhoades, K. Gilligan, G. Kirkner, C. Stever, A. Patel, M. E. Hughes, N. Priedigkeit, I. E. Krop, G. Curigliano, E. P. Winer, S. M. Tolaney, N. Tayob, H. Heiling, K. Xiong, N. U. Lin, V. A. Adalsteinsson, H. A. Parsons. Ultrasensitive ctDNA Tracking Predicts Exceptional Response in HER2+ Metastatic Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-11.