Bożena Bruhn-Olszewska

Cancer-related genomic instability (GI) may cause genetic alterations in spermatozoa, implying health issues not only in cancer survivors, but also in their children [1, 2]. We therefore studied Loss of Y chromosome (LOY), considered as hallmark of GI [3-15], in spermatozoa and blood from survivors of childhood and testicular cancer (CC, TC), and controls (CTRL). We found that LOY was statistically significantly more frequent in spermatozoa from cancer survivors than in controls (Odds Ratio [OR]=2.2 for CC vs. CTRL and OR=2.4 for TC vs. CTRL). Furthermore, LOY was about an order of magnitude more prevalent in spermatozoa than in blood among 18–53-year-old males within all cohorts. Our findings suggest that LOY in spermatozoa might be a clinically useful marker of GI, reduced fertility and disease predisposition in males. Introducing LOY in spermatozoa as a biomarker opens a new research avenue into disease prevention and the causes and consequences of LOY.

Loss of Y chromosome (LOY) and clonal hematopoiesis of indeterminate potential (CHIP) are common age-related events with implications for aging and Alzheimer disease (AD). LOY is linked to increased AD risk, whereas CHIP may be protective, and their co-occurrence remains unclear. We conducted whole-exome sequencing of CD4 ⁺ T cells, NK cells, and myeloid cells from AD patients and controls exhibiting LOY or retention of Y chromosome. We identified 39 variants in known myeloid and lymphoid driver genes, with up to 35% co-occurring with LOY in the same clone. In addition, we detected 192 unknown drivers of clonal hematopoiesis, enriched in AD-LOY individuals (odds ratio 4.8, P = 0.041). In myeloid cells, total driver burden correlated with LOY (ρ = 0.52, P = 0.00041). These results indicate that LOY is a primary driver of clonal hematopoiesis in AD, seeding myeloid clones that accumulate unknown driver variants, whereas most canonical CHIP mutations arise independently. Our study reveals distinct, partially overlapping clonal architectures for LOY and CHIP and highlights LOY-driven myeloid expansion as a contributor to AD pathogenesis.

Loss of Y chromosome (LOY) and clonal hematopoiesis of indeterminate potential (CHIP) are common age-related events associated with multiple adverse outcomes in the elderly. While LOY has been associated with higher risk of Alzheimer disease (AD), CHIP has been suggested to perform a protective role against AD. Moreover, the co-occurrence of CHIP and LOY is debated. We performed deep whole-exome sequencing of FACS-isolated CD4 ⁺ T lymphocytes, NK and myeloid cells from men with AD and controls exhibiting either LOY or retention of Y chromosome (ROY). We found 39 sequence variants in known (canonical) myeloid driver genes of clonal hematopoiesis (MD-CH) and known lymphoid driver genes (LD-CH), and maximally 14 (35%) of these could co-exist with LOY within the same clone. We further describe 192 unknown drivers of clonal hematopoiesis (UD-CH), which were markedly enriched in AD-LOY individuals (odds ratio=4.8, Benjamini-Hochberg-adjusted p=0.041), and over 20% of these variants were protein-truncating. In myeloid cells, the total burden of all detected drivers correlated with the percentage of LOY cells (Spearman ρ=0.52, adjusted p=0.00041). In conclusion, our findings suggest that LOY acts as the primary driver of clonal hematopoiesis in AD by seeding myeloid clones. These clones may subsequently accumulate additional, often truncating, UD variants, while most canonical CHIP mutations arise independently of LOY. Our study delineates distinct yet partially overlapping clonal architectures for LOY and CHIP in late-onset AD and underscores LOY-driven myeloid expansion as a potential contributor to disease pathogenesis.

Alzheimer’s disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of chromosome Y (LOY) in leukocytes is strongly associated with AD. We studied here DNA methylation and RNA expression in sorted monocytes and granulocytes with and without LOY from male AD patients. Through multi-omic analysis, we identified new candidate genes along with those previously associated with AD. Global analyses of DNA methylation in samples with LOY vs. normal state showed that hypomethylation dominated both in granulocytes and monocytes. Our findings highlight LOY-related differences in DNA methylation that occur in gene regulatory regions. Specifically, we observed alterations in key genes involved in leukocyte differentiation: FLI1, involved in early hematopoiesis; RUNX1, essential for blood cell development; RARA, regulating gene expression in response to retinoic acid; CANX, crucial for protein folding; CEBPB, a transcription factor important for immune responses; and MYADM, implicated in cell adhesion and migration. Moreover, protein–protein interaction analysis in granulocytes identified that products of two of these genes, CANX and CEBPB, are key hub proteins. This research underscores the potential of multi-omic approach in pure hematopoietic cell populations to uncover the molecular underpinnings of AD. Finally, our results link previous analysis showing impact of LOY on leukocyte differentiation, LOY-associated transcriptional dysregulation and GWAS studies of LOY.

Loss of Y chromosome (LOY) is the most commonly occurring post-zygotic (somatic) mutation in male individuals. The past decade of research suggests that LOY has important effects in shaping the activity of the immune system, and multiple studies have shown the effects of LOY on a range of diseases, including cancer, neurodegeneration, cardiovascular disease and acute infection. Epidemiological findings have been corroborated by functional analyses providing insights into the mechanisms by which LOY modulates the immune system; in particular, a causal role for LOY in cardiac fibrosis, bladder cancer and Alzheimer disease has been indicated. These insights show that LOY is a highly dynamic mutation (such that LOY clones expand and contract with time) and has pleiotropic, cell-type-specific effects. Here, we review the status of the field and highlight the potential of LOY as a biomarker and target of new therapeutics that aim to counteract its negative effects on the immune system.

Histologically normal mammary tissue from breast cancer patients can harbor significant genetic alterations. We analyzed the spectrum of DNA variants in 408 matched histologically normal tissue and tumors from 77 poor-prognosis patients, 49 patients recruited without prognosis bias, and mammary gland samples from 15 non-cancerous individuals. Whole exome sequencing revealed a higher prevalence of pathogenic post-zygotic variants in cancer-associated genes: AKT1, PIK3CA, PTEN, TBX3, and TP53, affecting poor-prognosis patients (29%), compared to 12.5% in those without prognosis criteria (p=0.0008578). PIK3CA variants were recurrent across patients, while TP53 variants were restricted to those with adverse prognoses. Duplex sequencing detected low-frequency pathogenic PIK3CA and TP53 variants in distant normal tissues of poor-prognosis patients. Disease recurrence significantly reduced survival rates, with poor prognosis patients experiencing higher mortality within 24 months (p=0.0088), further worsened by the presence of pathogenic post-zygotic variants. These findings highlight the importance of genetic monitoring even in microscopically normal mammary tissue.

Alzheimer’s disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of chromosome Y (LOY) in leukocytes is strongly associated with AD. We studied here DNA methylation and RNA expression in sorted monocytes and granulocytes with and without LOY from male AD patients. Through multi-omic analysis, we identified new candidate genes and confirmed the involvement of numerous genes previously associated with AD. Our findings highlight LOY-related differences in DNA methylation that occur in gene regulatory regions and are predominantly accompanied by down-regulation of affected genes. Specifically, we observed alterations in key genes involved in leukocyte differentiation: FLI1 , involved in early hematopoiesis; RUNX1 , essential for blood cell development; RARA , regulating gene expression in response to retinoic acid; CANX , crucial for protein folding; CEBPB , a transcription factor important for immune responses; and MYADM , implicated in cell adhesion and migration. Moreover, protein-protein interaction analysis in granulocytes identified that products of two of these genes, CANX and CEBPB , are key hub proteins. Thus, LOY appears to dysregulate genes involved in leukocyte differentiation and induce higher-level epigenetic changes. This research underscores the potential of multi-omic approaches in pure cell populations to uncover the molecular underpinnings of AD and reinforces the significance of LOY as a pathogenic factor in this disease. Overall, results support the hypothesis that age-related immune cell dysfunction contributes to AD development. Finally, our results link previous analysis showing impact of LOY on leukocyte differentiation, LOY-associated transcriptional dysregulation and GWAS studies of LOY.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Despite advances in early detection and treatment strategies, breast cancer recurrence and mortality remain a significant health issue. Recent insights suggest the prognostic potential of microscopically healthy mammary gland, in the vicinity of the breast lesion. Nonetheless, a comprehensive understanding of the gene expression profiles in these tissues and their relationship to patient outcomes is still missing. Furthermore, the increasing trend towards breast-conserving surgery may inadvertently lead to the retention of existing cancer-predisposing mutations within the normal mammary gland. This study assessed the transcriptomic profiles of 242 samples from 83 breast cancer patients with unfavorable outcomes, including paired uninvolved mammary gland samples collected at varying distances from primary lesions. As a reference, control samples from 53 mammoplasty individuals without cancer history were studied. A custom panel of 634 genes linked to breast cancer progression and metastasis was employed for expression profiling, followed by whole-transcriptome verification experiments and statistical analyses to discern molecular signatures and their clinical relevance. A distinct gene expression signature was identified in uninvolved mammary gland samples, featuring key cellular components encoding keratins, CDH1, CDH3, EPCAM cell adhesion proteins, matrix metallopeptidases, oncogenes, tumor suppressors, along with crucial genes (FOXA1, RAB25, NRG1, SPDEF, TRIM29 , and GABRP ) having dual roles in cancer. Enrichment analyses revealed disruptions in epithelial integrity, cell adhesion, and estrogen signaling. This signature, named KAOS for Keratin-Adhesion-Oncogenes-Suppressors, was significantly associated with reduced tumor size but increased mortality rates. Integrating molecular assessment of non-malignant mammary tissue into disease management could enhance survival prediction and facilitate personalized patient care.

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4+ T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY-cells (22%) in comparison to CD4+ T-lymphocytes and cytotoxic CD8+ T-lymphocytes. Using scRNA-seq LOY was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.