Arkadiusz Piotrowski

Background: In colorectal cancer (CRC), tumor-immune interactions are critical drivers of tumorigenesis, immune evasion, and therapeutic response. Therefore, a deeper spatially resolved understanding of these interactions is urgently needed. Methods: Spatial transcriptomics (ST) of CRC and matched normal samples was used to characterize cell populations and gene expression patterns associated with tumorigenesis and immune infiltration. An additional 41 publicly available ST slides, single-cell transcriptomics and bulk transcriptomics datasets from 472 and 617 CRC patients, respectively, were analyzed to validate the findings. Results: Cancer regions exhibited profound molecular alterations with increased oncogenic pathways, immunosuppressors, and tryptophan deprivation via KYNU-AHR axis, inducing the macrophage attractant CXCL5, contributing to immune evasion. In tumor boundaries, SPP1⁺ macrophages fostered an immunosuppressive microenvironment with regulatory T-cells (Tregs) recruited via CCL18-CCR8 and CCL22-CCR4. SIT1 emerged as a potential anti-tumor T-cells suppressor within tertiary lymphoid structures, suggesting a novel immune regulator. We also uncovered a previously uncharacterized population of tumor-infiltrating IFI6⁺ Tregs associated with unfavorable prognosis and therapy response, defined by elevated interferon-related genes and LGALS9, a potential immune checkpoint target. Conclusion: This study uncovered previously unrecognized immunosuppressive cell populations and key immune regulators that advance understanding of CRC immune evasion and highlight potential targets for personalized immunotherapy.

Background: Schwannomatosis (SWN) is a rare tumor predisposition syndrome caused by pathogenic variants in NF2 , SMARCB1 , or LZTR1 . Mosaicism contributes to up to 30% of de novo NF2- related cases, but its role in SMARCB1 - and LZTR1 -related SWN remains unclear. Accurate molecular classification is critical for clinical management, yet overlapping phenotypes complicate diagnosis. Because standard diagnostics have limited sensitivity and often require tumor tissue, we evaluated whether ultra-sensitive duplex sequencing of blood-derived DNA improves mosaic variant detection in SWN. Methods: We developed a duplex sequencing assay targeting NF2, SMARCB1, and LZTR1 and analyzed 102 individuals with suspected SWN previously negative for constitutional pathogenic variants, along with 35 controls. Results: Duplex sequencing identified low-level mosaic NF2 pathogenic variants in 11% of cases (11/102), with variant allele frequencies as low as 0.06%. In all patients with available tumor material (8/11), blood and tumor findings were concordant. No mosaic pathogenic variants were detected in SMARCB1 or LZTR1 . Controls showed no deleterious variants except one LZTR1 splice-site variant. Conclusions: Duplex sequencing enables robust detection of ultra-low-frequency NF2 variants in SWN and complements existing diagnostic workflows. This approach may reduce dependence on tumor tissue in NF2 -related SWN, improve differential diagnosis, and support patient management and genetic counseling.

Background: Histologically normal mammary tissue from breast cancer patients can harbor significant genetic alterations that could precede visible tumor development and influence disease progression. Methods: Whole-exome sequencing was performed on 408 samples from 77 breast cancer patients with poor prognosis, 49 patients recruited without prognosis-based selection, and 15 individuals undergoing non-cancer-related mammoplasty. Paired primary tumor and histologically normal mammary gland tissues were analyzed. Variant classification adhered to strict filtering criteria, incorporating allele frequency thresholds, multiple annotation databases, and in silico prediction tools. Duplex sequencing was employed to detect and confirm pathogenic PIK3CA and TP53 variants in normal mammary tissue samples from 11 breast cancer patients with unfavorable prognosis. Statistical analyses included hypergeometric testing, Kaplan–Meier survival analysis, and Cox proportional hazards modeling. Results: Post-zygotic pathogenic variants in cancer-associated genes were significantly more prevalent in normal mammary tissue of poor-prognosis patients (29%) than in unselected patients (12.5%) ( p = 0.0008578). Variant presence and per-individual burden were similar across age-matched cohorts and intrinsic subtypes, indicating that subtype composition, germline predisposition and age do not account for the excess UM variant load in BCAP. Truncating variants were exclusive to poor-prognosis cases. Frequently altered genes included AKT1 , PIK3CA , PTEN , TBX3 , and TP53 , with TP53 variants detected only in patients with adverse outcomes. Duplex sequencing confirmed the presence of low-frequency variants (as low as 1.34%) in regions of histologically normal breast tissue from patients with a poor prognosis. Notably, nearly one-quarter of all identified cases (24%, 12/49) harbored pathogenic variants in normal tissue absent from corresponding primary tumors, suggesting that at least some mosaic clones in uninvolved mammary tissue represent independent evolutionary events rather than residual tumor cells. Conclusions: Post-zygotic pathogenic variants are frequent in histologically normal mammary tissue from breast cancer patients, including alterations in key cancer-associated genes. These findings indicate that mosaic clonal changes outside the tumor are more common than previously appreciated and warrant further investigation. Assessing such variants in non-tumorous tissue may, in the future, help refine approaches to breast cancer risk evaluation and management.

Introduction: Idiopathic scoliosis (IS) is a complex spinal deformity affecting ~3% of the population, with a multifactorial and genetically heterogeneous origin. This study aimed to investigate the genetic origins of severe IS by examining both constitutional and post-zygotic alterations. Methods: We analyzed 70 unrelated IS-affected individuals using whole exome sequencing (WES) and SNP array approaches on intraoperatively collected articular processes and blood samples. Results: Two pathogenic constitutional copy number variants (CNVs) were identified – a 43.6 Mb duplication on chromosome 8p and trisomy X – along with eight regions of homozygosity (ROH) located on chromosomes 1, 2, 8, 12, 14, and 16, absent in ethnically matched controls. Additionally, a heterozygous DMD deletion (exons 17–36) was found in one female, and rare recurrent pathogenic single-nucleotide variants (SNVs) were detected in ENAM and FLNB genes. Notably, 13% (95% CI, 6.1–23%) of individuals harbored pathogenic variants, spanning CNVs, ROH, and SNVs, suggesting a genetic contribution to IS. Discussion: Our findings demonstrate that one in seven cases classified as idiopathic may have an underlying monogenic cause. This study underscores the polygenic and heterogeneous nature of IS and highlights the need for genetic testing by integrating WES and SNP array analyses into its diagnostic workflow. Our findings suggest that incorporating genetic testing into the diagnostic evaluation of severe IS patients may enable personalized genetic counseling and, consequently, improve clinical management.

Background: Adipose-derived mesenchymal stromal cells (AD-MSCs) and fibroblasts are both widely used in regenerative medicine, demonstrating significant potential for personalized cell therapy. A major challenge in their use lies in their high biological similarity, encompassing morphology, differentiation capabilities, and flow cytometric markers, making their distinction difficult. Methods: In our study, we aimed to compare AD-MSCs obtained from two types of adipose tissue, subcutaneous and visceral, alongside skin fibroblasts. Notably, all tissue samples were sourced from the same donors. We analyzed the cells for surface antigens via flow cytometry and conducted single-cell RNA sequencing, followed by verification with quantitative PCR (qPCR). Results: Our results revealed phenotypic similarities between the isolated AD-MSCs and dermal fibroblasts, particularly in the expression of markers characteristic of AD-MSCs. However, through in-depth analyses, we identified distinct differences between these cell types. Specifically, we pinpointed 30 genes exhibiting the most significant variations in expression between AD-MSCs and fibroblasts. These genes are associated with biological processes such as tissue remodeling, cell movement, and activation in response to external stimuli. Among them, MMP1, MMP3, S100A4, CXCL1, PI16, IGFBP5, COMP were further validated using qPCR, clearly demonstrating their potential to differentiate between AD-MSCs and fibroblasts. Conclusions: Our scRNA-seq analysis elucidates the transcriptional landscape of AD-MSCs and fibroblasts with unprecedented resolution, highlighting both the population-specific markers and the intrapopulation heterogeneity. Our findings underscore the importance of employing high-resolution techniques for cell identification.

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.

Recently, zebularine, a small-molecule epigenetic inhibitor and retinoic acid, acting as a transcriptional activator, have been found to induce tissue regeneration. In this study, the pro-regenerative properties of zebularine and retinoic acid were combined with the potential of the alginate carrier to expand its therapeutic possibilities. Alginate formulations of zebularine and retinoic acid were developed for subcutaneous administration to mice. Hydrophilic zebularine formed a homogenous formulation with extreme drug loadings reaching 240 mg of zebularine per 1 ml of 2% sodium alginate, while hydrophobic retinoic acid, 0.8 mg/ml, dispersed as fine crystals. Cell culture tests exhibited no significant cytotoxicity of the alginate formulations. Subcutaneous administration of zebularine and retinoic acid in 2% sodium alginate promoted regenerative responses in a mouse model of ear pinna punch wound mice involving the restoration of tissue architecture, nerve and vessel growth, and extensive epigenetic and transcriptional repatterning with no adverse effects observed in the animals. Significant trancriptomic responses to the epigenetic treatment included the induction of epithelium development genes contrasted with the downregulation of muscle development genes on day 7 post-injury. Among the remarkable changes in global gene methylation are those in neurodevelopmental genes. In vitro studies showed rapid zebularine but no retinoic acid discharge from the alginate formulations. Live ultrasound imaging demonstrated gradual absorption of the subcutaneously injected alginate formulations, which may explain the in vivo activity of retinoic acid following subcutaneous administration. Effective induction of tissue regeneration together with a high safety profile and of the subcutaneously administered pro-regenerative alginate formulations opens the way to testing further regenerative therapies for hard-to-reach lesions.

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 remain 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.

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.