Tanjina Kader

Supplementary Figures provide additional evidence and validation of copy number changes from the ORION-FISH assay.

Table S1 provides the details of antibodies (ORION) and FISH probes used in this manuscript.

Table S2 provides the cohort description.

Detecting chromosomal copy-number alterations together with protein-defined cell states in intact tissue is critical for understanding early clonal evolution and microenvironmental interactions in cancer. We developed ORION-FISH, which integrates high-plex tissue imaging with a morphology-preserving DNA fluorescence in situ hybridization (DNA-FISH) workflow and single-cell registration, yielding measurements concordant with clinical FISH. In high-grade serous ovarian carcinoma (HGSOC), ORION-FISH recapitulated known chromosomal changes while revealing subclonal heterogeneity missed by targeted sequencing. Applied to serous tubal intraepithelial carcinomas, precursors of HGSOC, ORION-FISH identified intermixed epithelial cells with MYC or CCNE1 copy-number gains, as well as concurrent alterations associated with distinct immune microenvironments. In addition, epithelial cells with MYC and CCNE1 copy-number gains were detected in morphologically normal fallopian tube epithelium, along with rare MDM4 increases across epithelial lineages. Together, ORION-FISH provides a framework linking chromosomal copy-number states to protein-defined phenotypes within preserved tissue architecture, enabling context-aware interrogation of early copy-number diversification at single-cell resolution. Significance:We introduce ORION-FISH, a spatially resolved workflow integrating multiplexed protein imaging with DNA-FISH to map genomic alterations within intact tissues. Applying this approach to ovarian cancer precursors reveals early copy-number diversification and associations with the local immune context, providing a foundation for studying how genomic and microenvironmental states coevolve during tumor initiation.

Table S4 provides the statistics summary.

Table S3 provides the FISH scores by BioView for HGSOC cases.

Detecting chromosomal copy-number alterations together with protein-defined cell states in intact tissue is critical for understanding early clonal evolution and microenvironmental interactions in cancer. We developed ORION-FISH, which integrates high-plex tissue imaging with a morphology-preserving DNA-FISH workflow and single-cell registration, yielding measurements concordant with clinical FISH. In High Grade Serous Ovarian Carcinoma (HGSOC), ORION-FISH recapitulated known chromosomal changes while revealing subclonal heterogeneity missed by targeted sequencing. Applied to serous tubal intraepithelial carcinomas (STICs), precursors of HGSOC, ORION-FISH identified intermixed epithelial cells with MYC or CCNE1 copy-number gains, as well as concurrent alterations associated with distinct immune microenvironments. In addition, epithelial cells with MYC and CCNE1 copy-number gains were detected in morphologically normal fallopian tube epithelium, along with rare MDM4 increases across epithelial lineages. Together, ORION-FISH provides a framework linking chromosomal copy number states to protein-defined phenotypes within preserved tissue architecture, enabling context-aware interrogation of early copy-number diversification at single-cell resolution.

Development of ipsilateral breast carcinoma following a diagnosis of breast ductal carcinoma in situ (DCIS) has been assumed to represent recurrence of the primary tumour. However, this may not always be the case, and it is important to determine how often such recurrences represent new tumours. Ipsilateral primary–recurrence pairs ( n = 78) were sequenced to test their clonal relatedness. Shared genetic events were identified from whole exome sequencing ( n = 54 pairs) using haplotype‐specific copy number and phylogenetic analysis. The remaining pairs were sequenced using a targeted panel or low‐coverage whole genome sequencing. We included 32 non‐recurrent DCIS to compare recurrent and non‐recurrent disease. We found that 7% of DCIS recurrences were non‐clonal by whole exome sequencing, indicative of a new breast carcinoma. Lower resolution methods detected a higher non‐clonality rate (29%). By comparing primary DCIS with their recurrence, we found that the evolution of DCIS to invasive disease was associated with increased ploidy and copy number events. TP53 mutations were enriched in DCIS with clonal recurrence compared with non‐recurrent DCIS. Our results verify that de novo ‘recurrent tumours’ of independent origin occur in patients who may be at high risk. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Supplementary Table S1 from Multimodal Spatial Profiling Reveals Immune Suppression and Microenvironment Remodeling in Fallopian Tube Precursors to High-Grade Serous Ovarian Carcinoma