Comprehensive molecular profiling is essential for modern precision oncology but remains hindered by prohibitive costs, specimen exhaustion, and protracted turnaround times. While pathology foundation models (PFMs) have demonstrated potential for inferring molecular phenotypes from routine hematoxylin and eosin (H&E) whole-slide images (WSIs), current architectures primarily rely on vision-centric self-supervised learning or vision-language alignment, lacking the spatially resolved molecular supervision required to connect subtle morphological features with underlying genomic alterations. Spatial transcriptomics (ST) emerges as a transformative technology that enables transcriptomic quantification within intact tissue sections, thereby preserving the precise spatial link between histology and molecular profiles. In this study, we present a Spatial Transcriptomics-guided Alignment framework for Molecular Profiling (STAMP), which endows PFMs with intrinsic molecular awareness. To support this paradigm, we curated HumanST-1k, a human ST dataset spanning diverse anatomical organs and sequencing platforms. This atlas yields 1.8 million pairs of H&E patches and corresponding transcriptomic profiles, providing a corpus that links histological structures with their molecular states. To mitigate the technical noise inherent to raw transcriptomics, STAMP applies a pathway-informed alignment strategy that aggregates transcriptomic data into biologically functional pathways, which are subsequently integrated into PFMs via parameter-efficient fine-tuning. This alignment enriches the representation space of PFMs and unlocks their capacity to resolve sub-visual molecular signatures. The clinical utility of these augmented representations was validated through a multi-tier evaluation framework.
Pathology foundation models (PFMs) have emerged as a core approach for learning transferable representations from whole slide images (WSIs), and they are typically benchmarked through downstream clinical endpoints. While such task level evaluations are indispensable, they offer limited insight into what the representations themselves encode, particularly whether PFM embeddings can distinguish meaningful tissue regions and capture their spatial relationships. We present SpaPath-Bench, a representation level benchmark designed to diagnose spatial representation capability in PFMs. SpaPath-Bench formulates spatial domain identification (SDI) on paired whole slide image and spatial transcriptomics (ST) data as a diagnostic task. It curates 42 public paired WSI and ST slides, enables large scale evaluation across 19 encoders and seven SDI methods, and measures partition quality using three complementary criteria: unsupervised spatial coherence, transcriptomics referenced agreement, and expert referenced agreement. Across 83K runs, SpaPath-Bench reveals that different pretraining paradigms capture distinct aspects of tissue spatial architecture, and it provides practical guidance for building the next generation of spatially aware computational pathology models. Code and data pipelines are publicly available at https://bokai-zhao.github.io/SpaPath-benchboard/.
H&E-stained whole-slide images offer cohort-scale availability and rich spatial context but lack molecular specificity, whereas bulk RNA-seq provides transcriptome-wide resolution at high cost with limited archival availability. We show that training a lightweight alignment module atop frozen histopathology and RNA-Seq foundation models enables open-vocabulary molecular prompting -- querying H&E slides with gene-set signatures to predict pathway activity without sequencing or end-to-end retraining. Using contrastive learning on a multi-cancer cohort (N=1,720), we achieve a 25-fold improvement in retrieval over baseline methods. Systematic analysis reveals a graduated predictability spectrum: morphologically grounded programs (cell-cycle programs, immune-related) are most reliably predicted (R^2>0.5), while predicting pathways with no morphological footprint remains challenging as expected. We validate clinical utility on the POSEIDON clinical trial: H&E-predicted squamous cell carcinoma scores recapitulate NSCLC subtype identity and predicted IFN-gamma mirror PD-L1 tumor-cell expression groups. Furthermore, genesets describing immune activation and fibrosis predict known tumor microenvironment archetypes from histology alone. We further validate generalization of our approach across unseen cohorts and demonstrate data-efficient domain adaptation, establishing a slide-native framework for molecular analysis on H&E images.
Dominik Winter, Dominik Vonficht, Loïc Le Bescond +6
Multiple instance learning (MIL) is the dominant framework for whole-slide image analysis in computational pathology, typically combining a frozen patch encoder, a projection layer, and a slide-level aggregator. While encoders and aggregators have been extensively studied, the projection layer remains a largely morphology-only bottleneck. This limits endpoints such as biomarker status and survival, which are governed by a molecular state that is not fully captured by H&E morphology. We introduce Molecularly Informed Staining Transform (MIST), a plug-in replacement for the MIL projection layer that uses paired spatial transcriptomics only during training to construct virtual molecular stains. MIST clusters gene expression profiles into cross-modal prototypes, anchors them in the frozen foundation model feature space, and uses them to reorganize H&E patch features along molecularly guided axes. It requires no transcriptomics at inference and can be inserted before standard MIL aggregators. We evaluate MIST across 23 downstream tasks and 8 MIL aggregators. MIST improves 240 of 256 configurations over the standard projection layer, with an average gain of +3.5%, observed consistently across endpoint types: +5.2% on survival prediction, +3.3% on tissue subtyping, and +2.6% on biomarker prediction. Ablations confirm that gene-derived prototypes are the primary source of the gains, while spatial, biological, and pathological analyses show that cross-modal prototype affinities capture spatially coherent molecular programs from H&E alone.