Cancer Genomics

This study explores the use of deep learning and explainable artificial intelligence to diagnose hepatocellular carcinoma (HCC) and define effective biomarkers across five different stages of disease development using a transcriptomic biomarker HCC dataset constructed via semi-supervised learning from three source datasets. Several deep learning experiments were conducted with different feature extraction techniques and gene sets to identify the most effective features for training high-accuracy models with minimal loss. The best-performing model, using 15 selected genes with the SelectKBest algorithm, achieved 90.74% accuracy, while the model with the lowest recorded loss of 0.3187 was obtained using 20 selected genes. To address the issue of class imbalance in the dataset, a weighted training approach was conducted, and for model transparency and interpretability a SHAP-based XAI analysis provided insights into the model's decision-making, consistently finding DNAJB14 as the most influential gene. Functional validation in this study has provided compelling evidence that DNAJB14 plays an important role in the adverse properties of HCC and that its inhibition effectively reverses tumour cell migration, invasion, colony and sphere formation. The main limitation of this study is the dataset's class imbalance, and while weighted training helped mitigate this, further research and additional data are needed to guarantee model generalizability. Future studies should also explore the influence of genetic variations, environmental factors, and clinical differences on model performance across diverse populations.

Artificial intelligence (AI) and natural language processing (NLP) are increasingly used to extract, integrate, and interpret biomedical knowledge relevant to cancer genomics, yet their translation into routine clinical oncology has been comparatively slow. The central challenge is not computational capability alone, but trustworthy integration into clinical workflows. This review examines how NLP and AI support the cancer genomics pipeline, from literature mining and automated variant interpretation to clinical trial matching, knowledge graph construction, and multimodal data integration. We identify four interrelated translational failure domains: evidence inconsistency, explainability and uncertainty, data governance and reproducibility, and interoperability. Rather than considering these challenges in isolation, we take a systems-level view, focusing on their interaction across the translational pathway. We propose a conceptual framework and roadmap for addressing these domains through rigorous validation, uncertainty-aware methods, interoperable infrastructures, regulatory alignment, and human oversight across the AI lifecycle. Progress toward routine clinical use will depend less on further improving model capability than on systematically addressing these interacting failure domains from development through deployment and post-deployment monitoring.

Orchestra composes two independently built bioinformatics MCP servers -- RegNetAgents, which infers gene regulatory network topology from ARACNe networks, and CASCADE, which supplies four independent evidence sources (LINCS knockdown, DepMap essentiality, super-enhancer status, DoRothEA transcription-factor confidence) -- into one multi-agent workflow exposed via the Model Context Protocol. Its central architectural claim is that requiring RegNetAgents' topology evidence and CASCADE's experimental evidence to agree on a candidate regulator yields a more trustworthy candidate than either alone -- not previously tested directly, since RegNetAgents' own validation asked only whether its candidate lists beat chance. We test this on the TCGA tumor-acquired regulator tier (regulators in a gene's tumor ARACNe network but absent from the GREmLN population-averaged baseline), selecting candidates by ARACNe mutual-information (MI) edge weight. On RegNetAgents' published BRCA/COAD focal-gene panel plus matched negative controls, agreement among at least 2 of the 4 CASCADE sources predicts OncoKB cancer-gene status among focal genes (odds ratio 2.89, Benjamini-Hochberg-adjusted p=0.0166) but not among negative controls (p=0.0721); a single source is not diagnostic for either group. The pattern replicates and strengthens in a third cancer type, STAD, on a separately constructed panel (odds ratio 5.82), and against an independently curated ground truth (the Sanger COSMIC Cancer Gene Census). MI edge weight is the strongest single predictor overall (p=0.0003); a logistic-regression likelihood-ratio test confirms corroboration adds value beyond it in both panels (p=0.0234; p=0.0001). Every experiment invokes Orchestra's real agentic entry point.

Lung cancer remains one of the leading causes of cancer- related mortality worldwide. Although targeted therapies have improved outcomes for patients with non-small cell lung cancer (NSCLC), they rely on mutation profiling through tissue biopsy, an invasive procedure with several limitations. This study investigates PET/CT-based radio- genomic prediction of epidermal growth factor receptor (EGFR), tumour protein 53 (TP53), and Kirsten rat sarcoma viral oncogene (KRAS) mutations using deep learning. We further evaluate whether pairwise multi-label learning improves mutation prediction compared with conventional single-gene classification. To the best of our knowledge, this is among the first studies to systematically investigate multi-label learning for PET/CT radiogenomic mutation prediction in NSCLC. Experiments were conducted on a novel UK-based radiogenomics cohort. Joint pre- diction of KRAS and TP53 improved AUC from 0.58 to 0.64 for KRAS and from 0.69 to 0.71 for TP53. For the EGFR/KRAS pair, only EGFR benefited from joint learning, while no improvement was observed for the EGFR/TP53 pair. These findings demonstrate that the effectiveness of multi-label learning depends on the specific combination of gene mutations being modelled, suggesting that mutation-specific modelling strategies may be preferable for PET/CT radiogenomic prediction.

CASCADE is an agentic framework that predicts downstream transcriptional effects of gene perturbation from precomputed ARACNe regulatory networks, exposed via MCP. Prior work validates such tools by checking whether predicted genes are known cancer genes (membership); we instead test whether the predicted direction of change matches reality, using focal-gene copy-number amplification as a dosage-based proxy for the inverse of knockdown against real TCGA patient tumor data. For MYC, CASCADE's predicted knockdown targets show strong concordance with real amplified-vs-non-amplified tumor expression across three cancer types (BRCA: 90.0%, COAD: 72.0%, STAD: 85.7%; all p<0.0013), well above permutation baselines, surviving a PAM50 subtype control and replicating in an independent cohort (METABRIC, 87.2%). Compared against curated MSigDB gene-set baselines via Fisher's exact test, CASCADE's accuracy is not shown to exceed existing public knowledge of MYC- or E2F-driven biology, though its gene-specific direction-calling clearly outperforms a naive uniform guess. Extending to fifteen additional genes, validation proves gene-specific rather than universal: proliferation-machinery regulators mostly replicate, while lineage-identity transcription factors and one cyclin-D paralog (CCND2) consistently fail, a pattern we discuss as a hedged, post-hoc hypothesis. We separately benchmark whether an LLM-based agent correctly grounds natural-language requests into CASCADE's real MCP tool calls. Across 35 queries, a documented local model reaches 71.4% exact match (85.7% for a larger model); schema and gene-alias failures are resolved by scale or server-side correction, but both models confidently default to the wrong perturbation type on ambiguous queries, a failure a targeted fix could not resolve because its trigger condition never occurs.

A natural language interface can be used to make cancer genomics databases easier to use, but even if a question is perfectly fluent, its scientific meaning can be ambiguous. We propose CLARA, a framework that represents a question as a typed scientific query specification, considers a few possible interpretations, executes them, and asks for clarification when the estimates diverge. CLARA was assessed on mutation-prevalence contrasts among eight TCGA PanCancer Atlas cohorts and a 30-gene panel. This benchmark consisted of 330 unique executable contrasts varying in mutation scope, assay denominator, and sample context; 115 contrasts were result-sensitive and 215 were result-stable, per the preregistered definition of relative divergence greater than 0.10 or absolute divergence greater than 5 percentage points. An independently implemented pandas execution engine perfectly replicated all 660 results from the SQLite engine. In a separate 120-question LLM-generated, manually vetted language stress test, CLARA recognized all 60 result-sensitive contrasts and needlessly clarified 13 of 60 stable contrasts (accuracy 89.2%, sensitivity/recall 100%, specificity 78.3%). Standalone machine learning had superior overall accuracy (97.5%) but missed one critical contrast. This demonstrates that downstream execution can distinguish consequential from inconsequential ambiguity and reveal an explicit trade-off between safety and burden.

The function of many genes is still unknown, and conventional driver-discovery methods, which rely on how frequently a gene is mutated, cannot assess genes that are only rarely affected. Here we pair Evo2-based genome analysis with routine clinical imaging to identify gene--phenotype associations at genome-wide scale. For every somatic mutation across three TCGA cohorts (cRCC=clear cell renal cell carcinoma, HCC=hepatocellular carcinoma, and BC=breast cancer; $n = 340$ total), Evo2 predicts a severity score, with no task-specific training. Per-gene severity summaries are then correlated with radiomic features extracted from paired tumor segmentations, controlling for total mutation burden. In TCGA-cRCC ($n = 162$), this sweep recovers established renal-cancer drivers and identifies 46 additional genes reaching false discovery rate (FDR) significance absent from curated cancer-gene panels, several of which are Mendelian ciliopathy and cytoskeletal-disease genes. These results demonstrate that pairing a genomic language model with widely available clinical imaging can serve as a hypothesis-free discovery tool for gene--imaging associations invisible to conventional approaches.

Accurate prediction of patient-specific therapeutic response from pre-treatment transcriptomes is hindered by the scarcity of matched clinical response labels and post-treatment molecular profiles. Preclinical transfer-learning models can simulate drug-induced expression changes but are often hard to interpret and unstable, whereas knowledge-graph methods provide mechanistic context yet remain static and fail to capture drug-induced transcriptomic perturbation dynamics. We propose PREDIKTOR, a patient-centered multi-view framework that aligns a personalized network view with a transferable transcriptomic perturbation view to predict clinical drug response. For each patient, we construct an individualized gene regulatory network from tumor expression using DysRegNet and augment it with drug-target links from DrugBank; a graph neural encoder yields a drug-centric, mechanistically grounded embedding. In parallel, a frozen condition-specific gene-gene attention model pretrained on LINCS L1000 generates a simulated post-perturbation transcriptomic profile for the same patient-drug pair. We align the two views in a shared latent space via a CLIP-style contrastive objective with drug-context hard negatives, then concatenate the representations for end-to-end response classification. On TCGA, PREDIKTOR consistently outperforms state-of-the-art baselines under patient-, drug-, and tissue-split evaluations, and transfers zero-shot to the I-SPY2 trial, improving AUROC by 5.6% over competing methods. The aligned embeddings yield stable gene and pathway attributions that recover known mechanisms, supporting actionable and interpretable precision oncology.

Genomic prediction models often fail to transfer across institutions because sequencing panels differ across sites, creating structural feature missingness at deployment. Existing approaches to this challenge typically restrict analysis to genes shared across cohorts, exclude patients with incomplete profiles, or rely on test-time imputation, all of which can reduce robustness and limit the use of multi-center data. We propose Survival prediction Handling Incomplete Features using Transformer (SHIFT), a missingness-aware survival model that directly predicts from incomplete genomic inputs without test-time imputation. SHIFT represents each genomic feature separately and uses masked self-attention, along with a feature-availability mask, so that predictions are based only on observed inputs. Further, we introduce variable-rate feature masking during training to improve robustness to heterogeneous missingness patterns. We evaluate the approach on glioblastoma and lung squamous cell carcinoma with external validation across multiple cohorts, including a challenging setting with severe cross-cohort panel mismatch. Across these settings, SHIFT shows strong generalization and compares favorably with standard survival baselines and imputation-based approaches, while using a single model across differing feature sets. We also find that incorporating patients from incomplete cohorts during development can improve performance on external data, suggesting that partially observed cohorts need not be excluded from model building. These results support missingness-aware modeling as a practical strategy for multi-center survival prediction in precision oncology.

Predicting cancer drug response from transcriptomic profiles is a cornerstone of precision oncology, yet the scientific value of machine learning models hinges not solely on predictive accuracy, but also on their capacity to generate reliable biological insights. Current explainability approaches in this setting are computationally costly, lack robustness, and reduce complex drug response to univariate gene importance scores, overlooking the coordinated gene activity that drives sensitivity and resistance. In this work, we present ILLUME+, a scalable post-hoc explainability framework that moves beyond single-gene assessments to capture multiple, complementary forms of explanation. Integrated into our end-to-end pipeline, ILLUME+ produces more stable gene importance scores than existing baselines, recovers established drug-gene associations and mechanisms of action, and enables AI-assisted hypothesis generation to uncover novel interaction-driven molecular signals in cancer biology.

Recent studies have shown that spatial properties of tumors are critical for understanding disease biology and predicting patient outcomes. These spatial properties are increasingly uncovered through complementary modalities: spatial transcriptomics (ST) captures spatially-resolved molecular states, while hematoxylin and eosin-stained whole slide images (HE) reveal tissue morphology. While approaches are emerging to fuse these modalities, effective methods that learn not only joint representations but also incorporate spatial context across modalities are lacking. Here, we present JASPR (Joint Spatial Representation learning), a self-supervised deep learning framework that integrates HE images and ST data through a cross-modal reconstruction objective that incorporates spatial context within HE images and ST profiles. It employs shared modules to capture universal spatial properties across modalities, while modality-specific experts encode features unique to morphological and genomic data. We train and validate JASPR on breast cancer datasets, demonstrating that its learned joint representation substantially improves HE-based prediction of 9,248 genes and provides prognostic value for breast cancer outcomes.

Binary data factorization is common, but real-valued methods ignore discreteness and yield hard-to-interpret factors. Boolean Matrix Factorization (BooMF) instead decomposes a binary matrix into two lower-rank binary matrices via logical AND and OR, expressing the data as a Boolean disjunction of interpretable patterns. In cancer genomics, BooMF can reveal coordinated feature changes that may drive tumor evolution, unlike rotational or additive decompositions. Most existing BooMF methods are heuristic, greedy, sensitive to initialization, prone to local optima, and do not support principled model selection or uncertainty quantification. We introduce Bayesian Boolean Matrix Factorization (BBMF), a fully conjugate generative model with sparsity-inducing priors. It enforces Boolean constraints, yields interpretable latent factors with coherent uncertainty quantification, and admits Gibbs sampling with closed-form full conditionals. Because cancer evolution often involves widespread, near-simultaneous chromosome-number changes (e.g., whole-genome duplication followed by instability and selection), Boolean factorizations capture these patterns more naturally than additive models. Applied to arm-level copy-number alteration data in multiple myeloma, where entries indicate presence/absence of chromosomal-arm amplifications, BBMF finds a small set of interpretable bicliques linking patient subsets to recurrently co-altered chromosomal arms, providing a compact, biologically meaningful summary of tumor heterogeneity and demonstrating BBMF's utility for uncovering discrete latent structure in complex binary data.

Resistance to first-line osimertinib in EGFR-mutant non-small-cell lung cancer (NSCLC) is the canonical example of predictable clonal evolution under therapeutic pressure, yet no public benchmark exists for training or evaluating computational models on the corresponding longitudinal patient trajectories. We introduce OncoTraj, a public benchmark of 813 EGFR-mutant NSCLC patients receiving first-line osimertinib, harmonized from three real-world clinical-genomic sources: MSK-CHORD (672 patients), AACR Project GENIE BPC NSCLC (34 patients), and the FLAURA molecular-resistance supplement (107 patients). OncoTraj defines three locked tasks: (A) binary classification of progression by a fixed 12-month landmark, (B) regression of time-to-first-progression in days, and (C) six-class classification of the dominant resistance mechanism. We release the harmonized dataset, patient-level train/validation/test splits with an audited no-leakage guarantee, an open-source evaluation harness, and six reference baselines spanning a majority-class predictor, logistic regression, random forest, XGBoost, an LSTM, and a multi-task transformer. With v1's single-timepoint snapshot features, no task clears chance on clean within-source evaluation: the uniformity of this ceiling across every model class localizes the limit to the input modality (single-snapshot tissue NGS rather than serial ctDNA), not the algorithm. The benchmark does recover a reproducible literature-consistent association: TP53 co-mutation raises the 12-month progression rate from 29% to 59% cohort-wide. OncoTraj establishes a reproducible, leakage-audited baseline and converts the modality limit into concrete design requirements for a serial-ctDNA-enriched v2.

Cancer treatment planning requires decisions across multiple clinical dimensions at once. Clinicians must determine whether a patient should receive targeted molecular therapy, radiation therapy, and whether they are likely to survive beyond six months. Existing pathway-informed deep learning models have been developed and tested in isolation, making fair comparison across architectures impossible. We present the first unified benchmark for pathway-guided therapy response modeling, evaluating three biologically informed architectures, BINN, GraphPath, and PATH, across five cancer cohorts drawn from The Cancer Genome Atlas, representing 2,622 patients encoded using Reactome pathway activity scores. Each model is trained jointly on all three clinical outcomes under identical data and evaluation conditions, the first study to treat pathway-structured deep learning as a combined therapy and survival prediction problem. Our results show that no single architecture wins across all tasks: PATH performs best for targeted molecular therapy prediction overall, BINN is most reliable for survival prediction, and no model produces useful predictions for radiation therapy, as the key drivers of that decision are clinical variables not captured in gene expression data. Most strikingly, GraphPath achieves an AUROC of 0.92 on prostate targeted molecular therapy prediction, the highest score in the entire benchmark, demonstrating that lateral co-regulation structure produces exceptional discriminative power when matched to a cohort with a narrow targetable driver programme, even under conditions of extreme class imbalance at only 11% positive prevalence.

Graph neural networks (GNNs) are increasingly used to model biological systems, yet the reliability of post-hoc explanation methods for recovering meaningful molecular mechanisms remains unclear. Here, we systematically evaluate four widely used approaches: Saliency Attribution (SA), Integrated Gradients (IG), GNNExplainer, and Layer-wise Relevance Propagation (LRP) for identifying disease-relevant structure in breast cancer RNA-seq data projected onto a protein-protein interaction network. Using synthetic benchmarks with known ground-truth motifs, we show that explanation methods recover distinct signal organizations: SA performs best for sparse single-node drivers, whereas IG and LRP preferentially recover distributed pathway-like and cascade-like signals. In TCGA BRCA data, we identify a consistent topological signature of disease-associated hubs in which attribution peaks in the immediate 1-hop neighborhood and decays across successive network shells, a pattern most pronounced for IG and LRP and associated with strong enrichment of known cancer hubs. We further observe a trade-off between local hub enrichment and global gene ranking performance, with IG optimizing local enrichment and SA achieving superior global discrimination. Motivated by these complementary behaviors, we introduce a framework combining a shell-based hub score with consensus ranking across explainers. Consensus scores improve prioritization of canonical cancer genes (TP53, BRCA1, ESR1, MYC), reduce dependence on node degree, and, especially when tuned, outperform individual methods. Pathway enrichment further reveals improved recovery of biologically coherent cancer programs, including ERBB2, RTK, MAPK, immune, and cytokine signaling. Together, these results demonstrate that topology-aware integration of graph explanations can improve biological interpretability and biologically relevant molecular recovery.

Understanding how molecular alterations propagate across biological systems to drive disease remains a central challenge. Although high-throughput profiling enables comprehensive characterization of tumor states, most models neglect structured biological relationships or lack interpretability across scales. Here we present PPI-Net, a hierarchical graph neural network that integrates protein-protein interaction (PPI) networks with pathway-level representations to model disease from molecular interactions to functional processes. Patient-specific molecular profiles are embedded within a shared interaction network from STRING and propagated through a multi-layer Reactome hierarchy using graph attention, enabling aggregation of gene-level signals into higher-order biological programs. Across RNA-seq data from ten cancer types from The Cancer Genome Atlas, PPI-Net achieves robust predictive performance, with balanced accuracy exceeding 90% in multiple cohorts. Comparative analysis on RNA-Seq data from breast cancer demonstrated that PPI-Net's integration of the Reactome hierarchy improved balanced accuracy by 6.7% relative to a PPI-only model, while hierarchical multi-level supervision improved balanced accuracy by 12.3% relative to using only a single top-level prediction head. Applying a multi-omics approach using RNA-seq and methylation data improves model interpretation, recovering canonical oncogenic modules, including TP53-AKT signaling and stress response pathways, while revealing convergence onto coherent programs such as ion signaling and cellular responses to stimuli. These results demonstrate that integrating interaction networks with pathway hierarchies enables accurate prediction while providing mechanistic insight into cancer biology.

Cancer survival prediction from multi-omics data remains challenging because prognostic signals are high-dimensional, heterogeneous, and distributed across interacting genes and pathways. We propose PathMoG, a pathway-centric modular graph neural network for multi-omics survival prediction. PathMoG reorganizes genome-scale inputs into 354 KEGG-informed pathway modules, introduces a Hierarchical Omics Modulation module to condition gene-expression representations on mutation, copy number variation, pathway, and clinical context, and uses dual-level attention to capture both intra-pathway driver signals and inter-pathway clinical relevance. We evaluated PathMoG on 5,650 patients across 10 TCGA cancer types and observed consistent improvements over representative survival baselines. The framework further provides gene-level, pathway-level, and patient-level interpretability, supporting biologically grounded and clinically relevant risk stratification.

Accurate prediction of cancer progression remains a challenge due to the high heterogeneity of molecular omics data across patients. While biologically informed models have improved the interpretability of these predictions, a persistent limitation lies in how they encode individual genes to construct pathway representations. Existing hierarchical models typically derive gene features by directly mapping raw molecular inputs, whereas integration frameworks often rely on simple statistical aggregations of patient-level signals. These approaches often fail to explicitly learn a shared base representation for each gene, thereby limiting the expressiveness and biological accuracy of downstream pathway embeddings. To address this, we introduce PATH, a modulation-based, patient-conditioned gene embedding strategy. PATH represents a paradigm shift by starting from a shared base embedding for each gene, preserving a stable biological identity across the population, and then dynamically adapting it using patient-specific copy number variation (CNV) and mutation signals. This allows the model to capture subtle individual molecular variations while maintaining a consistent latent understanding of the gene itself. We integrate PATH into a graph transformer framework that models interactions among biologically connected pathways through pathway-guided attention. Across pancancer metastasis prediction, PATH achieves an F1 score of 0.8766, representing an 8.8 percent improvement over the current SOTA multi-omics benchmarks. Beyond superior predictive accuracy, our approach identifies biologically meaningful pathways and, crucially, reveals disease-state-specific pathway rewiring, offering new insights into the evolving pathway-pathway interactions that drive cancer progression.

We introduce RegNetAgents, an AI-oriented multi-agent framework for structured, query-driven regulatory candidate identification across heterogeneous gene regulatory networks. It integrates bulk tumor (TCGA) and single-cell (GREmLN project) ARACNe networks and labels each candidate regulator by the network or networks in which it appears (Both, TCGA-only, GREmLN-only). For a given focal gene, the framework finds its regulators in both networks and labels each by source, flags those that are known cancer driver genes (IntOGen), and, for tumor-network regulators, gives the mode of action (MoA; activating or repressive). It is implemented as a multi-agent LangGraph state-graph workflow, accessible through a Python API and a Model Context Protocol (MCP) client, and operates as a downstream analytical layer over precomputed networks rather than a network inference method. For example, a single query for CTNNB1 in BRCA returns two tumor-specific (TCGA-only) driver-gene regulators, DDR2 and IL6ST, both with activating MoA. Across twelve breast cancer (BRCA) and thirteen colorectal cancer (COAD) driver genes, we compared each gene's tumor-network-only (TCGA-only) regulators with the TCGA-only regulators of random non-driver genes. Regulators as a group are about 2.7-fold richer in driver genes than genes overall, so almost any gene's regulators look enriched when tested against all genes. We therefore used random genes as the baseline. In COAD, the cancer genes' regulators include modestly but consistently more IntOGen driver genes than random genes' regulators do (nominal p = 0.012-0.036 across five random samples); in BRCA the difference is borderline (p = 0.048-0.083). Housekeeping and non-driver control genes show no such excess in the tumor-network tier, and the same comparison for GREmLN-only regulators shows none (p >= 0.20). Code: https://github.com/jab57/RegNetAgents

Machine learning models are primarily judged by predictive performance, especially in applied genomics, where explanations are read as biological findings. In practice, reported gene panels are stabilised by averaging, ranking, or taking consensus over the many models a pipeline produces across cross-validation folds, tuning grids, and repeated runs. This raises an overlooked question: when two models achieve high accuracy, do they rely on the same internal logic, or reach the same outcome via different mechanisms? We introduce EvoXplain, a diagnostic framework that measures whether a pipeline's explanation is uniquely determined across repeated training and model selection. Rather than analysing a single trained model, EvoXplain treats explanations as samples drawn from the training and model selection pipeline itself, without aggregating predictions or constructing ensembles, and examines whether they form a single coherent explanatory basin or separate into multiple structured basins. We evaluate EvoXplain on a TCGA pan-cancer cohort and a within-cancer breast-cancer subtype task, using elastic-net Logistic Regression and gradient-boosted trees. Although all models reach about 98% accuracy, explanation structure differs across pipelines. Holding the data split fixed and varying only the regularisation strength, equally accurate Logistic Regression models separate into a few discrete, reproducible basins that recur across 100 data splits and carry distinct biological content, while the gradient-boosted pipeline converges to one basin. The same multiplicity appears within a single cancer subtype, from the ordinary tuning step alone. EvoXplain makes explanatory structure visible, revealing when an averaged consensus corresponds to no single trained model, and reframes interpretability as a property of the training pipeline rather than of any single model.

Building trustworthy machine learning algorithms for biological applications requires adapting to data heterogeneity from different sources, batches, distributions, or studies. We propose the 'Cross-Cluster Weighted Forest' (CCWF), an ensembling approach that explicitly leverages heterogeneity in the feature distribution to produce more accurate and more generalizable predictors than the standard Random Forest in cases when data can be naturally clustered. CCWF generalizes the RF architecture to an outer unsupervised layer, supervised subtasks, and ensembling. Specifically it involves unsupervised clustering of the training data, fitting a Random Forest on each cluster, and combining the forests via stacked regression weights that reward cross-cluster generalizability. We provide a theoretical analysis of an analytically tractable forest model showing that cluster-based ensembling is asymptotically more accurate than training a single forest on the full data, with the gain driven by bias reduction. In simulations, we find that CCWF is robust across data-generating regimes and outcome models; furthermore, we explore the influence of data partitioning and ensemble weighting strategies on the benefits of our method. Finally, we apply our approach to cancer molecular profiling and gene expression datasets that are naturally divisible into clusters; in both simulations and real data examples, we illustrate that our approach outperforms classic Random Forest by margins of 30-40%, aligning with our theoretical results. Overall, we show that CCWF provides a statistically grounded prediction algorithm for data spanning multiple domains or sub-populations, a structure common in biological applications.