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A model in which tumours arise within the relevant host tissue rather than being introduced as an established graft. Defined engineered initiation still differs from a full human exposure history.

A fibroblast in a tumour-associated state. Some functions support tumour progression while other fibroblast populations or functions can restrain it; the label alone does not establish the causal effect.

An intervention intended to generate or strengthen immune recognition of tumour-associated targets. Successful priming does not guarantee entry or function within a tumour.

A malignant tumour of epithelial origin. Its cells can change phenotype without ceasing to belong to their original malignant lineage.

Colony-stimulating factor 1, a signal promoting survival and differentiation within monocyte/macrophage lineages and participating in some tumour–macrophage interactions.

A chemokine also called SDF-1, involved in cell recruitment and stromal–tumour signalling in several experimental settings.

A receptor tyrosine kinase gene also called HER2, expressed at different levels in some tumours and normal tissues. Target abundance alone does not establish a safe therapeutic window.

An assay introducing tumour biological cells at a selected point of dissemination, for example by vascular injection. Its interpretation must state which preceding barriers were bypassed.

Exit of circulating tumour cells through a vascular boundary into tissue, one step in a multistage metastatic process.

Avoidance or suppression of immune-mediated elimination. Normal immune-regulatory mechanisms can be repurposed in a tumour context.

Entry of tumour cells from tissue into a blood or lymphatic vessel, distinct from later survival or growth at a distant site.

A Janus kinase participating in cytokine-receptor signalling, including interferon responses relevant to tumour–immune interactions.

A RAS-family gene whose activating mutations can promote constitutive growth signalling in multiple tumour lineages.

The establishment of malignant growth at a site distant from the original tumour, requiring successful dissemination, survival and expansion. Circulating tumour cells alone do not establish that a metastasis has formed.

A sequence of conditional transitions from a primary tumour to a distant growing lesion. A bypass assay tests only selected transitions, and failure at one stage need not identify the slowest step in every setting.

A microRNA whose endogenous activity restrains tumour development or progression in a specified context. Specific loss, controlled restoration and target tests provide stronger evidence than an expression correlation.

A gene whose altered activity can promote tumour development, commonly through activating or expression-increasing changes that retain a useful protein function.

Strong dependence of an established tumour on an oncogenic activity. Dependence can change as the tumour acquires cooperating or bypass alterations.

Placement of a graft in the corresponding anatomical organ or site. It preserves more relevant local context than some ectopic grafts but still bypasses natural tumour initiation.

I would establish whether the alteration drives cancer, whether the established cancer depends on it, and whether that dependence offers a selective intervention point. These are different questions. A recurrent alteration can be a passenger; a genuine initiating driver can become dispensable after progression; and an essential protein can be equally essential in normal tissue.

First validate the genomic observation with an independent assay and matched normal DNA. Distinguish a somatic mutation from inherited variation, sequencing error and contamination. For gene amplification, resolve the altered interval, copy number and tumour purity, and determine whether the candidate is the focus of a recurrent, narrowly amplified segment or simply accompanies a neighbouring driver in a broad gain. Measure RNA, protein and relevant activity: increased DNA dosage need not increase active protein. For mutations, inspect the spectrum and allelic configuration. Recurrent clustered missense changes can suggest altered activity, whereas dispersed truncations and loss of the remaining allele can suggest tumour-suppressor inactivation. Neither pattern is conclusive without accounting for sequence context, background mutation rate and selection. Large genes, highly mutable sites and poorly repaired regions can recur without being selected; analyses of mutational heterogeneity and somatic selection show why a simple recurrence ranking is insufficient.^[ref][ref]

Relate the alteration to cancer type, stage, co-occurring lesions and clonal architecture. Single-cell or spatial assays can establish whether it is in the malignant compartment and whether every tumour biological cell carries it. Correct variant fractions for purity and copy number before inferring clonality. A clonal lesion may be early, but clonality alone does not prove causality; a passenger can hitchhike in an expanding clone. Association with outcome should be assessed within appropriate clinical and molecular strata rather than interpreted from a mixture of unrelated tumour types.

For functional necessity, use several independently altered cell lines and patient-derived organoids, together with lineage-matched unaltered cancers and nonmalignant controls. Reduce the candidate with orthogonal perturbations and rescue with a perturbation-resistant allele at a near-physiological level. Measure proliferation, cell-cycle distribution, apoptosis, differentiation and the proposed pathway output separately. In an amplified region, a particularly important control is to avoid equating CRISPR cutting toxicity with gene dependence: many DNA breaks at multiple copies can suppress growth independently of the encoded gene. Aguirre and colleagues demonstrated this copy-number-dependent response.^[ref] CRISPR interference or RNA interference, combined with rescue and appropriately matched cutting controls, helps distinguish protein dependence from this artefact. Guide-specific off-target effects and incomplete depletion also require direct checks.

Test sufficiency and allele specificity by introducing the observed allele into a relevant nonmalignant background, preferably through endogenous editing, or by increasing expression to the measured tumour level. Compare wild type, each recurrent mutant and appropriate inactive variants at matched abundance. For a suspected tumour suppressor, recreate the observed heterozygous or biallelic loss rather than assuming every mutation is equivalent to a knockout. A kinase-domain mutant might activate signalling, a truncation might remove negative regulation, and another allele might abolish function. Physiological assays of acinar organization, growth-factor dependence or invasion can reveal transformation phenotypes missed by a two-dimensional growth assay. Failure to transform one biological cell type alone does not exclude a driver requiring cooperating lesions or the correct developmental state.

Then connect genotype to mechanism. Identify affected enzymatic activity, binding partners, localization and downstream signalling, using time-resolved perturbation to separate primary changes from consequences of dying biological cells. Restore a downstream effector to test whether it bypasses loss of the candidate, or inhibit that effector to test whether it is required for the mutant phenotype. Rescue with an inhibitor-resistant target allele is strong evidence for a drug's intended mechanism. A correlation between mutation and a distant transcriptional signature is weaker. Genetic and pharmacological results should agree at exposures that establish target engagement rather than general cytotoxicity.

Finally test initiation and maintenance in suitable in vivo settings: tissue-appropriate engineered mice, orthotopic organoid transplantation or patient-derived xenografts, with the immune context chosen for the hypothesis. Inducing an allele before tumour development tests initiation; turning it off after established growth tests maintenance. Quantify tumour latency, burden, invasion and metastatic outcome with random allocation, blinded assessment and independent biological replicates. Measure effects on normal tissue to define a therapeutic window, and examine resistant outgrowth for bypass mechanisms. The strongest conclusion combines statistically credible human selection, allele-specific causal perturbation, mechanistic rescue and disease-selective dependence across appropriate models.