Table 2.

Criteria for evidence of oncogenicity of somatic variants

Category	Evidence	Criteria	Comments/Caveats
Very Strong (8 points)	OVS1	Null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a bona fide tumor suppressor gene.	Use caution interpreting predicted loss-of-function (pLOF) variants at the extreme 3′ end of a gene after the nonsense mediated decay site Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact (in frame events). Use caution if splice variant leads to expression of a well-known alternative isoform which preserves tumor suppressor functionality. Use caution in the presence of multiple transcripts.
Strong (4 points)	OS1	Same amino acid change as a previously established oncogenic variant (using this standard) regardless of nucleotide change. Example: Val→Leu caused by either G>C or G>T in the same codon.	Beware of changes that impact splicing rather than at the amino acid/protein level.
	OS2	Well-established in vitro or in vivo functional studies supportive of an oncogenic effect of the variant.	Functional studies that have been shown to be reproducible and robust are considered the most well established. If OS1 is applicable, this rule can be used only if functional studies are based on the particular nucleotide change of the variant.
	OS3	Located in one of the hotspots in cancerhotspots.org with at least 50 samples with a somatic variant at the same amino acid position, and the same amino acid change count in cancerhotspots.org in at least 10 samples.	Use caution with hotspots driven by truncating somatic variants. If the somatic variant is in a tumor type not well covered by cancerhotspots.org, resources such as COSMIC or a tumor type specific study could be used. This rule cannot be used if OS1 is applicable, unless it is possible to observe hotspots based on the particular nucleotide change.
Moderate (2 points)	OM1	Located in a critical and well-established part of a functional domain (e.g., active site of an enzyme).	This rule cannot be used if OS1 or OS3 is applicable.
	OM2	Protein length changes as a result of in-frame deletions/insertions in a known oncogene, or tumor suppressor gene or stop-loss variants in a known tumor suppressor gene.	This rule cannot be used if OVS1 is applicable.
	OM4	Missense variant at an amino acid residue where a different missense variant determined to be oncogenic (using this standard) has been documented. Amino acid difference from reference amino acid should be greater or at least approximately the same as for missense change determined to be oncogenic.	Example: p.Arg156His is oncogenic; now you observe p.Arg156Cys. This rule cannot be used if OS1 or OS3 or OM1 is applicable. Beware of changes that impact splicing rather than at the amino acid/protein level.
	OM3	Located in one of the hotspots in cancerhotspots.org with less than 50 samples with a somatic variant at the same amino acid position, and the same amino acid change count in cancerhotspots.org is at least 10.	This rule cannot be used if OM1 or OM4 is applicable. Use caution with hotspots driven by truncating somatic variants. If the somatic variant is in a tumor type which is not covered well by cancerhotspots.org, resources such as COSMIC or a tumor type specific study could be used.
Supporting (1 point)	OP1	All utilized lines of computational evidence support an oncogenic effect of a variant (conservation/evolutionary, splicing impact, etc.).	Because many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. Can be used only once in any evaluation of a variant.
	OP2	Somatic variant in a gene in a malignancy with a single genetic etiology. Example: retinoblastoma is caused by bi-allelic RB1 inactivation.	A small fraction of cases may be caused by an alternative mechanism; histological similarities may cause misdiagnosis.
	OP3	Located in one of the hotspots in cancerhotspots.org and the particular amino acid change count in cancerhotspots.org is below 10.	Use caution with hotspots driven by truncating somatic variants. If somatic variant is in a tumor type which is not covered well by cancerhotspots.org, resources such as COSMIC or a tumor type specific study could be used.
	OP4	Absent from controls (or at an extremely low frequency) in Genome Aggregation Database (gnomAD).	Population data for insertions/deletions may be poorly called by next-generation sequencing. Population data may contain somatic variants associated with clonal hematopoiesis.

OVS1: Oncogenic Very Strong-1, OS1: Oncogenic Strong-1, OS2: Oncogenic Strong-2, OS3: Oncogenic Strong-3, OM:1 Oncogenic Moderate-1, OM2: Oncogenic Moderate-2, OM3: Oncogenic Moderate-3, OM4: Oncogenic Moderate-4, OP1: Oncogenic Supporting-1, OP2: Oncogenic Supporting-1, OP3: Oncogenic Supporting-3, OP4: Oncogenic Supporting-4