Summary Box 1:

Expanded ACMG/AMP Criteria for Applying Tumor Evidence in the Classification of Variants in Cancer Genes

Very strong evidence of pathogenicity
PVS1	Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease
	The presence of loss of heterozygosity in a tumor, judged to be focal by experts, can increase the strength of a downgraded application of PVS1 by one level Caveat: intended for the context of a germline variant being interpreted with regard to cancer predisposition
Moderate evidence of pathogenicity
PM1	Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation
	Variant AA at the hotspot codon in question has a sample count in cancerhotspots.org that is equal or greater than 10 at that codon Reduce to PM1_Supporting if variant AA has a sample count that is greater than 1, and less than 10 (from 2–9) Caveat: intended for the context of a germline variant being interpreted with regard to cancer predisposition
Supporting evidence of pathogenicity
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology
	Mutation signatures, with statistically significant association to a single gene or small set of genes, all of which were sequenced in the individual(s) with the variant under investigation (e.g. BRCA2: signature 3, MLH1, MSH2, MSH6, PMS2: signature 6; TP53: signature 13) Caveat: intended for the context of a germline variant being interpreted with regard to cancer predisposition