The Oncogenicity Prediction feature estimates oncogenic potential of variants based on Standards for the classification of somatic variants in cancer (Horak et al., Genet Med. 2022). The standards include 17 criteria and allow to classify variants into 5 categories: Oncogenic, Likely Oncogenic, Variant of Unknown Significance (VUS), Likely Benign, and Benign. Connected Insights scores variants (only PASS small variants are currently supported) for 16 criteria (with exception of OP2) based on the implementation details provided below.

❗ Use predictions as a starting point for interpretation. Exercise judgement to determine if a greater or fewer number of criteria apply.

Estimated variant oncogenicity can be found:

• In the column “Oncogenicity Prediction” in the Variants tab available for sorting

• In the section “Oncogenicity Prediction” in the Biomarker page available for manual review and further interpretation

To view the evidence behind the estimated oncogenicity category and complete variant classification, follow the steps below:

1. Navigate to the "Oncogenicity Prediction" section in the Biomarker page

2. Review estimated oncogenicity category, score, and met criteria (displayed with filled checkboxes, see OS2, OM1, OM2, OP3, and OP4 on the figure below)

3. Review evidence for each criterion by clicking on it and displaying an evidence map. You can move objects on the map to facilitate review.

4. Select Report to edit and complete variant classification following Interpret a Variant. Information about met oncogenicity criteria is used to pre-populate variant summary.

Implementation Details

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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant is a “null variant”. We assess null variants as variants with consequences stop_gained, start_lost, frameshift_variant, splice_acceptor_variant, or splice_donor_variant as well as variants with a high splicing prediction (SpliceAI score > 0.8) regardless of their consequences

2. Variant is not in the last intron or exon

3. Variant is in a tumor suppressor gene, according to Cancer Genome Census, JAX-CKB, OncoKB or MyKB

OS1: “Same amino acid change as a previously established oncogenic variant regardless of nucleotide change. Example: Val→Leu caused by either G>C or G>T in the same codon.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant's consequences are not splice_donor_variant or splice_acceptor_variant

2. Variant’s splicing prediction is not high (SpliceAI score <= 0.8)

3. There is a previously established pathogenic / oncogenic or likely pathogenic / oncogenic variant with the same amino acid change but different nucleotide change in ClinVar (2-4 stars) or MyKB

OS2: “Well-established in vitro or in vivo functional studies supportive of an oncogenic effect of the variant.”

For this criterion, we are checking the fulfillment of the following conditions:

1. A variant with the same amino acid change is a gain-, loss-, or switch-of-function variant in MyKB, OncoKB, or JAX-CKB, is interpreted as pathogenic / oncogenic or likely pathogenic / oncogenic in MyKB, OncoKB, JAX-CKB, or ClinVar (2-4 stars), and OS1 is not applicable OR

2. A variant with the same nucleotide change is a gain-, loss-, or switch-of-function variant in MyKB, OncoKB, or JAX-CKB, is interpreted as pathogenic / oncogenic or likely pathogenic / oncogenic in ClinVar (2-4 stars), and OS1 is applicable

In this implementation, “Well-established in vitro or in vivo functional studies” is inferred based on knowledge bases having records of gain-, loss-, or switch-of-function, usually established based on functional studies. “... supportive of an oncogenic effect of the variant” is established based on the evidence of oncogenicity in knowledge sources.

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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. There are at least 50 samples with somatic variants at the same amino acid position in cancerhotspots.org, there are at least 10 samples with variants with the same amino acid change in cancerhotspots.org and OS1 is not applicable OR

2. There are at least 50 samples with somatic variants at the same amino acid position in COSMIC, there are less than 10 samples with variants with the same amino acid change in cancerhotspots.org and OS1 is not applicable OR

3. There are at least 50 samples with somatic variants at the same nucleotide position in cancerhotspots.org, there are at least 10 samples with a variant with the same nucleotide change in cancerhotspots.org and OS1 is applicable OR

4. There are at least 50 samples with somatic variants at the same nucleotide position in COSMIC, there are less than 10 samples with a variant with the same nucleotide change in cancerhotspots.org and OS1 is applicable

OM1: “Located in a critical and well-established part of a functional domain (e.g., active site of an enzyme).”

For this criterion, we are checking the fulfillment of the following conditions:

1. The variant is located in a region of a protein domain where pathogenic variants occur. The regions in each gene are defined by taking all protein domains from UniProt, mapping all pathogenic and likely pathogenic variants in ClinVar to each domain and defining the regions by using the positions of the first and last pathogenic and likely pathogenic variants from ClinVar and adding 2 bp padding on each side.

2. OS1 and OS3 are not 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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant’s consequence is inframe_deletion or inframe_insertion and it is located in an oncogene or a tumor suppressor gene (based on Cancer Genome Census, JAX-CKB, OncoKB or MyKB) or variant’s consequence is stop-lost and it is located in a tumor suppressor gene per the same sources

2. OVS1 is not applicable

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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. There are less than 50 samples with somatic variants at the same amino acid position in cancerhotspots.org, there are at least 10 samples with variants with the same amino acid change in cancerhotspots.org , and OM1 and OM4 are not applicable OR

2. There are 10 - 49 samples with somatic variants at the same amino acid position and change in COSMIC, there are less than 10 samples with variants with the same amino acid change in cancerhotspots.org , and OM1 and OM4 are not 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.”

For this criterion, we are checking the fulfillment of the following conditions:

1. There is a missense variant at the same amino acid position with a different amino acid change interpreted as pathogenic / oncogenic or likely pathogenic / oncogenic in ClinVar (2-4 stars), OncoKB or MyKB

2. OS1, OS3 and OM1 are not applicable

OP1: “All utilized lines of computational evidence support an oncogenic effect of a variant (conservation / evolutionary, splicing impact, etc.).”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant’s effect predicted to be damaging (REVEL > 0.75 and PrimateAI-3D score percentile > 0.8) or variant’s splicing prediction is high (SpliceAI score > 0.8)

OP2: “Somatic variant in a gene in a malignancy with a single genetic etiology.”

This criterion is not yet implemented.

OP3: “Located in one of the hotspots in cancerhotspots.org and the particular amino acid change count in cancerhotspots.org is below 10.”

For this criterion, we are checking the fulfillment of the following conditions:

1. There is at least 1 sample with a somatic variant at the same amino acid position in cancerhotspots.org or COSMIC

2. There are less than 10 samples with variants with the same amino acid change in both, cancerhotspots.org and COSMIC

OP4: “Absent from controls (or at an extremely low frequency) in Genome Aggregation Database (gnomAD).”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant’s allele count in the global population (gnomAD) is less than 10 if the variant is not in TP53, KRAS, or PTEN

2. Variant is absent in the global population (gnomAD) if the variant is in TP53 or KRAS

3. Variant’s frequency in the global population is less than 0.001% (gnomAD) if the variant is in PTEN

SBVS1: “Minor allele frequency is >5% in Genome Aggregation Database (gnomAD) in any of 5 general continental populations: African, East Asian, European (Non-Finnish), Latino, and South Asian.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant’s frequency in any one population is over 5% (gnomAD) if the variant is not in TP53, KRAS or PTEN

2. Variant’s frequency in the global population is equal or over 0.1% (gnomAD) and allele count in the global population is equal or over 5 (gnomAD) if the variant is in TP53

3. Variant’s frequency in the global population is equal or over 0.05% (gnomAD) if the variant is in KRAS

4. Variant’s frequency in the global population is equal or over 1% (gnomAD) and allele count in the global population is equal or over 5 (gnomAD) if the variant is in PTEN

SBS1: “Minor allele frequency is >1% in Genome Aggregation Database (gnomAD) in any of 5 general continental populations: African, East Asian, European (Non-Finnish), Latino, and South Asian.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant’s frequency in any one population is over 1% (gnomAD) if the variant is not in TP53, KRAS or PTEN

2. Variant’s frequency in the global population is over 0.03% (gnomAD) and allele count in the global population is equal or over 5 (gnomAD) if the variant is in TP53

3. Variant’s frequency in the global population is equal or over 0.025% (gnomAD) if the variant is in KRAS

4. Variant’s frequency in the global population is equal or over 0.1% (gnomAD) and allele count in the global population is equal or over 5 (gnomAD) if the variant is in PTEN

SBS2: “Well-established in vitro or in vivo functional studies show no oncogenic effects.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant is interpreted as benign or likely benign in ClinVar (2-4 stars), OncoKB or MyKB

SBP1: “All used lines of computational evidence suggest no effect of a variant (conservation / evolutionary, splicing effect, etc.).”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant’s effect predicted to be neutral (REVEL is equal or less than 0.75 and PrimateAI-3D score percentile is equal or less than 0.8)

2. Variant’s splicing prediction is low or unknown (SpliceAI score is equal or less than 0.2 or unknown)

SBP2: “A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.”

For this criterion, we are checking the fulfillment of the following conditions:

1. Variant has a consequence synonymous_variant

2. Variant’s conservation prediction score is not high (PhyloP < 0.1)

3. Variant’s splicing prediction is low or unknown (SpliceAI score <= 0.2)

This section presents a visualization of the variant in the genome with supporting annotations and surrounding variants.

Available Tracks

• Reference — Reference genome base pair proportions when zoomed out. Base pair letters and amino acids for a given reading frame when zoomed in.

• Genes — RefSeq gene track. Blue indicates the - strand and red indicates the + strand. Click a gene to show the reading frame of the selected transcript for the variant (amino acid and position).

• Protein Domains — Displays protein domains from UniProt. Blue indicates the - strand and red indicates the + strand.

• Small variants — Small variants shown with the reference allele on top and the two alt alleles below.

• Structural variants — Structural variants with VCF filter PASS by chromosomal position. The legend to the right of the genome view identifies the events (translocation, inversion, deletion, insertion, and tandem duplication). Translocations begin at the karyogram at the top of the track and connect to chromosomes identified at the bottom of the track.

• Coverage and copy number variants — The depth of coverage by chromosomal position. Data points are color-coded by presence of copy number variant calls. The legend identifies the events (gain, gain LOH, neutral LOH, loss, and no call). Hovering over a region will highlight the copy number variant call and clicking provides variant details.

• B-allele ratio — The minor allele frequency by chromosomal position.

Track availability depends on the VCFs provided by the secondary analysis pipeline.

For example,

• If there's no structural variant VCF provided, then the structural variant track will not be available.

• If there's no absolute copy number provided in the VCF, then the copy number variant track will not be available.

Navigation

• To pan, use the pan controls at the top of the section, or click and drag one of the tracks left or right.

• To zoom, use the zoom controls at the top of the section, or hold ctrl and scroll with your mouse or trackpad.

The Biomarker Details page brings together key information and actions related to variants. The page exists for individual variants and genome-wide biomarkers like TMB and MSI. You can use the Biomarker Details page to do the following:

• Interpret the variant and include it in the report.

• Review assertions already created for the variant. For more information, refer to Biological Classification Assertions.

• Review sign-off reports that included the variant. For more information, refer to Biological Classification Assertions.

• Review variant information related to oncology: – Knowledge bases. For more information, refer to Actionability. – Clinical trials. For more information, refer to Clinical Trials.

• Review variant information. For more information, refer to Variant Overview. – Annotation details – Gene information – Records in external sources – Functional impact predictions – Population frequency

• Set gene transcripts. For more information, refer to Transcripts.

To open the Biomarker Details page, select Interpret for a specific variant in the Overview or Variants tab.

This section provides details on the variant’s presence in different datasets.

Lab Frequency

Displays the number of samples with the variant in the given workgroup presented per disease. To be accounted for, the variant:

• Need to be present in any uploaded VCF or variant file

• Need to have a flag PASS in the VCF variant filters

• Doesn’t need to be interpreted or included in the report

• Can have any variant origin (predicted germline or suspected somatic)

❗ Cases processed prior to version 4.0 of Connected Insights will not appear in the lab frequency.

Cancer Hotspots

The section displays:

• Number of samples in the Cancer Hotspots database with the variant for the selected transcript

• Highest number of samples in the Cancer Hotspots database for the variant across transcripts

COSMIC

The section displays:

• Genomic Mutation ID in the COSMIC database

• Number of samples with the variant in the COSMIC database displayed per cancer site, for example, breast

• Number of samples with the variant in the COSMIC database displayed per histology, for example, carcinoma

Clinical trials are listed in this when one of the knowledge bases determines the trial to be a match for the variant. The information in this table is from the clinical trial database.

By default, the assertions are filtered by the case and ancestor diseases. Ancestor diseases are determined using SNOMEDCT. These diseases can be viewed at the SNOMED CT Browser website, with the following exceptions:

• Descendents of neoplastic disease do not include ancestors beyond neoplastic disease.

• Non-small cell lung cancer has been added as an ancestor of adenocarcinoma of lung.

You can also filter by other diseases, title, city, state, and country.

Filter changes are saved and applied across variants within a case and can be reset if needed.

Clinical Trials Legend

Available Clinical Trials Knowledge Bases

Connected Insights provides access to the National Library of Medicine Clinical Trials website, which provides information on publicly and privately supported clinical studies on a wide range of diseases and conditions.

The following from JAX-CKB are not currently supported:

• Clinical trials with a recruitment status of completed, withdrawn, or active, not recruiting.

• Variants that have the excluded requirement type.

The software does not yet determine if a case has all the requirements for a clinical trial (for example, a clinical trial requires two variants).

Diagnostic

A single table in the biomarker details provides a list of all the diagnostic assertions from all included sources that have content about the biomarker (e.g., My Knowledge Base).

You can filter by other diseases or by summary. When filtering by summary for guideline evidence, specify the website (for example, ESMO.org or NCCN.org).

Filter changes are saved and applied across variants within a case and can be reset if needed.

Therapeutic, Prognostic

A single table in the biomarker details provides a list of all the therapeutic and prognostic assertions from all included sources that have content about the biomarker (e.g., My Knowledge Base).

By default, the assertions are filtered by the case and ancestor diseases. Ancestor diseases are determined using SNOMEDCT. These diseases can be viewed at the , with the following exceptions:

• Descendents of neoplastic disease do not include ancestors beyond neoplastic disease.

• Non-small cell lung cancer has been added as an ancestor of adenocarcinoma of lung.

You can filter by other diseases, by summary, or by approval. When filtering by summary for guideline evidence, specify the website (for example, ESMO.org or NCCN.org).

Filter changes are saved and applied across variants within a case and can be reset if needed.

Actionability Legend

Available Knowledge Bases

Memorial Sloan Kettering OncoKB

Connected Insights provides the Memorial Sloan Kettering OncoKB. For more information, refer to the OncoKB website.

The following from OncoKB are not supported:

• Non-compliant HGVS

• Unspecified positions: Epigenetic Silencing, ARv567es, AR-V7, DNMT3B7, vIII, vII, vV, TGFBR1*6A, Overexpression, Wildtype, Promoter Hypermethylation, Hypermethylation, Kinase Domain Duplication, Internal Tandem Duplication, Partial Tandem Duplication

Jackson Clinical Knowledge Base (JAX-CKB)

Connected Insights provides the Jackson Clinical Knowledge Base (JAX-CKB). For more information, refer to the Jackson CKB website.

The following from JAX-CKB are not supported:

• Complex molecular profiles (for example, co-occuring biomarker assertions)

• Emerging and risk factor evidence types

• Class #, over exp, dec exp, hypermethylation, hypomethylation, dup exonX, dup exonX-X, LOH, loss, negative (except for HRD andMSI), positive (except for HRD), and wild type variants.

Clinical Interpretation of Variants in Cancer (CIViC)

Connected Insights also provides the Clinical Interpretation of Variants in Cancer (CIViC) knowledge base. For more information, refer to the CIViC website.

The following from CIViC are not supported:

• Assertions

• Functional and oncogenic evidence types

• Splice Site (c.3028G>A), Boolean-like evidence (for example, (V600E or V600K) and Amplification), Underexpression, Overexpression, Expression, Decreased Peri-therapeutic Expression, Cytoplasmic Expression, Loss, Biallelic Inactivation, Alu Insertion, Alternative Transcript, RSID, Homozygosity, Copy-neutral Loss of Heterozygosity, Cytoplasmic Mislocation, Deleterious Mutation, Double Ph, Wildtype, Domain, Exon-specific fusions

This section is only available for variants.

Common Information

External Links

This section is only available for variants that overlap a transcript.

Gene symbols

A list of all genes overlapping the variant are provided. Select one to view the details below.

Gene Descriptions

Available Knowledge Bases

Memorial Sloan Kettering OncoKB

Connected Insights provides the Memorial Sloan Kettering OncoKB. For more information, refer to the OncoKB website.

Jackson Clinical Knowledge Base (JAX-CKB)

Connected Insights provides the Jackson Clinical Knowledge Base (JAX-CKB). For more information, refer to the Jackson CKB website.

Clinical Interpretation of Variants in Cancer (CIViC)

Connected Insights also provides the Clinical Interpretation of Variants in Cancer (CIViC) knowledge base. For more information, refer to the CIViC website.

Gene-Related Diseases

The associated phenotypes in ClinGen and OMIM databases and links to open the entries in the databases in a new browser tab.

External Links

The following external links are included in this section:

• PubMed

• MGI

• DECIPHER

• GeneReviews

• Monarch

• ZFIN

• STRING

The following is a list of computational tools Connected Insights uses to understand the functional impact of the variant on the protein function. This sectionis only available for variants.

Computational Tools

Lists the allele count, allele number, number of homozygotes, and allele frequency for each population from gnomAD for small variants and 1000 Genomes for copy number variants and structural variants. This section is only available for variants.

The Biological Classification section of the Biomarker Details page summarizes all biological assertions previously created for the variant across all sources (e.g., My Knowledge Base or JAX-CKB). If you have existing knowledge to upload to the system, refer to Upload Assertions.

You can also filter by other diseases and by summary.

Filter changes are saved and applied across variants within a case and can be reset if needed.

Assertion Details

Available Knowledge Bases

Memorial Sloan Kettering OncoKB

Connected Insights provides the Memorial Sloan Kettering OncoKB. For more information, refer to the OncoKB website.

The following from OncoKB are not supported:

• Non-compliant HGVS

• Unspecified positions: Epigenetic Silencing, ARv567es, AR-V7, DNMT3B7, vIII, vII, vV, TGFBR1*6A, Overexpression, Wildtype, Promoter Hypermethylation, Hypermethylation, Kinase Domain Duplication, Internal Tandem Duplication, Partial Tandem Duplication

Jackson Clinical Knowledge Base (JAX-CKB)

Connected Insights provides the Jackson Clinical Knowledge Base (JAX-CKB). For more information, refer to the Jackson CKB website.

The following from JAX-CKB are not supported:

• Complex molecular profiles (for example, co-occuring biomarker assertions)

• Emerging and risk factor evidence types

• Class #, over exp, dec exp, hypermethylation, hypomethylation, dup exonX, dup exonX-X, LOH, loss, negative (except for HRD andMSI), positive (except for HRD), and wild type variants.