3.7.2. VSClinical AMP

The AMP Guidelines provide a set of standards for the interpretation of somatic mutations in cancer. These mutations can include single nucleotide variants, copy number variants, gene fusions, and considerations for wild type genes. This workflow supports the user in determining the oncogenicity of variants and aids the user in the identification of clinical evidence, including drug sensitivity, drug resistance, prognostic evidence and diagnostic evidence. The AMP Guidelines provide a rubric to grade the summation of this clinical evidence and categorize each biomarker into a given tier as follows [4]:

  • Tier I: Variants of strong clinical significance with known FDA-approved therapies or well powered studies with expert consensus.

  • Tier II: Variants with known FDA-approved therapies for different a tumor type, investigational therapies, multiple published studies with consensus, or preclinical trials with case reports.

  • Tier III: Variants of unknown clinical significance which are not observed at a significant allele frequency in the general population, but have convincing published evidence of cancer association.

  • Tier IV: Variants that are classified as benign or likely benign due to high allele frequency in population databases and have no published evidence of cancer association.

While the capture of all relevant evidence to determine the appropriate classification tier is a difficult undertaking, VSClinical guides the user through the classification process and automates the presentation of all relevant content in the final report.

This workflow requires the AMP Guidelines product to be enabled as an add-on to your VarSeq license.

Project Options

When starting a VSClinical workflow, you will be greeted by the VSClinical Project Options dialog, which allows you to configure your workflow. These options are grouped into four tabs. The first tab allows you to select the database catalogs used by VSClinical to store interpretation data.

Project Options

The Internal Database of Cancer Interpretations stores interpretation information for reported genes and biomarkers. The Internal Database of Classified Somatic Variants stores information about classified somatic variants. The Internal Database of Classified Germline ACMG Variants stores information about germline variants classified using the ACMG Guidelines. See Assessment Catalog View for more information on assessment catalogs.

The second tab is used to specify consortium sources. These sources contain previously classified variants and are used to determine if a variant has been previously classified as pathogenic or benign. The third tab is used to specify population frequency sources. These sources provide the variant-level allele frequencies used for recommending the population-based criteria such as BA1, BS1, and PM2. The final tab is used to specify control sources. These sources are assumed to contain only healthy adult individuals and are used to recommend the BS2 criterion.

Evaluation Options

The Evaluation Options can be accessed from the top right corner of the VSClinical starting page. These options allow you to configure the settings used by each new evaluation. These settings are organized into four tabs:

  • Report Sections: Used to to specify the various variant and coverage region record sets.

  • General: Used to specify the preferred HGVS representation for each variant’s protein description and the classification system for germline variants.

  • Gene List/Target Panel: Used to specify the name of the gene panel and a list of genes in the target panel used for filtering and reporting for the current test.

  • Gene Thresholds: Used to specify allele count and frequency thresholds

For more details on Evaluation Options, see the Evaluation Options section of the ACMG Guidelines documentation.

Source Versions

The annotation source versions used by VSClinical can be configured by clicking Source Versions in the upper right corner of the VSClinical starting page. This dialog allows you to select the currently used version of each annotation source by clicking on the source version in the rightmost column.

For more details on configuring Source Versions, see the Source Versions section of the ACMG Guidelines documentation.

Evaluation Tab

The Evaluation Tab is the first tab you will see when opening a new AMP Guideline evaluation. This tab is broken into seven sections. The first section is the Evaluation Summary. This section shows sample information, report information, data sources, and a summary of variants and biomarkers. At the bottom of this section are three buttons which can be used to finalize, delete, or close the current evaluation.

Evaluation Summary

The second section is used to edit sample and patient information. This includes samples details such as the record number and tumor purity, patient details such as the ordering physician and facility, along with information such as the patient name and sex.

The next section is titled Tumor Type. The tumor type can be selected by first narrowing down the tissue type on the left side of the selection menu, by searching for the tumor type directly in the Search Tumor Type search bar, or by using the tumor type acronym (e.g. NSCLC for Non-Small Cell Lung Cancer). It is important to select the tumor type before adding biomarkers, as the biomarker analysis will use this information to collect relevant data. The right side of the screen also has a quick access list of common tumor types to select.

Tumor Type

The third section is used to manage somatic variants within the current evaluation. This section displays variants to be evaluated in a table, which includes summary information about each variant. Variants can be added to the evaluation by clicking Manually Enter Variants or Add Variants From Project. For additional details on these dialogs, refer to the Variants to Evaluate section of the ACMG Guidelines documentation.

The next section displays biomarkers and genes with significant wild-types. This section contains summary information for each biomarker including the current clinical evidence tier.

Biomarkers and Genes with Significant Wild-Types

The final sections consist of the NGS Sequencing Summary and the Changelog History. Details on these sections can be found in the NGS Sequencing Summary section of the ACMG Guidelines documentation.

Genes Tab

The Genes Tab displays information about target-level coverage and reported genes. This tab requires the Coverage Statistics algorithm, which can be added to a VarSeq project by clicking Add -> Secondary Tables -> Coverage Region Annotation, provided that the samples in your project have associated BAM files. This tab includes three sections:

  • NGS Coverage Summary

  • Reported Genes

  • Reported Failed Targets and Hotspots

A detailed description of these sections can be found in the Genes Tab section of the ACMG Guidelines documentation.

Variants Tab

The Variants Tab uses VSClinical’s oncogenicity scoring system to assess whether a variant is likely to be a driver mutation. This system relies on an additive oncogenicity score, where scores exceeding ‘3’ indicate an ‘oncogenic’ or ‘likely oncogenic’ effect. While most of the individual criteria used to evaluate a variant’s oncogenicity are based directly on criteria specified in the ACMG Guidelines, there are several criteria that are specific to somatic variant interpretation.

Scoring and Evidence Summary

This section shows a brief summary of the variant under evaluation along with details about the variant’s position, origin, and allele frequency. The right sidebar can be used to switch the evaluation to other variants as well as see summary information about the current variant. Each variant is represented with a box with the variant names available on hover. The color of the outline and the fill-in color correlate to the classification of each variant. As you answer criteria questions, the right bar will update to show the current oncogenicity score for the variant, along with the applied criteria.

Scoring and Evidence Summary

Oncogenicity Scoring Recommendations

This section displays a list of the currently applied criteria along with the current classification based on these criteria. On the right side of this section is a plot showing the current oncogenicity score for the variant. The codes for the recommended criteria are shown in colored boxes with arrow links to navigate to the section containing the criteria questions and supporting evidence.

Oncogenicity Scoring Recommendations

Variant Interpretation for Sample

The Variant Interpretation for Sample section is used to enter the variant interpretation along with other relevant details including:

  • Report Section (Primary Findings, Secondary Findings, or Uncertain Significance)

  • Variant Set

  • Classification (Pathogenic, Likely Pathogenic, Uncertain Significance, Benign, or Likely Benign)

  • Tumor Type

  • Interpretation Notes

  • Flags or Comments

A more detailed description of this section can be found in the Variant Interpretation for Sample section of the ACMG Guidelines documentation.

Somatic Catalogs

The Somatic Catalogs section asks how frequently the variant appears in COSMIC. Variants appearing in at least one sample in COSMIC are assigned between 1 and 3 points, based on the number of samples containing the variant. The card on the right of this section shows the tumor type frequency for different catalogs like COSMIC and ICGC.

Somatic Catalogs

Germline Population Catalogs

This section is similar to the Population Frequency section of the ACMG Guidelines. It displays population frequency information from gnomAD and 1kG, and assigns negative points for variants that occur frequently in the general populations.

Relevant Clinical Assessments

This section displays previously classified variants in the same codon as the variant of interest. Positive points are applied if the codon contains a previously classified pathogenic variant, while negative points are applied if the variant has been previously classified as benign.

Relevant Clinical Assessments

Gene and Transcript

This section is similar to the Gene and Transcript section of the ACMG Guidelines. It displays details about the gene, including the NCBI gene description and the variant’s predicted effect on the protein. The Gene Impact card on the right side of this section presents a summary of the variant’s impact on the gene. This includes the overlapping exon number and the predicted effect along with the HGVS protein and coding change notation.

Nearby Pathogenic

This section displays a table of all previously classified nearby variants along with details about each variant. The variant table can be filtered by type (Missense or LoF), genomic region (Gene, Exon, or Codon), classification (Benign or Pathogenic), and star rating. This section also shows gene-level counts for each classification across variant types. The card for criteria NP+1 is also shown in this section, which awards one point for missense variants in a region containing multiple pathogenic variants but no nearby benign variants.

Nearby Pathogenic

Hotspots and Active Binding Sites

This section shows nearby cancer hotspots and active binding sites, along with a plot showing the variant’s coding change in the context of the surrounding region. Criteria HR+1 and AR+1 are recommended in this section if the variant occurs in a cancer hotspot or active binding site respectively.

Hotspots and Active Binding Sites

Computational Evidence

This section displays all computational evidence associated with the variant and contains the In-silico Predictions (IP) and Splice Predictions (SP) criteria cards. The information shown in this section includes the results of the functional prediction algorithms SIFT and PolyPhen2, along with the results for the splice prediction algorithms. This section is similar to the Computational Evidence section of the ACMG Guidelines.

Biomarkers Tab

The Biomarkers Tab is used to write interpretations and evaluate available clinical evidence related to the current gene and biomarker. The top of this tab displays information about the current gene, including links to external gene resources and descriptions about the gene as it relates to the hallmarks of cancer. The sidebar to the right of this tab provides an overview of the current biomarkers and associated clinical evidence. The top of the sidebar contains small cards for the biomarkers in the evaluation which can be used to change the currently selected biomarker. As the you scroll down the body of the tab, the sections become more specific, moving from more general gene level information to data about the specific biomarker of interest.

Biomarkers Tab

The Gene Summary section is used to write the Gene Interpretation and displays data about the current gene from various databases such as COSMIC and CIViC. The descriptions in this section can be expanded and copied using the plus signs at the end of each entry and added to the Interpretation on the left. Note that the Gene interpretation is often not specific to a given tumor type and will be re-used for all biomarkers in this gene.

Gene Summary

The Alteration Frequency and Outcomes section is used to analyze the gene in terms of the related tissues and tumor types and describes the frequency of mutation and prognostic outcomes related to biomarkers in this gene. Note that this section’s interpretation is specific to tumor type and will only be applied to samples with the same tumor type. The drop-down menu at the top of this section allows users to switch to a more broad tumor type if the selected one is too specific for the interpretation being written. Once changes are made to any of these interpretation sections, it is important to select the Review & Save Now button to compare and save any changes.

Alteration Frequency and Outcomes

The Biomarker Summary section is used to enter an interpretation for the biomarker as it relates to the specific tumor type. The scope can be changed at this point if the clinical interpretation is not specific to the exact variant but is instead associated with a class of variants in a region or for the entire gene. For instance, a loss-of-function variant in a tumor supressor gene should be interpreted at the broadest scope of “Gene LoF” so future variants in this gene that are loss-of-function share the same interpretation. On the other hand, missense variants generally have specific clinical evidence and are typically interpreted in scope of the specific codon change. This section also includes the Variant Classification and Details card, which presents various sources of information about the current biomarker to assist in writing the biomarker interpretation. If there are entries for this variant or other related variants in CancerKB or your own interpretation catalog, they will be displayed in the Related Interpretations cards.

CancerKB is a manually curated dataset containing assessments of biomarkers and genes in the context of specific cancers, including information on the Gene, Biomarker and available treatments. This catalog is built by an expert panel of curators and professionals in the clinical context that aggregate and write up interpretations for the most commonly seen biomarkers and genes. Interpretations provided by CancerKB are a great starting point, and as you save your own lab interpretations, your internal knowledgebase will grow to cover more and more of the biomarkers seen in each new sample. Additionally, users of the AMP feature can choose to share their interpretations back to the GHI curators anonymously. CancerKB will be updated on a regular basis to serve as an ever-growing cancer resource.

Biomarker Summary

The final section in the biomarker tab is the Clinical Evidence section. This section includes tables displaying information about Drug Sensitivity, Drug Resistance, Prognostic Data, and Diagnostic Data. These tables can be sorted by drug name or level of evidence and can be filtered by tissue type, region, and evidence type.

Clinical Evidence

This information is used to write the clinical evidence interpretation for the final report. This process includes constructing a list of relevant treatments and specifying the clinical evidence tier for the biomarker. The clinical evidence card on the right contains information on relevant clinical sources, including DrugBank, PMKB and CIViC. These are sorted by the strength of their clinical assertions.

Clinical Trials Tab

The Clinical Trials Tab provides a simple and efficient means to search for relevant clinical trials associated with the reported biomarkers. At the top of this tab, the user may specify the country and zip code over which to search for clinical trials.

Clinical Trials Tab

The Therapeutic Options section is used to search for nearby clinical trials associated with reported therapies. To search for clinical trials, click on the Matching Trials button to the right of the drug of interest. This will open the clinical trials search window, where you can search for clinical trials based on disease and location. The checkbox next to the clinical trial ID can be used to add the clinical trial to the report.

Clinical Trials Search

The Selected Clinical Trials section at the bottom of this tab is used edit the summary and relevant inclusion criteria for each selected clinical trial. The card on the right side of this section displays information about the currently selected clinical trial and is broken into four tabs:

  • Summary/Diseases: Displays summary information about the clinical trial.

  • Description: Contains a detailed description of the clinical trial.

  • Inclusion Criteria: Displays a list of all inclusion criteria. Each entry in the list can be added to the relevant inclusion criteria by clicking the blue button next to each criterion.

  • Sites: Shows a list of all clinical trials sites.

Selected Clinical Trials

Report Tab

The report tab is used to edit report details, generate reports, and finalize the evaluation. It shows all reportable fields and is organized into the following sections:

  • Report: Includes the result summary, along with patient and sample information.

  • Biomarker Results: Includes information on reported biomarkers.

  • Drugs: Includes information on reported drugs.

  • Clinical Trials: Includes information on selected clinical trials.

  • Secondary Germline Findings: Includes information on any reported germline mutations.

  • Variants of Unknown Significance: Includes information on variants of uncertain significance.

  • NGS Coverage Report: Includes information on coverage statistics, including any failed target or hotspot regions.

  • Inline References: Includes all inline PubMed and clinical trial references with hyperlinks to the source data.

When you click on a group of fields in the report data display, a form will be shown on the right side of the tab, allowing you to edit any of the fields in the selected group. When the report is ready to be finalized, select the green Sign Out & Finalize button. The report can be exported in Microsoft Word or PDF format by clicking on either the Microsoft Word or PDF icon on the right side of the report tab. The first time you select the Word file item, you will need to create a new Report Template. The software comes shipped with two System Templates that can serve as a starting point for creating custom reports: * Cancer Report Template: This template is the standard cancer report template and includes details on all reported biomarkers, including secondary germline findings. * Cancer Drugs and Trials Template: This template is a modification of the Cancer Report Template which includes information about FDA approved drugs and reported clinical trials. See Customizing VSClinical Reports for more information on how to configure custom reports to match your needs. For more details on report generation in VSClinical, please see the Report Tab section of the ACMG Guidelines documentation.

References

  1. Richards, Sue, et al. “Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.” Genetics in Medicine 17.5 (2015): 405-423.

  2. Abou Tayoun, Ahmad N., et al. “Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion.” Human Mutation 39.11 (2018): 1517-1524.

  3. Ellard, Sian, et al. “ACGS best practice guidelines for variant classification 2017.” ACGS Guidelines (2017).

  4. Li, Marilyn M., et al. “Standards and guidelines for the interpretation and reporting of sequence variants in cancer: a joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists.” The Journal of Molecular Diagnostics 19.1 (2017): 4-23.