Table 3.

Technical recommendations for genetic testing of somatic variants

Technical aspect	Recommendation of the VASCERN-VASCA expert group
Tissue sampling	4 mm of affected tissue specimen from debulking/reduction surgery, or skin biopsy of affected tissue containing abnormal vasculature is the minimum tissue size recommended for adequate DNA yield Fresh/frozen and FFPE samples can be considered Blood samples are generally not recommended, as the diagnostic yield is low. Cell-free DNA from blood plasma or lymphatic fluid are a promising alternative source, but need further evaluation
Histologic examination	Recommended for differential diagnostics and/or estimation of the representativeness of the sample. Dissection (removal of epidermis) can be performed to increase the percentage of affected tissue
Tissue culturing	Tissue culturing is not necessary, but culturing endothelial cells from affected tissue may aid subsequent variant detection
DNA extraction	Standard DNA extraction protocols
DNA quality	Low-quality samples can be analysed; however, it reduces sensitivity and may introduce sequencing artefacts. Cost-effectiveness of prior DNA quality control must be determined in each laboratory
Gene-disease validity	Core gene list (Table 1) and updates thereof https://vascern.eu/
Expected VAFs	Mosaic variants can be present at multiple frequencies, expected VAFs are mostly lower than 10%
Target region	Minimum required regions that must be covered for the detection of somatic variants associated with vascular malformation are reported in Table 2. Recommended optional regions are also listed in Table S2
Sequencing analysis by NGS	Various platforms can be used UMI-based sequencing is recommended especially for amplicon-based, but also for hybridisation-based capture technologies. For the latter, if UMIs are not used, read deduplication is recommended, which can be based on read start and end coordinates; Strand-specific amplification or capture can help to discriminate genuine C:G > T > A variants from deamination artefacts in FFPE samples
Technical sensitivity and specificity	Sequencing depth and minimum number of variant reads should be empirically defined. Detection threshold of 1% is recommended. Including common SNPs in the library preparation is advised to detect cross-contamination from tumour samples when handled in parallel
Read alignment, variant detection, and variant filtering	Diagnostically validated pipelines for somatic calling are recommended. Prefiltering is advised to remove benign variants and artefacts
Validation of NGS findings	Recommended for all variants with VAF less than 5% using dPCR or a second NGS analysis
Alternative methods of somatic variant detection	dPCR can be used for the detection of recurrent variants associated with specific phenotypes
Variant classification	ACMG classification with modifications suggested in PMID: 35,997,716 and PMID: 34,040,190 are recommended. Literature/database searches should extend beyond cancer variant data annotation and interpretation
Reporting	AMP/ACMG/ASCO recommendations are largely applicable (PMID: 27,993,330). Variants should be annotated using the HGVS standard nomenclature. Reports should provide VAF, coverage, regions that do not meet quality standards, description of methods, limitations and list of genes analysed. Pathogenic/likely pathogenic variants should be disclosed. Recommendations for supplemental testing should be made, such as germline panel testing, based on the indication and clinical context
Identification of potential germline variants	Potential pathogenic/likely pathogenic germline variants should be included in the report and referral for germline testing and genetic counselling should be advised
Data sharing	Submission of variants with phenotypic, clinical significance, and classification criteria to DNA variant databases, such as ClinVar and LOVD, is recommended to increase knowledge of the genetics of vascular anomalies