Abstract
Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype–phenotype associations and guide medical therapy in individuals with vascular anomalies.
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Data availability
De-identified next-generation sequencing data for participants are available through controlled access at dbGaP with accession number phs003197.v1.p1. Data will be made available for secondary research only after investigators have obtained approval from the NIH to use the requested data for a particular project. Source data are provided with this paper.
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Acknowledgements
We thank the patients and their families for participating in the research. We also thank the Comprehensive Vascular Anomaly Program for clinical care. The work was supported by a Children’s Hospital of Philadelphia Frontier Program Grant (D.M.A., H.H.), Children’s Hospital of Philadelphia K-Readiness Grant (S.E.S.), the National Center for Advancing Translational Sciences of the National Institutes of Health (NIH) under award number 5R21TR003331 (D.L.), a research grant from the Lymphatic Malformation Institute (D.L.), and the Eunice Kennedy Shriver National Institute of Child Health and Human Development under award number ZIA-HD009003-01 (S.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank A. Hoofring of NIH Medical Arts for his assistance with the preparation of Fig. 2c.
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Authors and Affiliations
Contributions
D.L., S.E.S. and H.H. conceived the study. D.L., S.E.S., A.J.B., F.W., M.E.M., C.S. designed the methodology. M.E.M., M.R.B., L.S.M., C.S., M.C.M. and S.M.P. performed functional investigations. D.L., S.E.S., M.E.M., L.S.M., L.F.S., A.S.S., G.K., E.P., A.J.B., A.D.B., L.B., M.Q., J.R.R., J.A.N., D.W.L., S.V., J.T., C.L.S., A.M.C., K.M.S., D.M.A., Y.D. and H.H. carried out formal analysis. D.L., S.E.S., M.E.M., L.S.M., L.F.S., A.S.S., A.J.B., E.P., A.D.B., M.Q., J.R.R., J.A.N., D.W.L., S.V., J.T., C.L.S., A.M.C., K.M.S., D.M.A., Y.D., H.H., L.T., F.W., J.D., R.P.S., S.N.C., C.H., C.K. and K.N. conducted investigation. D.L., S.E.S., M.E.M., L.S.M., L.F.S., A.S.S., E.P., A.J.B., M.Q., J.R.R., J.A.N., D.W.L., S.V., J.T., C.L.S., A.M.C., K.M.S., D.M.A., Y.D., H.H. and J.D. performed data curation. D.L., S.E.S., L.F.S., A.S.S., A.J.B., S.R., J.D., M.E.M. and C.S. provided visualization. B.N., T.S., N.O., A.T. and L.W. carried out project administration. D.L. and S.E.S. wrote the original draft. All authors performed reviewing and editing. D.L., S.E.S., M.C., D.M.A., Y.D. and H.H. provided supervision. D.L., S.E.S., D.M.A., Y.D. and H.H. acquired funding. D.M.A., Y.D. and H.H. contributed equally to this manuscript.
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Competing interests
H.H. and CHOP are equity holders in Nobias Therapeutics, which is developing MEK inhibitor therapy for complex lymphatic anomalies. D.M.A. is a consultant for Novartis Pharmaceuticals and Nobias. K.M.S. is a consultant for Novartis Pharmaceuticals, which makes Vijoice (alpelisib), a selective PI3Ka inhibitor. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Functional characterization of BRAF-F486S, RAF1-T145P, and KRAS-A146T.
Transduction of BRAF-F486S (a) or RAF1-T145P (c) significantly increased the level of p-ERKs and Trametinib treatment led to a significant reduction of p-ERKs. Three-dimensional lymphatic spheroid sprouting assay showed elevated sprouting activity in HDLECs expressing BRAF-F486S (b) or RAF1-T145P (d) compared to its WT as measured by both cumulative sprout length and number of sprouts. Trametinib treatment led to a significant reduction of both cumulative sprout length and number of sprouts. For a-d, three independent experiments were performed. P values were calculated using 2-sided t-tests and included in each panel (degree of freedom was included in the Source Data for Extended Data Fig. 1), and corrected for multiple testing using the FDR (Benjamini and Hochberg) method. Bar graphs in panels a and c represent mean fold change in the ratio of pERK to GAPDH, normalized to the untreated wild type. Error bars represent standard deviations. In the box and whisker plots in panels b and d, the line in the middle of the box represents the medians, tops and bottoms of the boxes represent the 25th and 75th quartiles respectively, and the whiskers extend to 1.5 times the interquartile range beyond the 25th and 75th quartiles. All the data points that are summarized by the boxplots are superimposed as dots on the plots; minima and maxima can be determined by the highest and lowest dots. e, e’, Expression of KRASWT had no impact on lymphatic vessel morphology in trunk. f, f’, Expression of KRASA146T resulted in lymphatic tissue expansion (asterisks) and dilation of the thoracic duct (dotted line). Red: Expression of transgene in trunk of zebrafish at 5dpf, Green: mrc1a:GFF labeling lymphatic vessels. g, Quantitation of WT EK larvae that were assayed for pericardial edema at 5 dpf. Injected embryos were screened for transgenic mCherry expression in endothelial cells prior to quantitation so that only transgenic expressing embryos were counted. The p-values in the graph were calculated via Fisher’s Exact tests (two-sided) between samples as indicated, followed by Bonferroni correction. Indicated n are the total number of larvae assayed per condition.
Supplementary information
Supplementary Table 1
Molecular findings for the cohort and validation.
Supplementary Table 2
Molecular findings in blood or saliva samples in patients who typically have germline variants.
Supplementary Table 3
Molecular findings from deep exome sequencing.
Supplementary Table 4
Molecular findings from UMI panel ultra-deep sequencing.
Supplementary Table 5
Molecular findings from FFPE samples.
Supplementary Table 6
Molecular findings from body fluid samples.
Supplementary Table 7
Molecular findings from cfDNA.
Supplementary Table 8
PIK3CA variants were identified in 45 patients with blood and lymphatic malformations.
Supplementary Table 9
BDA and qPCR oligonucleotide sequences.
Source data
Source Data Extended Data Fig. 1
Unprocessed western blots.
Source Data Extended Data Fig. 1
Statistical source data.
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Li, D., Sheppard, S.E., March, M.E. et al. Genomic profiling informs diagnoses and treatment in vascular anomalies. Nat Med 29, 1530–1539 (2023). https://doi.org/10.1038/s41591-023-02364-x
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DOI: https://doi.org/10.1038/s41591-023-02364-x
