Precision molecular diagnosis and treatment of vascular anomalies via ultra-deep genomic sequencing of affected tissue and a multidisciplinary care model
Clinical Genetics and Therapeutics
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Primary Categories:
- Clinical- Pediatric
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Secondary Categories:
- Clinical- Pediatric
Introduction:
Several molecular mechanisms have been identified in vascular anomalies (VAs); however, a precision diagnosis remains elusive for many patients. Multidisciplinary clinics are not standard of care for VAs nor has the acquisition of VA tissue for genetic testing historically been routine in clinical practice. Moreover, typical molecular diagnostic methods may not reliably detect the low-level somatic mosaicism relevant for many VAs. This investigation demonstrates that ultra-deep sequencing of VA affected tissue or other appropriate sample types increases the yield of precision molecular diagnosis in a large cohort of patients from a multidisciplinary clinic and enables individualized treatment in some cases.
Methods:
The VA clinic triaged pediatric and adult patients based on their individual condition, severity and treatment options and developed a coordinated care plan for each patient’s visit. Patients who were surgical candidates underwent retrieval of cyst fluid or affected tissue via core biopsy, surgical debulking, or other excisional procedure as guided by interventional radiology and/or surgery. For patients without invasive procedures, biopsy of affected skin, stored pathological specimens or cfDNA from blood was collected. Ultra-deep sequencing was performed at a partner laboratory. This involved next generation sequencing of up to 47 genes known to be associated with the pathogenesis of VAs at an average coverage of >1,000x.
Results:
To date, 56 patients in our multidisciplinary VA clinic have undergone sample acquisition and have results from ultra-deep sequencing. Forty-three of these patients had positive findings (diagnostic yield = 76.8% or 43/56 patients), eight of which had previously normal germline genetic testing. A patient with a central conducting lymphatic anomaly, had a paternally inherited variant of uncertain significance (VUS) in EPHB4, which is suspicious for the patient’s phenotype and their father’s history of venous insufficiency. RNA sequencing is pending to further characterize the EPHB4 VUS. The most identified causative genes in our cohort were PIK3CA, TEK and PIK3R1 with a range of variant allele fractions (VAF) of 2.0%-35.5%, 2.0%-21.0% and 2.0%-4.0%, respectively. The most common PIK3CA variant identified in our population was c.3140A>G (p.His1047Arg), which is also frequent in cancers. This variant was identified in 5 patients with the following phenotypes: large macrocystic lymphatic malformation, mediastinal venolymphatic malformation, clinical diagnosis of Klippel-Trenaunay syndrome, clinical diagnosis of congenital lipomatous overgrowth with vascular anomalies, epidermal nevi and scoliosis (CLOVES) syndrome, and venous malformation. The presence of this variant in these 5 patients demonstrates the phenotypic spectrum of vascular anomalies and the importance of a molecular diagnosis, which has further treatment implications. Patients with activating variants in PIK3CA may benefit from utilization of the PI3K inhibitor, alpelisib. A single patient was identified to harbor a RASA1 germline variant as well as a somatic RASA1 variant at 3.0% VAF, which is consistent with the second-hit hypothesis. In our patient population, the diagnostic yield for testing was lower for patients with primarily lymphatic malformations (11% or 1/9) versus those who had venolymphatic malformations or other vascular malformations (89.3% or 42/47).
Conclusion:
Ultra-deep sequencing of VA affected tissue and other appropriate sample types increases precision molecular diagnosis of VAs and provides opportunities for targeted therapeutics. Importantly, in this cohort we found 8 positive results in patients who were previously negative by standard NGS sequencing. Additional investigation is needed to understand molecular mechanisms involved in patients with primarily lymphatic malformations given the low yield in our patient population. These results emphasize that the combination of ultra-deep sequencing with multiple sample procurement methods and sample types is effective in establishing a molecular diagnosis for patients with VAs. Further, utilization of a multidisciplinary approach assists in achieving an individualized care plan for each patient.
Several molecular mechanisms have been identified in vascular anomalies (VAs); however, a precision diagnosis remains elusive for many patients. Multidisciplinary clinics are not standard of care for VAs nor has the acquisition of VA tissue for genetic testing historically been routine in clinical practice. Moreover, typical molecular diagnostic methods may not reliably detect the low-level somatic mosaicism relevant for many VAs. This investigation demonstrates that ultra-deep sequencing of VA affected tissue or other appropriate sample types increases the yield of precision molecular diagnosis in a large cohort of patients from a multidisciplinary clinic and enables individualized treatment in some cases.
Methods:
The VA clinic triaged pediatric and adult patients based on their individual condition, severity and treatment options and developed a coordinated care plan for each patient’s visit. Patients who were surgical candidates underwent retrieval of cyst fluid or affected tissue via core biopsy, surgical debulking, or other excisional procedure as guided by interventional radiology and/or surgery. For patients without invasive procedures, biopsy of affected skin, stored pathological specimens or cfDNA from blood was collected. Ultra-deep sequencing was performed at a partner laboratory. This involved next generation sequencing of up to 47 genes known to be associated with the pathogenesis of VAs at an average coverage of >1,000x.
Results:
To date, 56 patients in our multidisciplinary VA clinic have undergone sample acquisition and have results from ultra-deep sequencing. Forty-three of these patients had positive findings (diagnostic yield = 76.8% or 43/56 patients), eight of which had previously normal germline genetic testing. A patient with a central conducting lymphatic anomaly, had a paternally inherited variant of uncertain significance (VUS) in EPHB4, which is suspicious for the patient’s phenotype and their father’s history of venous insufficiency. RNA sequencing is pending to further characterize the EPHB4 VUS. The most identified causative genes in our cohort were PIK3CA, TEK and PIK3R1 with a range of variant allele fractions (VAF) of 2.0%-35.5%, 2.0%-21.0% and 2.0%-4.0%, respectively. The most common PIK3CA variant identified in our population was c.3140A>G (p.His1047Arg), which is also frequent in cancers. This variant was identified in 5 patients with the following phenotypes: large macrocystic lymphatic malformation, mediastinal venolymphatic malformation, clinical diagnosis of Klippel-Trenaunay syndrome, clinical diagnosis of congenital lipomatous overgrowth with vascular anomalies, epidermal nevi and scoliosis (CLOVES) syndrome, and venous malformation. The presence of this variant in these 5 patients demonstrates the phenotypic spectrum of vascular anomalies and the importance of a molecular diagnosis, which has further treatment implications. Patients with activating variants in PIK3CA may benefit from utilization of the PI3K inhibitor, alpelisib. A single patient was identified to harbor a RASA1 germline variant as well as a somatic RASA1 variant at 3.0% VAF, which is consistent with the second-hit hypothesis. In our patient population, the diagnostic yield for testing was lower for patients with primarily lymphatic malformations (11% or 1/9) versus those who had venolymphatic malformations or other vascular malformations (89.3% or 42/47).
Conclusion:
Ultra-deep sequencing of VA affected tissue and other appropriate sample types increases precision molecular diagnosis of VAs and provides opportunities for targeted therapeutics. Importantly, in this cohort we found 8 positive results in patients who were previously negative by standard NGS sequencing. Additional investigation is needed to understand molecular mechanisms involved in patients with primarily lymphatic malformations given the low yield in our patient population. These results emphasize that the combination of ultra-deep sequencing with multiple sample procurement methods and sample types is effective in establishing a molecular diagnosis for patients with VAs. Further, utilization of a multidisciplinary approach assists in achieving an individualized care plan for each patient.