In cancer medicine, next generation sequencing (NGS) has emerged as a practical method to generate patient- and tumor-specific genetic data for optimal selection of targeted therapies. Targeted ...sequencing allows clinical testing to focus on cancer-related genes, thus maximizing the test’s sensitivity and specificity for actionable variants. In this review, we summarize the current regulatory environment surrounding clinical NGS, including regulations and professional opinions established by the College of American Pathologists, the Centers for Disease Control and Prevention, the Clinical Laboratory Improvement Amendments, the Clinical and Laboratory Standards Institute, the Association for Molecular Pathology, the New York State Department of Health, and the American College of Medical Genetics. We outline practical considerations for the design of targeted NGS assays, with an emphasis on capture-based methods. Finally, we discuss components of the validation process for clinical NGS assays as well as challenges that still remain for clinical NGS.
IMPORTANCE: Patients with somatic overgrowth commonly require surgical intervention to preserve function and improve cosmesis. To our knowledge no observation of scarring outcomes in this population ...has been published to date. OBJECTIVE: To observe the frequency of abnormal scarring in patients with somatic overgrowth and sequencing-verified mutations in the PIK3CA gene. DESIGN, SETTING, AND PARTICIPANTS: This retrospective study evaluated scarring outcomes in patients with PIK3CA-related overgrowth. Samples of affected tissue were sequenced between July 2015 and October 2016. Medical records from multiple large academic tertiary care centers were reviewed for surgical history and scar descriptions, and clinical photographs were assessed by 2 surgeons (J.N.J. and D.M.K.) to confirm abnormal scarring. Analysis of medical records and photographs was performed between April 2017 and June 2017 by a multidisciplinary team from dermatology, plastic surgery, orthopedic surgery, radiology, and genetics departments. All patients considered for the study were diagnosed with somatic overgrowth and previously had affected tissue sent for next-generation sequencing. Those with pathogenic PIK3CA variants and 1 or more prior surgical procedures were reviewed. MAIN OUTCOMES AND MEASURES: Presence of excessive scarring in patients with PIK3CA overgrowth. RESULTS: A total of 57 patients with segmental overgrowth syndromes were sequenced. Of the 57 patients, 25 (44%) had pathogenic or likely pathogenic variants in PIK3CA. Of those with pathogenic PIK3CA variants, 6 (24%) had past surgical procedures, all with preoperative and postoperative photographs. Of 6 patients with PIK3CA-related overgrowth and a history of 1 or more surgical procedure, 4 (67%) developed excessive scarring. The cohort with abnormal scarring comprised 3 females and 1 male, with a median age of 8.5 years. All abnormal scarring occurred in affected overgrowth tissue. Three of the 4 patients developed the excessive scarring after debulking procedures for overgrowth and/or vascular malformations of the upper or lower extremity. CONCLUSIONS AND RELEVANCE: Excessive scarring occurred frequently in patients with PIK3CA-related overgrowth syndromes. The risk of abnormal scarring should therefore be discussed preoperatively. Given the activating nature of these PIK3CA variants, we suggest that the excessive scarring may be owing in part to up-regulation of the PI3K-Akt-mTOR pathway. Additional studies are needed to assess scarring outcomes in patients with other types of overgrowth.
Cerebral cavernous malformations (CCM) may present in sporadic or familial forms, with different cutaneous manifestations including deep blue nodules, capillary malformations, and hyperkeratotic ...cutaneous capillary venous malformations (HCCVM). We report the case of an infant with a KRIT1‐positive HCCVM associated with familial CCM. Moreover, histopathology showed positive immunohistochemical stain with GLUT1, further expanding the differential diagnosis of GLUT1‐positive vascular anomalies.
The 2007 World Health Organization Classification of Tumours of the Central Nervous System classifies lower-grade gliomas LGGs (grades II to III diffuse gliomas) morphologically as astrocytomas or ...oligodendrogliomas, and tumors with unclear ambiguous morphology as oligoastrocytomas. The World Health Organization's newly released (2016) classification incorporates molecular data. A single, targeted next-generation sequencing (NGS) panel was used for detecting single-nucleotide variation and copy number variation in 50 LGG cases originally classified using the 2007 criteria, including 36 oligoastrocytomas, 11 oligodendrogliomas, 2 astrocytomas, and 1 LGG not otherwise specified. NGS results were compared with those from IHC analysis and fluorescence in situ hybridization to assess concordance and to categorize the tumors according to the 2016 criteria. NGS results were concordant with those from IHC analysis in all cases. In 3 cases, NGS was superior to fluorescence in situ hybridization in distinguishing segmental chromosomal losses from whole-arm deletions. The NGS approach was effective in reclassifying 36 oligoastrocytomas as 30 astrocytomas (20 IDH1/2 mutant and 10 IDH1/2 wild type) and 6 oligodendrogliomas, and 1 oligodendroglioma as an astrocytoma (IDH1/2 mutant). Here we show that a single, targeted NGS assay can serve as the sole testing modality for categorizing LGG according to the World Health Organization's 2016 diagnostic scheme. This modality affords greater accuracy and efficiency while reducing specimen tissue requirements compared with multimodal approaches.
Closed spinal dysraphism (SD) is a type of neural tube defect originating during early embryonic development whereby the neural tissue of the spinal defect remains covered by skin, often coinciding ...with markers of cutaneous stigmata. It is hypothesized that these events are caused by multifactorial processes, including genetic and environmental causes. We present an infant with a unique congenital midline lesion associated with a closed SD. Through comprehensive molecular profiling of the intraspinal lesion and contiguous skin lesion, an internal tandem duplication (ITD) of the kinase domain of the fibroblast growth factor receptor 1 (
) gene was found. This ITD variant is somatic mosaic in nature as supported by a diminished variant allele frequency in the lesional tissue and by its absence in peripheral blood.
ITD results in constitutive activation of the receptor tyrosine kinase to promote cell growth, differentiation, and survival through RAS/MAPK signaling. Identification of
ITD outside of central nervous system tumors is exceedingly rare, and this report broadens the phenotypic spectrum of somatic mosaic
related disease.
Syringocystadenocarcinoma papilliferum (SCACP) is a rare malignant neoplasm arising from adnexal tissues and is the malignant complement to the benign neoplasm syringocystadenoma papilliferum (SCAP). ...SCACP lesions appear as raised nodules or inflammatory plaques and can be associated with SCAP or nevus sebaceous. There have been fewer than 100 described cases of this neoplasm in the literature, and all previously published cases have been described in adults, with the majority occurring in the elderly. We present a case of an adolescent female with a syringocystadenocarcinoma papilliferum arising from a large thigh mass harboring an in-frame alteration in MAP2K1 along with a brief review of the literature.
Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple ...complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case-control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.
Abstract
Retinoblastoma is an ocular cancer associated with genomic variation in the RB1 gene. In individuals with bilateral retinoblastoma, a germline variant in RB1 is identified in virtually all ...cases. We describe herein an individual with bilateral retinoblastoma for whom multiple clinical lab assays performed by outside commercial laboratories failed to identify a germline RB1 variant. Paired tumor/normal exome sequencing, long-read whole genome sequencing, and long-read isoform sequencing was performed on a translational research basis ultimately identified a germline likely de novo Long Interspersed Nuclear Element (LINE)-1 mediated deletion resulting in a premature stop of translation of RB1 as the underlying genetic cause of retinoblastoma in this individual. Based on these research findings, the LINE-1 mediated deletion was confirmed via Sanger sequencing in our clinical laboratory, and results were reported in the patient's medical record to allow for appropriate genetic counseling.
Alterations in the
gene have recently emerged as the cause of developmental delay with or without intellectual impairment or behavioral abnormalities (MIM # 619575). The 32 cases currently described ...in the literature have predominantly de novo alterations in
and a wide spectrum of neurodevelopmental abnormalities. Here, we report four patients with novel pathogenic
variants identified by research genome sequencing, clinical exome sequencing, and international matchmaking. The overlapping clinical features of our patients are consistent with the emerging core phenotype of
-associated syndrome: facial dysmorphism, feeding difficulties, global developmental delay, joint laxity, and hypotonia. However, behavioral abnormalities and gastrointestinal issues are more common in our cohort than previously reported. Two patients have de novo
variants (one missense, one splice site) consistent with most known alterations in this gene. However, we also report the first sibling pair who both inherited a
frameshift variant from a mildly affected mother. Our findings suggest that incomplete penetrance and variable expressivity are relatively common in
-associated syndrome, which holds important implications for clinical genetic testing.
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