Validation of a next-generation sequencing oncology panel optimized for low input DNA.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p < 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

Histologic and Molecular Profile of Pediatric Insulinomas: Evidence of a Paternal Parent-of-Origin Effect.

CONTEXT: Acquired insulinomas are rare causes of hyperinsulinemic hypoglycemia in children and are much less common than focal lesions of congenital hyperinsulinism. The latter are known to be associated with isodisomy for paternally transmitted ATP-sensitive potassium channel mutations on 11p15; however, the molecular basis for pediatric insulinomas is not well characterized. OBJECTIVE: The purpose of this study was to characterize the histopathological and molecular defects in a large group of 12 pediatric insulinomas seen at The Children's Hospital of Philadelphia. RESULTS: Twelve children with insulinomas were seen between 1971 and 2013, compared to 201 cases with focal congenital hyperinsulinism seen between 1997 and 2014. The age of insulinoma patients ranged from 4-16 years at the time of surgery. Features of MEN1 syndrome were present in five of the 12, including four cases with heterozygous mutations of MEN1 on 11q. Immunohistochemical analysis revealed nuclear loss of p57 staining consistent with loss of the maternal 11p15 allele in 11 of the 12 insulinomas, including all five MEN1-associated tumors. Imbalance of the paternal 11p allele was confirmed by single nucleotide polymorphism genotyping and methylation assays of the 11p imprinting control loci in four of five MEN1-associated tumors and six of seven sporadic insulinomas. In addition, single nucleotide polymorphism genotyping revealed extensive tumor aneuploidy beyond chromosome 11. CONCLUSIONS: These data indicate that MEN1 mutations are more common in insulinomas in children than in adults. Aneuploidy of chromosome 11 and other chromosomes is common in both MEN1 and non-MEN1 insulinomas. The novel observation of a paternal parent-of-origin effect in all MEN1 and most non-MEN1 tumors suggests a critical role for imprinted growth-regulatory genes in the 11p region in the genesis of ß-cell endocrine tumors in children.

Dominant form of congenital hyperinsulinism maps to HK1 region on 10q.

BACKGROUND/AIMS: In a family with congenital hyperinsulinism (HI), first described in the 1950s by McQuarrie, we examined the genetic locus and clinical phenotype of a novel form of dominant HI. METHODS: We surveyed 25 affected individuals, 7 of whom participated in tests of insulin dysregulation (24-hour fasting, oral glucose and protein tolerance tests). To identify the disease locus and potential disease-associated mutations we performed linkage analysis, whole transcriptome sequencing, whole genome sequencing, gene capture, and next generation sequencing. RESULTS: Most affecteds were diagnosed with HI before age one and 40% presented with a seizure. All affecteds responded well to diazoxide. Affecteds failed to adequately suppress insulin secretion following oral glucose tolerance test or prolonged fasting; none had protein-sensitive hypoglycemia. Linkage analysis mapped the HI locus to Chr10q21-22, a region containing 48 genes. Three novel noncoding variants were found in hexokinase 1 (HK1) and one missense variant in the coding region of DNA2. CONCLUSION: Dominant, diazoxide-responsive HI in this family maps to a novel locus on Chr10q21-22. HK1 is the more attractive disease gene candidate since a mutation interfering with the normal suppression of HK1 expression in beta-cells could readily explain the hypoglycemia phenotype of this pedigree.

An unusual presentation of obstructive sleep apnoeas.

We report the case of a 69-year-old married man who presented with features of irritability, characterised by outbursts of anger, short-term memory deficits and clumsiness, which progressed over a period of some 20 years. A detailed review elicited motor and verbal agitation during sleep, a history that was only available from his wife. He had excessive daytime sleepiness. A parasomnia in association with his possible neurological disorder was suspected and a referral made to the sleep disorders clinic. Further investigation with polysomnography determined that the abnormal behaviours during the night were secondary to arousals caused by obstructive sleep apnoea. Treatment with continuous positive airways pressure therapy prevented the abnormal behaviours at night, improved his daytime sleepiness but also led to improvements in his clumsiness, short-term memory and temper, all corroborated by his wife.