Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms.

Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9-99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.

Randomized trial of DVD, telephone, and usual care for increasing mammography adherence.

The purpose of this study was to test an intervention to increase mammography screening in women 51-75 years of age who had not received a mammogram in the last 15 months. A total of 1681 women were randomized to (1) a mailed tailored interactive DVD, (2) a computer-tailored telephone counseling, or (3) usual care. Women with income below US$75,000 who were in the interactive DVD group had significantly more mammograms than women in usual care. Women with income above US$75,000 had significantly fewer mammograms than women with income less than US$75,000 regardless of group. Further investigation is needed to understand why women with income above US$75,000 did not show the same benefit of the intervention.

Microcystic Stromal Tumor of the Ovary: A Case Report of a Newly Described Ovarian Neoplasm With a ß-catenin (CTNNB1) G34E Mutation.

Microcystic stromal tumor of the ovary (MSTO) is an exceedingly rare, unusual, and recently described entity with unique genetic alterations that assist in its diagnosis. We describe the case of a 50-year-old woman who presented with a complex right ovarian mass. A hysterectomy with bilateral salpingo-oophorectomy was performed and revealed an ovarian mass consistent with MSTO by histomorphology and immunohistochemical studies. Tumor cells were immunohistochemically reactive for vimentin, CD10, ß-catenin, and Wilms tumor 1. In addition, we detected a missense mutation c.101 G>A, p.G34E in exon 3 of the ß-catenin (CTNNB1) gene, which leads to an amino acid substitution of glycine at codon 34 by glutamic acid. The utility of genetic testing of this tumor and additional reporting of alterations detected is needed to verify pathogenicity of variants detected, as well as their potential roles with prognosis, behavior, and therapeutic targets. The overall clinical course of MSTO appears to be nonaggressive, although the number of reported cases are limited thus far.

Unique cerebrovascular anomalies in Noonan syndrome with RAF1 mutation.

Noonan syndrome is a common autosomal dominant neurodevelopmental disorder caused by gain-of-function germline mutations affecting components of the Ras-MAPK pathway. The authors present the case of a 6-year-old male with Noonan syndrome, Chiari malformation type I, shunted benign external hydrocephalus in infancy, and unique cerebrovascular changes. A de novo heterozygous change in the RAF1 gene was identified. The patient underwent brain magnetic resonance imaging, computed tomography angiography, and magnetic resonance angiography to further clarify the nature of his abnormal brain vasculature. The authors compared his findings to the few cases of Noonan syndrome reported with cerebrovascular pathology.

Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection.

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).

Improving the limit of detection for Sanger sequencing: a comparison of methodologies for KRAS variant detection.

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).

Identification and characterization of a novel homozygous deletion in the alpha-N-acetylglucosaminidase gene in a patient with Sanfilippo type B syndrome (mucopolysaccharidosis IIIB).

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Over 100 different mutations in the NAGLU gene have been identified in Sanfilippo syndrome type B patients; however, no large deletions have been reported. Here we present the first case of a large homozygous intragenic NAGLU gene deletion identified in an affected child of consanguineous parents. Long range and multiplex PCR methods were used to characterize this deletion which encompasses exons 3 and 4 and is 1146 base pairs long. We propose that Alu element-mediated unequal homologous recombination between an Alu-Y in intron 2 and an Alu-Sx in intron 4 is the likely mechanism for this deletion, thereby contributing further insight into the molecular etiology of this disorder and providing additional evidence of its allelic heterogeneity.

Endothelial function of von Hippel-Lindau tumor suppressor gene: control of fibroblast growth factor receptor signaling.

von Hippel-Lindau (VHL) disease results from germline and somatic mutations in the VHL tumor suppressor gene and is characterized by highly vascularized tumors. VHL mutations lead to stabilization of hypoxia-inducible factor (HIF), which up-regulates proangiogenic factors such as vascular endothelial growth factor (VEGF). This pathway is therefore believed to underlie the hypervascular phenotypes of the VHL tumors. However, recent studies have identified novel VHL functions that are independent of the HIF-VEGF pathway. In addition, a potential role of VHL in the tumor microenvironment, which carries heterozygous VHL mutations in VHL patients, has been overlooked. Here, we report a novel HIF-independent VHL function in the endothelium. VHL knockdown in primary human microvascular endothelial cells caused defective turnover of surface fibroblast growth factor (FGF) receptor, increased extracellular signal-regulated kinase signaling, and ETS1 activation, leading to increased cell motility in response to FGF and three-dimensional cord formation in vitro. HIF-alpha knockdown in VHL loss-of-function endothelial cells does not impede their elevated in vitro angiogenic activity. Importantly, the elevated angiogenic response to FGF is recapitulated in Vhl-heterozygous mice. Thus, partial loss of function of VHL in endothelium may be a contributing factor in tumor angiogenesis through a HIF-VEGF-independent mechanism.

Cyr61/CCN1 and CTGF/CCN2 mediate the proangiogenic activity of VHL-mutant renal carcinoma cells.

The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia-inducible factor (HIF)-alpha subunits. Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the proangiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor upregulated in VHL-mutant cells, the efficacy of antiangiogenic therapy specifically targeting VEGF signaling remains modest. In this study, we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared with VHL wild-type cells, VHL-mutant RCC cells demonstrated a significantly increased proangiogenic activity, which correlated with increased secretion of cysteine-rich 61 (Cyr61)/cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed (CCN) 1, connective tissue growth factor (CTGF)/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNA interference knockdown experiments. Importantly, the proangiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2alpha function, the dominant HIF-alpha isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in RCC tissue samples showed that increased expression of these proteins correlates with the loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple proangiogenic factors that function in parallel pathways.