Germline and sporadic mTOR pathway mutations in low-grade oncocytic tumor of the kidney.

Low-grade oncocytic tumor (LOT) of the kidney is a recently described entity with poorly understood pathogenesis. Using next-generation sequencing (NGS) and complementary approaches, we provide insight into its biology. We describe 22 LOT corresponding to 7 patients presenting with a median age of 75 years (range 63-86 years) and male to female ratio 2:5. All 22 tumors demonstrated prototypical microscopic features. Tumors were well-circumscribed and solid. They were composed of sheets of tumor cells in compact nests. Tumor cells had eosinophilic cytoplasm, round to oval nuclei (without nuclear membrane irregularities), focal subtle perinuclear halos, and occasional binucleation. Sharply delineated edematous stromal islands were often observed. Tumor cells were positive for PAX8, negative for CD117, and exhibited diffuse and strong cytokeratin-7 expression. Six patients presented with pT1 tumors. At a median follow-up of 29 months, four patients were alive without recurrence (three patients had died from unrelated causes). All tumors were originally classified as chromophobe renal cell carcinoma, eosinophilic variant (chRCC-eo). While none of the patients presented with known syndromic features, one patient with multiple bilateral LOTs was subsequently found to have a likely pathogenic germline TSC1 mutation. Somatic, likely activating, mutations in MTOR and RHEB were identified in all other evaluable LOTs. As assessed by phospho-S6 and phospho-4E-BP1, mTOR complex 1 (mTORC1) was activated across all cases but to different extent. MTOR mutant LOT exhibited lower levels of mTORC1 activation, possibly related to mTORC1 dimerization and the preservation of a wild-type MTOR copy (retained chromosome 1). Supporting its distinction from related entities, gene expression analyses showed that LOT clustered separately from classic chRCC, chRCC-eo, and RO. In summary, converging mTORC1 pathway mutations, mTORC1 complex activation, and a distinctive gene expression signature along with characteristic phenotypic features support LOT designation as a distinct entity with both syndromic and non-syndromic cases associated with an indolent course.

Hb Alcorn County: A ß-Globin Variant [ß40(C6)Arg→Thr; HBB: c.122G>C (p.Arg41Thr)] with Increased Oxygen Affinity.

We describe Hb Alcorn County, a heterozygous hemoglobin (Hb) variant, in a 6-month-old Hispanic male and his mother. DNA sequencing demonstrated a mutation on the HBB gene [ß40(C6)Arg→Thr; HBB: c.122G>C (p.Arg41Thr)], predictive of a substitution of arginine to threonine at position 40 of the ß-globin protein. This amino acid substitution involves the α1ß2 contact and occurs at the same position as Hb Austin [ß40(C6)Arg→Ser; HBB: c.[123G>C or 123G>T] (p.Arg41Ser)] and Hb Athens-GA [ß40(C6)Arg→Lys; HBB: c.122G>A (p.Arg41Lys)], both of which show increased oxygen affinity.

Helicobacter pylori Clarithromycin Resistance and Treatment Failure Are Common in the USA.

BACKGROUND: Helicobacter pylori antibiotic resistance leads to frequent treatment failure. However, the current US prevalence of H. pylori clarithromycin resistance and treatment failure is unknown. AIMS: To determine the prevalence of clarithromycin-resistant H. pylori and its impact on treatment failure in the USA. METHODS: A multicenter, retrospective, cohort study for clarithromycin-resistant H. pylori was conducted over four academic medical centers in different geographic regions of the USA. Gastric biopsy material, residual from standard clinical pathologic examination, was examined for clarithromycin resistance by DNA sequencing of H. pylori 23S rRNA. RESULTS: One hundred and twenty-four cases of H. pylori gastritis were examined from medical centers in four different geographic regions of the USA. The overall prevalence of clarithromycin resistance was 32.3 % (range 23.1-45.8 %). There was no significant difference in the prevalence of clarithromycin resistance by study site, gender, age, or race/ethnicity. In a subset of 67 patients that had clinical follow-up data, the overall prevalence of clarithromycin resistance was 31.3 %. There was a 2.9-fold increase (p = 0.002) in treatment failure for cases with clarithromycin resistance (57.1 %) compared to wildtype H. pylori (19.6 %). CONCLUSIONS: H. pylori clarithromycin resistance in the USA exceeds the estimated 20 % prevalence compatible with successful empiric antibiotic therapy. This resistance resulted in a significant rate of treatment failure in all sites surveyed. Empiric therapy in the USA should be used with caution until there is better regional or local determination of H. pylori antibiotic resistance.

Characterization of the HBB: c.*233G > C Variant: No Evidence of a ß-Thalassemic Phenotype.

ß-Thalassemia (ß-thal) results from homozygous or compound heterozygous inheritance of ß-globin alleles that yield decreased or absent synthesis of the ß chain. Disease is frequently severe, requiring lifelong transfusion therapy. Heterozygosity for a ß-thal allele results in an asymptomatic carrier state with mild but characteristic hematological findings. More than 200 ß-globin alleles have been demonstrated to produce ß-thal. For populations with a high prevalence of ß-thal, screening for carrier status, genetic counseling and prenatal diagnosis are important components of efforts to both reduce disease incidence and provide early diagnosis and treatment. It is therefore important to define and characterize potential ß-thal alleles. We sought to further characterize the previously reported ß-thal allele, HBB: c.*233G > C. This variant is provisionally included in the HbVar database based on a study of Palestinians in the Gaza Strip with ß-thal disease or carrier status (known or suspected) where 4.2% of subjects were found to have HBB: c.*233G > C. In our patient population, we detected the HBB: c.*233G > C variant in 17.3% of individuals (17 heterozygotes, one homozygote) undergoing ß hemoglobin (Hb) gene sequencing at our laboratory over a 25-month period. Hematological parameters were analyzed to determine if these individuals demonstrated findings consistent with inheritance of a ß-thal allele. Individuals with the HBB: c.*233G > C variant did not demonstrate any abnormalities in hematological parameters characteristic of ß-thal carrier state (17 heterozygotes) or clinical evidence of disease (homozygote). Our data demonstrate no evidence for pathogenicity of the HBB: c.*233G > C variant but rather demonstrate that this variant is a common benign polymorphism.

Novel Helicobacter pylori sequencing test identifies high rate of clarithromycin resistance.

OBJECTIVES: Eradication therapy selection for Helicobacter pylori gastritis requires knowledge of the local resistance rate to clarithromycin. There is minimal population-based or regional data in the United States on pediatric clarithromycin resistance. Although commercial methods such as fluorescence in situ hybridization and DNA probe assays are available in Europe for the evaluation of H pylori 23S rRNA mutations associated with resistance, clinical testing for 23S rRNA in the United States is not widely available. This study examined a single pediatric institution's clarithromycin resistance rate by a DNA polymerase chain reaction/sequencing assay applied to archived gastric biopsy specimens. METHODS: From the period 2010 to 2012, 38 H pylori-infected gastric biopsies were examined from archived formalin-fixed paraffin-embedded (FFPE) material. The 23S rRNA gene of H pylori was polymerase chain reaction amplified and sequenced for the identification of point mutations that are associated with clarithromycin therapeutic resistance. RESULTS: By 23S rRNA gene sequencing, 50% (n=19) of the specimens contained H pylori with mutations significant for clarithromycin resistance. CONCLUSIONS: This study is consistent with other pediatric reports suggesting significant H pylori clarithromycin resistance in the United States. Furthermore, the method used in this study can be used by hospital-based clinical laboratories to assess local clarithromycin resistance from archived biopsy material.

Long-range PCR based sequencing of the highly homologous genes, SFTPA1 and SFTPA2.

In humans, Surfactant Protein A exists as two highly homologous genetic isoforms, SFTPA1 and SFTPA2. Mutations in these two genes are associated with idiopathic pulmonary fibrosis (IPF) and lung cancer. We have developed a Sanger DNA sequencing assay which utilizes long-range PCR to detect mutations in these two genes.

Development and validation of a quantitative real time PCR assay for BK virus.

Several studies have shown that BK viral load in plasma and urine are reliable markers for the detection of BK virus associated nephropathy (BKVAN) in renal transplant patients. We developed a quantitative real time PCR assay based on TaqMan technology for the measurement of BK viral load in plasma and urine. Considering the high similarity of the nucleotide sequence of the BK virus (BKV) with the JC virus (JCV), we designed this assay to specifically amplify BKV. We determined the viral DNA recovery rate on manual (QIAGEN's QIAamp DNA Blood Mini Kit) and automated (BioMerieux's NucliSENS EasyMAG) extraction methods. The comparison showed a higher viral DNA recovery rate on the automated extraction (61-76% in plasma and 52-65% in urine) as compared to the manual method (49-52% in plasma and 33-56% in urine). Quantitation of the viral load was performed using an external standard curve that was constructed with serial dilution of a plasmid containing the full length of the BKV genome. Commercially available quantitative BKV standards showed good correlation with the plasmid standard. The reproducibility of the assay was determined based on the Ct values of the amplified products as well as in BK copies per milliliter of sample. This assay is linear over a 7 log range (10 to 1 × 10(7) copies per reaction), no cross-reactivity was detected with the closest-related polyomavirus JCV, as well as other viruses that may be found in immunocompromised patients, and human genomic DNA. The limit of detection of the assay is 300 copies per milliliter in both plasma and urine and the limit of quantitation is 1000 copies per milliliter using the NATtrol BK Virus Linearity Panel (ZeptoMetrix). This real time PCR assay provides a reliable and sensitive method for the quantitation of BKV in plasma and urine samples.

TP53 codon 72 polymorphisms in favorable histology Wilms tumors.

In Wilms tumor (WT), mutations in the gene encoding p53, TP53, are correlated with anaplasia; however TP53 variants have not been studied in favorable histology (FH) WTs. A single nucleotide polymorphism of TP53 encoding either arginine or proline at codon 72 is suggested to alter in vitro p53 behavior. Therefore, we analyzed tissue from 23 consecutive patients with FHWT to determine allelic and genotypic frequencies of Pro72 and Arg72 variants and correlate this with clinical outcomes. Interestingly, our cohort showed a statistically significant over-representation of the Arg allele and Arg/Arg genotype. However, the genotypic and allelic frequencies showed no significant correlation with age, stage, or disease recurrence.

Cytoplasmic P53 Immunostaining With N-Terminus P53 Antibody And Absence Of Staining With C-Terminus P53 Antibody: A Report Of Two Pediatric Sarcomas With Distal Truncating TP53 Mutations Affecting Nuclear Localization Domain.

P53 immunohistochemical staining with antibodies targeted to epitopes at or near the N-terminus are commonly used in diagnostic pathology practice as a surrogate for TP53 mutations. The abnormal staining patterns indicating TP53 mutations include nuclear overexpression, null, and the recently described cytoplasmic staining. The latter staining pattern occurs with the less common TP53 mutations affecting its nuclear localization and/or tetramerization domains that are located toward the C-terminus. Here we describe the first two cases of pediatric sarcomas with cytoplasmic staining with P53 antibody against N-terminus epitope and the absence of staining with P53 antibody against C-terminus epitope. We propose that a more precise description of P53 immunohistochemical staining patterns should include the nature of the antibody used.

Papillary thyroid carcinoma shows elevated levels of 2-hydroxyglutarate.

Elevated levels of D: -2-hydroxyglutarate (D: -2-HG) occur in gliomas and myeloid leukemias associated with mutations of IDH1 and IDH2. L: -2-Hydroxyglutaric aciduria, an inherited metabolic disorder, predisposes to brain tumors. Therefore, we asked whether sporadic cancers, without IDH1 or IDH2 hot-spot mutations, show elevated 2-hydroxyglutarate levels. We retrieved 15 pairs of frozen papillary thyroid carcinoma (PTC) and adjacent non-neoplastic thyroid, and 14 pairs of hyperplastic nodule (HN) and adjacent non-hyperplastic thyroid. In all lesions, exon 4 sequencing confirmed the absence of known mutations of IDH1 and IDH2. We measured 2-hydroxyglutarate by liquid chromatography-tandem mass spectrometry. Compared to normal thyroid, PTCs had significantly higher D: -2-HG and L: -2-hydroxyglutarate (L: -2-HG) levels, and compared to HNs, PTCs had significantly higher D: -2-HG levels. D: -2-HG/L: -2-HG levels were not significantly different between HNs and normal thyroid. Further studies should clarify if elevated 2-hydroxyglutarate in PTC may be useful as cancer biomarker and evaluate the role of 2-hydroxyglutarate in cancer biology.

Isocitrate dehydrogenase 1/2 mutational analyses and 2-hydroxyglutarate measurements in Wilms tumors.

BACKGROUND: L-2-Hydroxyglutaric aciduria (L-2-HGA) is an uncommon inborn error of metabolism, in which the patients are predisposed to develop brain tumors. Elevated levels of D-2-hydroxyglutarate have been demonstrated with malignant gliomas and myeloid leukemias associated with somatic mutations of the genes encoding NADP(+)-dependent isocitrate dehydrogenases (IDH1 and IDH2, respectively). Recently, we noted a Wilms tumor in a child with L-2-HGA. Given the accumulating evidence that both enantiomers of 2-hydroxyglutarate are associated with cellular transformation, we investigated if sporadic Wilms tumors are associated with IDH1 or IDH2 mutations or with elevated levels of 2-hydroxyglutarate. PROCEDURE: We retrieved 21 frozen Wilms tumor tissues. In 20 cases, we sequenced exon 4 and flanking intronic regions of IDH1 and IDH2. In all 21 cases, we measured 2-hydroxyglutarate levels by liquid chromatography-tandem mass spectrometry. RESULTS: We did not find mutations at the hot spots IDH1 codon 132 or IDH2 codon 172. Two cases (1 with favorable histology and 1 with unfavorable histology) showed heterozygous change c.211G>A (p.Val71Ile) in IDH1, a change previously reported as a mutation but listed as a single nucleotide polymorphism in the NCBI SNP database. We did not find increased levels of 2-hydroxygluatric acid in any sample. CONCLUSIONS: Our results suggest that IDH1 codon 132 or IDH2 codon 172 mutations or elevated 2-hydroxyglutarate levels do not play a role in the biology of sporadic Wilms tumors. The significance of heterozygous change c.211G>A (p.Val71Ile) in IDH1, seen in two tumors, is not clear.

Evaluation of Maxwell® 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue.

BACKGROUND: The aim of the study was to assess the performance of Promega, Maxwell® 16 for the extraction of genomic DNA from whole blood and FFPE tissue. METHODS: DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits. RESULTS: For whole blood, the mean DNA concentration obtained by Maxwell® 16 was significantly greater than either easyMAG® (p<0.0001) or QIAamp® Blood DNA kit (p<0.001). For FFPE, the mean DNA concentration obtained by the AllPrep® FFPE specific DNA/RNA kit was significantly greater than either the Maxwell® 16 (p<0.0001) or the general AllPrep® DNA/RNA kit (p<0.0001). CONCLUSIONS: Comparative evaluation of the six DNA extraction kits indicated that the semi-automated Maxwell® 16 was superior for whole blood extraction while the manual AllPrep® FFPE DNA/RNA kit (Qiagen) performed better for FFPE DNA extraction in terms of quantity of DNA obtained. All six extraction methods (blood and FFPE) performed well in terms of purity. Although there were variances in the quantity of DNA obtained, there were no significant differences in the efficiency of these methods in yielding amplifiable DNA extracts, as demonstrated by ß-actin for whole blood specimens. In evaluation of FFPE DNA extraction methods, the Qiagen AllPrep® FFPE DNA/RNA Mini Kit was the best for applications requiring larger amplicons, but for smaller amplicons the Maxwell was most consistent.

Control Charting Genomic Data.

BACKGROUND: Control charting is routine in the quality assurance of traditional clinical laboratory testing. Genomic tests are not typically managed by control charting. We examined control charting to monitor the performance of a clinical next-generation sequencing (NGS) assay. METHODS: We retrospectively examined 3 years of control material (NA12878) data from clinical genomic epilepsy testing. Levey-Jennings plots were used to visualize changes in control material depth of sequencing coverage in genomic regions of an epilepsy genomic panel. Changes in depth of coverage were correlated with changes in the manufactured lot of capture probe reagent. Depth of coverage was also correlated between quality control material and clinical samples. RESULTS: Fifty-seven sequencing runs of NA12878 were analyzed for 1811 genomic regions targeting 108 genes. Manufactured probe lot changes were associated with significant changes in the average coverage of 537 genomic regions and the lowest coverage of 173 regions (using a critical cut-off of P < 5.52 x 10-6). Genomic regions with the highest sensitivity to lot-to-lot variation by average sequencing depth of coverage were not the same regions with the highest sensitivity by lowest sequencing depth of coverage. Levey-Jennings plots displayed differences in genomic depth of coverage across capture probe reagent lot changes. There was moderate correlation between the changes in depth of sequencing across lot changes for control material and clinical cases (r2 = 0.45). CONCLUSIONS: Genomic control charting can be used routinely by clinical laboratories to monitor assay performance and ensure the quality of testing.

Clinical Validation of a SARS-CoV-2 Real-Time Reverse Transcription PCR Assay Targeting the Nucleocapsid Gene.

BACKGROUND: Detection of SARS-CoV-2 viral RNA is important for the diagnosis and management of COVID-19. METHODS: We present a clinical validation of a reverse transcription PCR (RT-PCR) assay for the SARS-CoV-2 nucleocapsid (N1) gene. Off-board lysis on an automated nucleic acid extraction system was optimized with endemic coronaviruses (OC43 and NL63). Genomic RNA and SARS-CoV-2 RNA in a recombinant viral protein coat were used as control materials and compared for recovery from nucleic acid extraction. RESULTS: Nucleic acid extraction showed decreased recovery of endemic Coronavirus in vitro transcribed RNA (NL63) compared with attenuated virus (OC43). SARS-CoV-2 RNA had more reliable recovery from extraction through amplification than genomic RNA. Recovery of genomic RNA was improved by combining lysis buffer with clinical matrix before adding RNA. The RT-PCR assay demonstrated 100% in silico sensitivity and specificity. The accuracy across samples was 100% (75 of 75). Precision studies showed 100% intra-run, inter-run, and inter-technologist concordance. The limit of detection was 264 copies per milliliter (estimated 5 copies per reaction; 35.56 mean threshold cycle value). CONCLUSIONS: This SARS-CoV-2 assay demonstrates appropriate characteristics for use under an Emergency Use Authorization. Endemic coronavirus controls were useful in optimizing the extraction procedure. In the absence of live or attenuated virus, recombinant virus in a protein coat is an appropriate control specimen type for assay validation during a pandemic.

Comparison of automated nucleic acid extraction methods with manual extraction.

Automated nucleic acid extractors can improve workflow and decrease variability in the clinical laboratory. We evaluated Qiagen EZ1 (Valencia, CA) and bioMérieux (Durham, NC) easyMAG extractors compared with Qiagen manual extraction using targets and matrices commonly available in the clinical laboratory. Pooled samples were spiked with various organisms, serially diluted, and extracted in duplicate. The organisms/matrices were Bordetella pertussis/bronchoalveolar lavage, herpes simplex virus II/cerebrospinal fluid, coxsackievirus A9/cerebrospinal fluid, BK virus/plasma, and Mycoplasma pneumoniae/endotracheal tube samples. Extracts were amplified in duplicate using real-time PCR assays, and amplification of the target at a cycle threshold of 35 using the manual method was used for comparison. Amplification efficiency of nucleic acids extracted by automated methods was similar to that by the manual method except for a loss of efficiency for M. pneumoniae in endotracheal tube samples. The EZ1 viral kit 2.0 gave better results for coxsackievirus A9 than the EZ1 viral kit version 1.0. At the lowest limit of detection (past a cycle threshold of 35), the easyMAG was more likely to produce amplifiable nucleic acid than were either the EZ1 or manual extraction. Operational complexity, defined as the number of manipulations required to obtain an extracted sample, was the lowest for the easyMAG. The easyMAG was the most expensive of the methods, followed by the EZ1 kit and manual extraction.

A subset of cranial fasciitis is associated with dysregulation of the Wnt/beta-catenin pathway.

Cranial fasciitis, an unusual fibroproliferative lesion that occurs in the scalp of infants, is considered a posttraumatic reactive process similar to nodular fasciitis. Its pathobiology has not been investigated. Over the last 15 years, we diagnosed cranial fasciitis in six children; in one case, the lesion recurred after 4 years. This lesion and two others showed aberrant, diffuse nuclear reactivity for beta-catenin. One of the lesions with aberrant nuclear beta-catenin occurred in a child with a history of familial adenomatous polyposis (FAP) and a germline frameshift adenomatous polyposis coli (APC) mutation, c.878delG. The other APC allele in this tumor showed an acquired nonsense mutation, c.4132C --> T. Both these mutations lead to translation of a truncated APC protein. The other two cases of cranial fasciitis with aberrant nuclear beta-catenin occurred sporadically. One of these showed a point mutation, c.122C --> T, in exon 3 of CTNNB1. This mutation causes replacement of threonine with isoleucine at codon 41, leading to loss of a phosphorylation site in the beta-catenin protein. The third case with nuclear beta-catenin staining was the single one that showed recurrence. This tumor did not show mutations in exon 3 of CTNNB1 or in exons 8/9/16 of APC. The results of this small study indicate a dysregulation of the Wnt/beta-catenin pathway in a subset of cranial fasciitis, suggesting that this subset is pathobiologically related to desmoid fibromatoses rather than to nodular fasciitis. Occasional cases of cranial fasciitis may be associated with FAP and serve as an early indicator of this disease, information that would be important in the early diagnosis of FAP in patients without a family history of polyposis.

Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation.

Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.

ATM mutations on distinct SNP and STR haplotypes in ataxia-telangiectasia patients of differing ethnicities reveal ancestral founder effects.

Due to the large size (150 kb) of the ataxia-telangiectasia mutated (ATM) gene and the existence of over 400 mutations, identifying mutations in patients with ataxia-telangiectasia (A-T) is labor intensive. We compared the SNP and STR haplotypes of A-T patients from varying ethnicities who were carrying common ATM mutations. We used SSCP to determine SNP haplotypes. To our surprise, all of the most common ATM mutations in our large multiethnic cohort were associated with specific SNP haplotypes, whereas the STR haplotypes varied, suggesting that ATM mutations predated STR haplotypes but not SNP haplotypes. We conclude that these frequently observed ATM mutations are not hot spots, but have occurred only once and spread with time to different ethnic populations. More generally, a combination of SNP and STR haplotyping could be used as a screening strategy for identifying mutations in other large genes by first determining the ancestral SNP and STR haplotypes in order to identify specific founder mutations. We estimate this approach will identify approximately 30% of mutations in A-T patients across all ethnic groups.