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.

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.

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.

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.

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.

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.

ß-Globin gene sequencing of hemoglobin Austin revises the historically reported electrophoretic migration pattern.

CONTEXT: Hemoglobin (Hb) Austin was defined in 1977, using amino acid sequencing of samples from 3 unrelated Mexican-Americans, as a substitution of serine for arginine at position 40 of the ß-globin chain (Arg40Ser). Its electrophoretic migration on both cellulose acetate (pH 8.4) and citrate agar (pH 6.2) was reported between Hb F and Hb A, and this description persists in reference literature. OBJECTIVES.-To review the clinical features and redefine the diagnostic characteristics of Hb Austin. DESIGN: Eight samples from 6 unrelated individuals and 2 siblings, all with Hispanic surnames, were submitted for abnormal Hb identification between June 2010 and September 2011. High-performance liquid chromatography, isoelectric focusing (IEF), citrate agar electrophoresis, and bidirectional DNA sequencing of the entire ß-globin gene were performed. RESULTS: DNA sequencing confirmed all 8 individuals to be heterozygous for Hb Austin (Arg40Ser). Retention time on high-performance liquid chromatography and migration on citrate agar electrophoresis were consistent with that identification. Migration on IEF, however, was not between Hb F and Hb A, as predicted from the report of cellulose acetate electrophoresis. By IEF, Hb Austin migrated anodal to ("faster than") Hb A. CONCLUSIONS: Hemoglobin Austin (Arg40Ser) appears on IEF as a "fast," anodally migrating, Hb variant, just as would be expected from its amino acid substitution. The cited historic report is, at best, not applicable to IEF and is probably erroneous. Our observation of 8 cases in 16 months suggests that this variant may be relatively common in some Hispanic populations, making its recognition important. Furthermore, gene sequencing is proving itself a powerful and reliable tool for definitive identification of Hb variants.

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.

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.

Immunohistochemical expression of GLUT1 and its correlation with unfavorable histology and TP53 codon 72 polymorphism in Wilms tumors.

Reprogramming of energy metabolism, such as increased glycolysis, is a hallmark of cancer cells. One mechanism by which cancer cells fuel glycolysis is through increased uptake of glucose across cell membranes via the glucose transporter GLUT1. One of the transcriptional repressors of GLUT1 is wild-type TP53, and cancer-associated loss of function mutations within the DNA-binding domain of TP53 impairs the repressive effect of TP53 on transcriptional activity of the GLUT1 gene promoter. Because TP53 mutations are associated with unfavorable histology (diffuse anaplasia) in Wilms tumors, we hypothesized increased expression of GLUT1 in these tumors. To evaluate this hypothesis, we performed tissue microarray-based immunohistochemistry for GLUT1 in a set of 50 Wilms tumors, including 5 with unfavorable histology. In a subset of 16 favorable histology Wilms tumors, we compared the GLUT1 immunoexpression with TP53 codon 72 polymorphism status. We found consistently stronger immunoexpression of GLUT1 in unfavorable histology Wilms tumors compared to favorable histology Wilms tumors (P  =  0.04). We noted that the favorable histology Wilms tumors with a proline residue at position 72 of TP53 tended to have higher immunoexpression of GLUT1, although this immunoexpression did not reach statistical significance in this small set of cases. In summary, our finding of strong GLUT1 immunoexpression in unfavorable histology Wilms tumors indicates that these tumors are likely to be 2-deoxy-2-((18)F)fluoro-d-glucose avid and that GLUT1 should be evaluated as a therapeutic target for these tumors that otherwise show resistance to conventional therapy.

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.

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.

A lean laboratory: operational simplicity and cost effectiveness of the Luminex xTAG™ respiratory viral panel.

During certain months of the year, viral respiratory infections lead to a dramatic increase in pediatric emergency room visits and hospital admissions. Rapid identification of the infectious organism results in timely treatment and reductions in hospital cost and length of stay. Before the introduction of molecular testing to the virology laboratory, diagnosis relied on the standard methods of immunofluorescence and culture. These tests can be labor-intensive and costly. Recent studies have demonstrated the higher sensitivity, faster turnaround, and broader diagnostic spectrum provided by multiplexed RT-PCR assays. Data comparing the laboratory cost and labor efficiency of the tests are lacking. To address this issue, we chose to implement the principles of operational workflow analysis using lean methodology to critically evaluate the potential advantages of a multiplexed RT-PCR assay both in terms of workflow and cost effectiveness. Our results indicated that the implementation of the Luminex xTAG Respiratory Viral Panel (RVP) resulted in a standardized workflow with decreased requirements in laboratory cost as well as improvement in efficiency. In summary, we demonstrate that, in our laboratory, the Luminex xTAG RVP is more operationally streamlined and cost-effective than standard viral direct fluorescent antibody and culture. Further studies are needed to highlight additional benefits of the test, including shortened hospital stay and improved patient outcome.

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.

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.