Guideline-Adherent Clinical Validation of a Comprehensive 170-Gene DNA/RNA Panel for Determination of Small Variants, Copy Number Variations, Splice Variants, and Fusions on a Next-Generation Sequencing Platform in the CLIA Setting.

We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/µl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.

Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes.

T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3(+)CD8(+) cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P(5) is the predominant S1P receptor in leukemic LGLs, whereas S1P(1) is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Ischaemic injury to femoral head induces apoptotic and oncotic cell death.

AIMS: The mechanism of cell death in ischaemic osteonecrosis of the femoral head is not clear. Therefore, this study was designed to clarify the mode of cell death following ischaemic osteonecrosis of the femoral head in an established pig model. METHODS: Morphological assessment, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, detection of DNA laddering and transmission electron microscopy studies were performed to determine whether apoptosis is one of the pathways of cell death following ischaemic osteonecrosis of the femoral head. RESULTS: Mode of cell death was investigated from 2 to 14 days following the surgical induction of ischaemia. Ischaemic femoral heads showed morphological evidence of cell death by oncotic and apoptotic pathways in earlier stages of osteonecrosis. TUNEL positive cells were seen from 2 to 14 days following the induction of ischaemia. DNA samples obtained from ischaemic femoral heads following the induction of ischaemia showed nucleosomal ladders indicating apoptotic cell death. Electron micrographs also showed morphological changes associated with apoptosis. CONCLUSIONS: This study demonstrates that oncosis is not the sole mechanism of cell death following ischaemic injury of the femoral head. Both apoptosis and oncosis are involved as a result of ischaemic injury to the femoral head.

Constitutive production of proinflammatory cytokines RANTES, MIP-1beta and IL-18 characterizes LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disease often associated with autoimmune disorders such as rheumatoid arthritis. High levels of soluble Fas ligand have been implicated in development of chronic neutropenia. However, a comprehensive analysis of constitutive chemokine and lymphokine production in LGL leukemia has not previously been reported. Here, we utilized RNase protection assays and enzyme-linked immunosorbent assays (ELISAs) to address this question. RANTES, IL-8, MIP-1alpha, MIP-1beta, IL-1beta, IL-10, IL-12 p35, IL-18, IFN-gamma and IL-1Ra were the cytokine transcripts expressed in elevated levels from RNA of peripheral blood mononuclear cells of LGL leukemia patients. Confirmatory ELISAs indicated that sera from LGL leukemia patients have elevated levels of RANTES, MIP-1beta, IL-18, and to a lesser extent IL-8 and IL-1Ra. This pattern of cytokine upregulation is similar to that seen in some chronic infections or in autoimmune diseases, thus characterizing LGL leukemia as a proinflammatory disorder.

Characterization of a variant of PAC-1 in large granular lymphocyte leukemia.

Phosphatase in activated T cells (PAC-1) is a mitogen-induced early responsive gene. It encodes a 32 kDa tyrosine-threonine dual specificity phosphatase. Constitutive expression of PAC-1 leads to an inhibition of MAP kinase activity in vivo. Such constitutive expression was reported in HTLV-1 infected cell lines. In the present study, we observed the constitutive over-expression of two transcripts related to PAC-1 in large granular lymphocyte (LGL) leukemia. By screening a LGL leukemia cDNA library using the 3' end of a PAC-1 probe, we obtained a clone (clone 8) which retains one and one half introns, excludes two exons, and matches one hundred percent with a DNA sequence on chromosome 2. The deduced amino acid sequence of the predicted protein contains 170 amino acids and is 144 amino acids shorter than PAC-1. When we expressed this protein in Escherichia coli as a GST-fusion protein, a 45 kDa (19 kDa PAC-1 variant+26 kDa GST protein) protein was obtained. The expressed protein was purified to near homogeneity by using a glutathione affinity column. The purified protein did not have any intrinsic phosphatase activity when assayed in vitro. But when this purified protein was added to a phosphatase assay system in combination with a recombinant dual specificity phosphatase, CL100, enhanced phosphatase activity was observed. The significance of the constitutive over-expression and its physiological role of this protein remain to be established in leukemic LGL.

Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template.

In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template. All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay. Therefore the validity of the assay was questioned. Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA. DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly. Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template. These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses.

Constitutive expression of cytotoxic proteases and down-regulation of protease inhibitors in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with rheumatoid arthritis. The etiology of LGL leukemia is not known. In order to better understand the pathogenesis of LGL leukemia, we analyzed differential gene expression using microarray technology. We found that approximately 80 genes were up-regulated and 12 genes were down-regulated when compared to normal peripheral blood mononuclear cells (PBMC). In the present study, we were interested in a group of genes involved in cytotoxic function. The up-regulated genes involved in cytotoxic function were serine proteinases (granzymes A, B, H and K) cysteine proteinases [cathepsin C, cathepsin W (lymphopain)], calpain small subunit and caspase-8. In addition, a pore-forming protein perforin, was also up-regulated. Northern blot analysis and RNase protection assays (RPA) confirmed that these genes were over-expressed in the majority of samples from LGL leukemia patients. Of interest, proteolytic inhibitors such as cystatin C, A, alpha-1 antitrypsin and metalloproteinase inhibitors were down-regulated in leukemic LGL when compared to normal peripheral blood mononuclear cells. Importantly, the pattern of gene expression in leukemic LGL resembles that seen in activated cytotoxic T cells (CTL).

Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with autoimmune disease. A central feature of this disease is dysregulation of apoptosis. In order to identify differentially expressed genes in LGL leukemia, microarray analysis was performed. We found many differentially expressed genes including several expression sequence tags (ESTs). As a systematic study, we selected one up-regulated EST (GenBank Accession number N47089) and further investigated. An LGL leukemia library was screened using this EST as a probe and a full-length sequence for a novel gene was identified. The deduced amino acid sequence revealed that the novel gene encodes a G-protein-coupled receptor gene that exhibits 86% identity with rat sphingosine-1-phosphate receptor (edg-8/nrg-1). This gene is present in brain, spleen, and peripheral blood mononuclear cells (PBMC) and is overexpressed in leukemic LGL.

Microarray results: how accurate are they?

BACKGROUND: DNA microarray technology is a powerful technique that was recently developed in order to analyze thousands of genes in a short time. Presently, microarrays, or chips, of the cDNA type and oligonucleotide type are available from several sources. The number of publications in this area is increasing exponentially. RESULTS: In this study, microarray data obtained from two different commercially available systems were critically evaluated. Our analysis revealed several inconsistencies in the data obtained from the two different microarrays. Problems encountered included inconsistent sequence fidelity of the spotted microarrays, variability of differential expression, low specificity of cDNA microarray probes, discrepancy in fold-change calculation and lack of probe specificity for different isoforms of a gene. CONCLUSIONS: In view of these pitfalls, data from microarray analysis need to be interpreted cautiously.