Synthesis and Assessment of Novel Probes for Imaging Tau Pathology in Transgenic Mouse and Rat Models.

Aggregated tau protein is a core pathology present in several neurodegenerative diseases. Therefore, the development and application of positron emission tomography (PET) imaging radiotracers that selectively bind to aggregated tau in fibril form is of importance in furthering the understanding of these disorders. While radiotracers used in human PET studies offer invaluable insight, radiotracers that are also capable of visualizing tau fibrils in animal models are important tools for translational research into these diseases. Herein, we report the synthesis and characterization of a novel library of compounds based on the phenyl/pyridinylbutadienylbenzothiazoles/benzothiazolium (PBB3) backbone developed for this application. From this library, we selected the compound LM229, which binds to recombinant tau fibrils with high affinity (Kd = 3.6 nM) and detects with high specificity (a) pathological 4R tau aggregates in living cultured neurons and mouse brain sections from transgenic human P301S tau mice, (b) truncated human 151-351 3R (SHR24) and 4R (SHR72) tau aggregates in transgenic rat brain sections, and (c) tau neurofibrillary tangles in brain sections from Alzheimer's disease (3R/4R tau) and progressive supranuclear palsy (4R tau). With LM229 also shown to cross the blood-brain barrier in vivo and its effective radiolabeling with the radioisotope carbon-11, we have established a novel platform for PET translational studies using rodent transgenic tau models.

Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients.

We used next-generation sequencing (NGS) of the immunoglobulin genes to evaluate residual disease in 153 specimens from 32 patients with adult B cell acute lymphoblastic leukemia enrolled in a single multicenter study. The sequencing results were compared with multiparameter flow cytometry (MFC) data in 66 specimens (25 patients) analyzed by both methods. There was a strong concordance (82%) between the methods in the qualitative determination of the presence of disease. However, in 17% of cases, leukemia was detected by sequencing but not by MFC. In 54 bone marrow (BM) and peripheral blood (PB) paired specimens, the burden of leukemia detected by NGS was lower in PB than in BM, although it was still detectable in 68% of the 28 paired specimens with positive BM. Lastly, patients without disease detected by NGS or MFC had a 5-year relapse free survival of > 80%. The results suggest that residual disease detection by immunoglobulin gene sequencing is an extremely sensitive technique and may identify patients that might benefit from transplantation. Moreover, the increased sensitivity of the method may allow frequent peripheral blood testing to supplement marrow sampling to measure disease response.

TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.

Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin.

High-throughput pairing of T cell receptor α and ß sequences.

The T cell receptor (TCR) protein is a heterodimer composed of an α chain and a ß chain. TCR genes undergo somatic DNA rearrangements to generate the diversity of T cell binding specificities needed for effective immunity. Recently, high-throughput immunosequencing methods have been developed to profile the TCR α (TCRA) and TCR ß (TCRB) repertoires. However, these methods cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications. We describe and validate a method called pairSEQ, which can leverage the diversity of TCR sequences to accurately pair hundreds of thousands of TCRA and TCRB sequences in a single experiment. Our TCR pairing method uses standard laboratory consumables and equipment without the need for single-cell technologies. We show that pairSEQ can be applied to T cells from both blood and solid tissues, such as tumors.

Using synthetic templates to design an unbiased multiplex PCR assay.

T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.

High-throughput sequencing of T-cell receptors reveals a homogeneous repertoire of tumour-infiltrating lymphocytes in ovarian cancer.

The cellular adaptive immune system mounts a response to many solid tumours mediated by tumour-infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma, with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood, including specificity, clonality, and spatial heterogeneity of the T-cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T-cell development, the TCR beta chain sequence serves as a molecular tag for each T-cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumours and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumour and are distinct from the circulating T-cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T-cell compartment of peripheral blood.

A novel bioinformatics pipeline for identification and characterization of fusion transcripts in breast cancer and normal cell lines.

SnowShoes-FTD, developed for fusion transcript detection in paired-end mRNA-Seq data, employs multiple steps of false positive filtering to nominate fusion transcripts with near 100% confidence. Unique features include: (i) identification of multiple fusion isoforms from two gene partners; (ii) prediction of genomic rearrangements; (iii) identification of exon fusion boundaries; (iv) generation of a 5'-3' fusion spanning sequence for PCR validation; and (v) prediction of the protein sequences, including frame shift and amino acid insertions. We applied SnowShoes-FTD to identify 50 fusion candidates in 22 breast cancer and 9 non-transformed cell lines. Five additional fusion candidates with two isoforms were confirmed. In all, 30 of 55 fusion candidates had in-frame protein products. No fusion transcripts were detected in non-transformed cells. Consideration of the possible functions of a subset of predicted fusion proteins suggests several potentially important functions in transformation, including a possible new mechanism for overexpression of ERBB2 in a HER-positive cell line. The source code of SnowShoes-FTD is provided in two formats: one configured to run on the Sun Grid Engine for parallelization, and the other formatted to run on a single LINUX node. Executables in PERL are available for download from our web site: http://mayoresearch.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm.

Glucuronic acid epimerase is associated with plasma triglyceride and high-density lipoprotein cholesterol levels in Turks.

We narrowed chromosome 15q21-23 linkage to plasma high-density lipoprotein cholesterol (HDL-C) levels in Turkish families by fine mapping, then focused on glucuronic acid epimerase (GLCE), a heparan sulfate proteoglycan (HSPG) biosynthesis enzyme. HSPGs participate in lipid metabolism along with apolipoprotein (apo) E. Of 31 SNPs in the GLCE locus, nine analyzed by haplotype were associated with HDL-C and triglyceride levels (permuted p = 0.006 and 0.013, respectively) in families. Of five tagging GLCE SNPs in two cohorts of unrelated subjects, three (rs16952868, rs11631403, and rs3865014) were associated with triglyceride and HDL-C levels in males (nonpermuted p < 0.05). The association was stronger in APOE 2/3 subjects (apoE2 has reduced binding to HSPGs) and reached multiple-testing significance (p < 0.05) in both males and females (n= 2612). Similar results were obtained in the second cohort (n= 1164). Interestingly, at the GLCE locus, bounded by recombination hotspots, Turks had a minor allele frequency of SNPs resembling Chinese more than European ancestry; adjoining regions resembled the European pattern. Studies of glce(+/-) apoe(-/-) mice fed a chow or high-fat diet supported a role for GLCE in lipid metabolism. Thus, SNPs in GLCE are associated with triglyceride and HDL-C levels in Turks, and mouse studies support a role for glce in lipid metabolism.

Polymorphisms in the hepatic lipase gene affect plasma HDL-cholesterol levels in a Turkish population.

We investigated the effects of single nucleotide polymorphisms (SNPs) of the hepatic lipase gene (LIPC) on plasma HDL-cholesterol (HDL-C) levels in Turks, a population with low levels of HDL-C. All exons and six evolutionarily conserved regions from 28 Turkish subjects were sequenced. We found 51 SNPs, nine of which were novel. Those 51 SNPs and SNPs from the National Center for Biotechnology Information dbSNP were evaluated by bioinformatics approaches. The population frequencies and linkage disequilibrium among SNPs from HapMap were combined with results from transcriptional factor prediction tools and the literature to select SNPs for genotyping. We found that five tagging LIPC SNPs, two reported here for the first time, were significantly associated with plasma HDL-C levels in both men and women (n = 2,612). These results were replicated in a separate Turkish cohort (n = 1,164). Plasma HDL-C levels were higher in subjects homozygous for the minor alleles of rs4775041, rs1800588 (-514C>T), and rs11858164 and lower in subjects homozygous for the minor alleles of rs11856322 and rs2242061. These SNPs seemed to have independent and additive effects on plasma HDL-C levels (1.5-5.2 mg/dl). Hepatic lipase activity in a subset (n = 260) of the main cohort was also significantly associated with all five SNPs. Thus, five LIPC SNPs, two novel, are associated with plasma HDL-C levels and hepatic lipase activity in two cohorts of Turkish subjects.

Prevalence of CXCR4 tropism among antiretroviral-treated HIV-1-infected patients with detectable viremia.

Although CXCR4-tropic viruses are relatively uncommon among untreated human immunodeficiency virus (HIV)-infected individuals except during advanced immunodeficiency, the prevalence of CXCR4-tropic viruses among treated patients with detectable viremia is unknown. To address this issue, viral coreceptor usage was measured with a single-cycle recombinant-virus phenotypic entry assay in treatment-naive and treated HIV-infected participants with detectable viremia sampled from 2 clinic-based cohorts. Of 182 treated participants, 75 (41%) harbored dual/mixed or X4-tropic viruses, compared with 178 (18%) of the 976 treatment-naive participants (P<.001). This difference remained significant after adjustment for CD4+ T cell count and CCR5 Delta 32 genotype. Enrichment for dual/mixed/X4-tropic viruses among treated participants was largely but incompletely explained by lower pretreatment nadir CD4 + T cell counts. CCR5 inhibitors may thus be best strategically used before salvage therapy and before significant CD4 + T cell depletion.

Apolipoprotein A-V: a potential modulator of plasma triglyceride levels in Turks.

The apolipoprotein A-V gene (APOA5) plays an important role in determining plasma triglyceride levels. We studied the effects of APOA5 polymorphisms on plasma triglyceride levels in Turks, a population with low levels of HDL cholesterol and a high prevalence of coronary artery disease. We found 15 polymorphisms, three of which were novel. Seven haplotype-tagging single nucleotide polymorphisms (SNPs) were chosen and genotyped in approximately 3,000 subjects. The rare alleles of the -1464T>C, -1131T>C, S19W, and 1259T>C SNPs were significantly associated with increased triglyceride levels (19-86 mg/dl; P < 0.05) and had clear gene-dose effects. Haplotype analysis of the nine common APOA5 haplotypes revealed significant effects on triglyceride levels (P < 0.001). Detailed analysis of haplotypes clearly showed that the -1464T>C polymorphism had no effect by itself but was a marker for the -1131T>C, S19W, and 1259T>C polymorphisms. The -1131T>C and 1259T>C polymorphisms were in a strong but incomplete linkage disequilibrium and appeared to have independent effects. Thus, the APOA5 -1131T>C, S19W, and 1259T>C rare alleles were associated with significant increases in plasma triglyceride levels. At least one of these alleles was present in approximately 40% of the Turks. Similar associations were observed for -1131T>C and S19W in white Americans living in San Francisco, California.

Common polymorphisms of ATP binding cassette transporter A1, including a functional promoter polymorphism, associated with plasma high density lipoprotein cholesterol levels in Turks.

The role of high levels of high density lipoprotein cholesterol (HDL-C) in protection against development of atherosclerosis is generally attributed to its role in reverse cholesterol transport, and the ATP binding cassette transporter A1 (ABCA1) is a key element of this process. We examined polymorphisms in ABCA1 in Turks, a population characterized by very low HDL-C levels. We discovered 36 variations in ABCA1 and genotyped informative polymorphisms in over 2,300 subjects. The rare alleles of C-14T and V771M polymorphisms were associated with higher HDL-C levels in men and, in combination with the rare alleles of R219K and I883M, respectively, with higher HDL-C in both sexes. Rare alleles of the C-14T and V771M polymorphisms were more frequent in the high HDL-C (>OR=40mg/dl) than in the low HDL-C group (