HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP

Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue, we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment, whole-genome amplification, and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen''s kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n = 131) and the HC2 assay (n = 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies.Infection with a carcinogenic type of human papillomavirus (HPV) is necessary for development of cervical cancer.^1,2 There are 13 types of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) confirmed to be of high or moderate risk for cervical cancer and four types (26, 73, 66 and 82) that are probable high-risk. Other HPV types are low-risk and may be associated with genital warts, such as HPV 6 and 11. Molecular-based screening for the most oncogenic types of HPV is being used more frequently to diagnose risk for cervical cancer because it is easier and more reliable than cytological screening.³ For example, the digene HC2 HPV DNA Test (HC2) is widely used for screening⁴ because it has exceptional clinical performance for a cancer and high-grade disease end point (ie, negative predictive value). Molecular screening, however, is not designed to determine which HPV type is present in an infection. The type of HPV in an infection is important to monitor because the risk of cancer and high-grade lesions increases for those whose HPV infection type is the same over time, especially for HPV 16 and 18.⁵Because of the numerous HPV types, genotyping assays require a high level of multiplex. The development of PCR-based assays for HPV genotyping is useful to identify infections with carcinogenic HPV-types, but they have some technical limitations. Many genotyping assays involve PCR with consensus primers that amplify conserved regions of the targeted HPV types.^6,7 The efficiency of consensus-PCR is limited because primers have a variable number of nucleotide matches to the various HPV targets. An alternate approach of using multiple primer sets in a single tube is also limited due to interference among multiple primer sets. Multiplex real-time PCRs in a single reaction is also limited by the number of dyes that may be discriminated by the PCR detector instrument. Furthermore, currently described PCRs amplify a relatively small region of HPV, most in the L1 region, so they may miss HPV that has been disrupted due to viral integration.⁸ Besides integration, many point mutations in the HPV genome have been described, which may decrease the efficiency of any specific primer or probe set.⁹ The efficiency of HPV genotyping assays may also be reduced if a multitude of human DNA and nontargeted DNA is included in the reaction. This occurs for DNA isolation methods that are not specific to the targeted HPV sequences.Our HPV genotyping assay was developed to permit rapid and accurate genotyping with a high level of multiplex, while avoiding or mitigating the stated drawbacks of PCR. The assay is called Hybrid Capture (HC)-whole genome amplification (WGA)-Luminex (LMX) and is described as follows. First, there is a sequence-specific sample enrichment process to isolate single-stranded HPV DNAs while removing much of the nonspecific genomic DNA that may interfere with some assays. This sample preparation was devised from HC technology used in the HC2 Test^10,11 but requires less time and captures the RNA:DNA hybrids on magnetic beads instead of an assay plate.¹² The second, WGA step amplifies the enriched single-stranded DNA using isothermal amplification with φ-29 DNA polymerase. This polymerase is highly processive and has strand-displacement activity, which creates multiple, long linear strands of target in the presence of short random primers or where nicks occur in the DNA.^13,14 This step amplifies all sequences present, which permits a high level of multiplex. The long amplicon size allows use of several probes for simultaneous detection of multiple regions of the same HPV target. The final, multiplex detection step utilizes Luminex XMAP technology¹⁵ combined with HC signal amplification. LMX technology consists of capturing HPV target amplicons onto oligonucleotides that are fixed to fluorescent beads. For this assay, there are two specific oligonucleotide sequences per bead per HPV target, and each bead has a distinct fluorescent emission. Long complementary HPV RNA probes are bound to the denatured captured DNA creating hybrids on the LMX bead complex. The hybrids are detected using a fluorescent HC antibody (reporter). Multiple reporter antibodies are bound to each hybrid, which provides signal amplification and improves the analytical sensitivity of the assay. The bead and the reporter emissions are analyzed by a LMX flow cytometer instrument. The assay was designed to detect the 17 most common high-risk types detected in cervical cancers and the two most common low-risk types found in genital warts. The assay was tested on samples of HPV DNA plasmids and with clinical specimens. Results were compared with the widely-used clinical diagnostic assay, HC2 test, and with a PCR-based genotyping assay.     

Comparative Analysis of Human Serum Albumin Adsorption and Complement Activation for Intraocular Lenses

Intraocular liquid, in contrast to blood, has no cellular components; therefore, proteins (human serum albumin [HSA], and [alpha, beta, gamma] globulins) are the major components that determine patients' response to the intraocular lens (IOL) surface. In addition to the amount of adsorbed proteins, the possibility of its conformational changes, including conformational changes of globulins C1 and C3 that respond for the activation of the complements system by the classical and alternative pathways, cannot be excluded. The interaction between IOLs and protein components of intraocular liquid directly influences the ocular exudative reaction in the early postoperational period, the intensity of cellular and pigmental scurf on the surface of the IOLs, and the state of endothelial cells of the cornea in the distant postoperational period. Our goal was to compare the interaction of commercial IOLs made from polymethylmethacrylate, silicone, poly-2-hydroxyethyl methacrylate (p-HEMA), and copolymer p-HEMA with collagen with HSA and the complement system. The total internal reflection fluorescence (TIRF) method and hemolytic assay were used for this task, respectively. It has been demonstrated that the probability of biocompatibility of commercially produced IOLs on the stage of protein adsorption can be evaluated using the kinetic of HSA-fluorescein isothiocyanate adsorption onto the IOL surface by the TIRF METHOD: In the case of IOLs from p-HEMA, a negative correlation was shown between the degree of irreversible adsorption of HSA and the minimum relative rate constant of the surface-induced complement activation. We did not find any correlation between hydrophilicity/hydrophobicity of lenses and their adsorptional properties including complement activation. From suggested adsorptional criteria in vitro for biocompatible surfaces, the hydrogel lens from p-HEMA has a lower probability of biocompatibility in comparison with other IOLs.     

Myocardial perfusion quantitation with 15O-labelled water PET: high reproducibility of the new cardiac analysis software (Carimas™)

Purpose

Carimas? (Cardiac Image Analysis System) is a new software package developed at the Turku PET Centre for the quantitation of PET studies of the heart with a broad range of tracers. The goal of this study was to assess the reproducibility of results the package provides for myocardial perfusion (MP) quantitation using ¹⁵O-labelled water.

Methods

Four observers with various levels of experience in nuclear medicine independently analysed 20 MP studies (10 rest flow: “rest”, 10 adenosine-induced hyperaemia: “stress”). Each study was analysed twice. The linear mixed model for repeated measures was fitted to the data to calculate intraclass correlation coefficients (ICC), differences between the repeats (the intraobserver differences) and differences between the observers (the interobserver differences). Also, Pearson correlation coefficients (r) were calculated and Bland-Altman plots were drawn. The reproducibility of MP was assessed on global, regional and segmental levels. Thereafter, this analysis was applied in 48 consecutive clinical patients with suspected coronary heart disease (CHD).

Results

For the experienced observer the Pearson r for all segments was 0.974 at rest and 0.978 at stress (p?<?0.0001), and the repeatability coefficients were 0.145 ml/g per min (15.5% of the average) and 0.389 ml/g per min (14.9%), correspondingly. The ICC reflected very good overall reproducibility. The intraobserver and interobserver differences were small, and the difference between the most and the least experienced observers at stress was 8.5% for the global MP. The clinical accuracy of the perfusion in the detection of CHD was excellent (positive predictive value 91% and negative predictive value 88%) against invasive angiography.

Conclusion

The results demonstrate high reproducibility of myocardial perfusion quantitation with ¹⁵O-labelled water PET using Carimas?. The results support the feasibility of robust analysis and good clinical accuracy.     

Asciminib and ponatinib exert synergistic anti-neoplastic effects on CML cells expressing BCR-ABL1T315I-compound mutations

Ponatinib is a tyrosine kinase inhibitor (TKI) directed against BCR-ABL1 which is successfully used in patients with BCR-ABL1 ^T315I+ chronic myeloid leukemia (CML). However, BCR-ABL1 compound mutations may develop during therapy in these patients and may lead to drug resistance. Asciminib is a novel drug capable of targeting most BCR-ABL1 mutant-forms, including BCR-ABL1^T315I, but remains ineffective against most BCR-ABL1^T315I+ compound mutation-bearing sub-clones. We demonstrate that asciminib synergizes with ponatinib in inducing growth-arrest and apoptosis in patient-derived CML cell lines and murine Ba/F3 cells harboring BCR-ABL1 ^T315I or T315I-including compound mutations. Asciminib and ponatinib also produced cooperative effects on CRKL phosphorylation in BCR-ABL1-transformed cells. The growth-inhibitory effects of the drug combination ‘asciminib+ponatinib’ was further enhanced by hydroxyurea (HU), a drug which has lately been described to suppresses the proliferation of BCR-ABL1 ^T315I+ CML cells. Cooperative drug effects were also observed in patient-derived CML cells. Most importantly, we were able to show that the combinations ‘asciminib+ponatinib’ and ‘asciminib+ponatinib+HU’ produce synergistic apoptosis-inducing effects in CD34⁺/CD38^- CML stem cells obtained from patients with chronic phase CML or BCR-ABL1 ^T315I+ CML blast phase. Together, asciminib, ponatinib and HU synergize in producing anti-leukemic effects in multi-resistant CML cells, including cells harboring T315I+ BCR-ABL1 compound mutations and CML stem cells. The clinical efficacy of this TKI combination needs to be evaluated within the frame of upcoming clinical trials.     

The separation of processing stages in a lexical interference fMRI-paradigm

In picture–word interference paradigms, the picture naming process is influenced by an additional presentation of linguistic distractors. Naming response times (RTs) are speeded (facilitation) by associatively-related and phonologically-related words when compared to unrelated words, while they are slowed down by categorically-related words (inhibition), given that distractor onsets occur at appropriate stimulus onset asynchronies (SOAs). In the present study with healthy subjects, we for the first time integrated all four auditorily presented distractor types into a single paradigm at an SOA of ? 200 ms, in order to directly compare behavioral and neural interference effects between them. The behavioral study corroborated results of previous studies and revealed that associatively-related distractors speeded RTs even more than phonologically-related distractors, thereby becoming equally fast as naming without distractors. Distractors were assumed to specifically enhance activation of brain areas corresponding to processing stages as determined in a cognitive model of word production (Indefrey, P., Levelt, W.J.M., 2004. The spatial and temporal signatures of word production components. Cognition 92, 101–144.). Functional magnetic resonance imaging (fMRI) at 3 T revealed activation of left superior temporal gyrus exclusively for phonologically-related distractors, and activation of left or right lingual gyrus exclusively for associatively-related and categorically-related distractors, respectively. Moreover, phonologically-related distractors elicited phonological–phonetic networks, and both semantic distractors evoked areas associated with mental imagery, semantics, and episodic memory retrieval and associations. While processes involved in distractor inhibition (e.g., conflict/competition monitoring) and high articulatory demands were observed for categorically-related distractors, priming of articulatory planning was revealed for associatively-related distractors. We conclude that activations of neural networks as obtained by the fMRI interference paradigm can be predicted from a cognitive model.     

Oncolytic Rat Parvovirus H-1PV, a Candidate for the Treatment of Human Lymphoma: In Vitro and In Vivo Studies

The incidence of lymphomas developing in both immunocompetent and immunosuppressed patients continues to steadily increase worldwide. Current chemotherapy and immunotherapy approaches have several limitations, such as severe side toxicity and selection of resistant cell variants. Autonomous parvoviruses (PVs), in particular the rat parvovirus H-1PV, have emerged as promising anticancer agents. Although it is apathogenic in humans, H-1PV has been shown to infect and suppress various rat and human tumors in animal models. In this study, we demonstrate the capacity of H-1PV for efficiently killing, through necrosis, cell cultures originating from Burkitt''s lymphoma (BL), while sparing normal B lymphocytes. The cytotoxic effect was generally accompanied by a productive H-1PV infection. Remarkably, parvovirus-based monotherapy efficiently suppressed established BL at an advanced stage in a severe combined immunodeficient (SCID) mouse model of the disease. The data show for the first time that an oncolytic parvovirus deserves further consideration as a potential tool for the treatment of some non-Hodgkin B-cell lymphomas, including those resistant to apoptosis induction by rituximab.