Nanopanel2 calls phased low-frequency variants in Nanopore panel sequencing data.

MOTIVATION: Clinical decision making is increasingly guided by accurate and recurrent determination of presence and frequency of (somatic) variants and their haplotype through panel sequencing of disease-relevant genomic regions. Haplotype calling (phasing), however, is difficult and error prone unless variants are located on the same read which limits the ability of short-read sequencing to detect, e.g. co-occurrence of drug-resistance variants. Long-read panel sequencing enables direct phasing of amplicon variants besides having multiple other benefits, however, high error rates of current technologies prevented their applicability in the past. RESULTS: We have developed Nanopanel2, a variant caller for Nanopore panel sequencing data. Nanopanel2 works directly on base-called FAST5 files and uses allele probability distributions and several other filters to robustly separate true from false positive (FP) calls. It effectively calls SNVs and INDELs with variant allele frequencies as low as 1% and 5%, respectively, and produces only few low-frequency false-positive calls (∼1 FP call with VAF<5% per kb amplicon). Haplotype compositions are then determined by direct phasing. Nanopanel2 is the first somatic variant caller for Nanopore data, enabling accurate, fast (turnaround <48 h) and cheap (sequencing costs ∼10$/sample) diagnostic workflows. AVAILABILITYAND IMPLEMENTATION: The data for this study have been deposited at zenodo.org under DOIs accession numbers 4110691 and 4110698. Nanopanel2 is open source and available at https://github.com/popitsch/nanopanel2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Detection and Monitoring of Lineage-Specific Chimerism by Digital Droplet PCR-Based Testing of Deletion/Insertion Polymorphisms.

Analysis of specific leukocyte subsets for post-transplantation monitoring of chimerism provides greater sensitivity and clinical informativeness on dynamic changes in donor- and recipient-derived cells. Limitations of the most commonly used approach to chimerism testing relying on PCR-based analysis of microsatellite markers prompted us to assess the applicability of digital droplet (dd) PCR amplification of deletion/insertion polymorphisms (DIPs) for lineage-specific chimerism testing in the related stem cell transplantation setting, where the identification of informative markers facilitating the discrimination between donor-derived and recipient-derived cells can be challenging. We analyzed 100 genetically related patient-donor pairs by ddPCR analysis using commercially available DIP kits including large sets of polymorphic markers. At least 1 informative marker was identified in all related pairs analyzed, and 2 or more discriminating markers were detected in the majority (82%) of instances. The achievable detection limit is dependent on the number of cells available for analysis and was as low as 0.1% in the presence of ≥20,000 leukocytes available for DNA extraction. Moreover, the reproducibility and accuracy of quantitative chimerism analysis compared favorably to highly optimized microsatellite assays. Thus, the use of ddPCR-based analysis of DIP markers is an attractive approach to lineage-specific monitoring of chimerism in any allogeneic transplantation setting.

Risk assessment of relapse by lineage-specific monitoring of chimerism in children undergoing allogeneic stem cell transplantation for acute lymphoblastic leukemia.

UNLABELLED: Allogeneic hematopoietic stem cell transplantation is required as rescue therapy in about 20% of pediatric patients with acute lymphoblastic leukemia. However, the relapse rates are considerable, and relapse confers a poor outcome. Early assessment of the risk of relapse is therefore of paramount importance for the development of appropriate measures. We used the EuroChimerism approach to investigate the potential impact of lineage-specific chimerism testing for relapse-risk analysis in 162 pediatric patients with acute lymphoblastic leukemia after allogeneic stem cell transplantation in a multicenter study based on standardized transplantation protocols. Within a median observation time of 4.5 years, relapses have occurred in 41/162 patients at a median of 0.6 years after transplantation (range, 0.13-5.7 years). Prospective screening at defined consecutive time points revealed that reappearance of recipient-derived cells within the CD34(+) and CD8(+) cell subsets display the most significant association with the occurrence of relapses with hazard ratios of 5.2 (P=0.003) and 2.8 (P=0.008), respectively. The appearance of recipient cells after a period of pure donor chimerism in the CD34(+) and CD8(+) leukocyte subsets revealed dynamics indicative of a significantly elevated risk of relapse or imminent disease recurrence. Assessment of chimerism within these lineages can therefore provide complementary information for further diagnostic and, potentially, therapeutic purposes aiming at the prevention of overt relapse. This study was registered at clinical. TRIALS: gov with the number NC01423747.

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML.

PURPOSE: Ponatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1T315I-mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1T315I-mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1T315I+ sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro. METHODS: Viability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting. FINDINGS: HU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1T315I+ cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1T315I-associated TKI resistance. INTERPRETATION: HU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of `ponatinib+HU´ and ´ponatinib+palbociclib´ combinations in advanced CML. FUNDING: This project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.

Large granular lymphocyte proliferation and revertant mosaicism: two rare events in a Wiskott-Aldrich syndrome patient.

We report on a 6 year old patient with an unusual clinical presentation of WAS and oligoclonal proliferation of TCR+ large granular lymphocytes (LGL). Flow cytometry demonstrated two distinct populations of lymphocytes with strongly decreased (WASP-) or normal expression levels of WASP (WASP+), respectively. Molecular analysis confirmed a splice site mutation in intron 2 of the WASP gene in the WASP- cells but not in WASP+ cells. LGL cells were WASP+, suggesting that two independent rare events, somatic revertant mosaicism and LGL expansion, have occurred in a child with WAS. Our report points to diagnostic difficulties in the presence of partial WASP reversions and LGL.

The Pan-AC assay: a single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species.

In view of the growing incidence and the high mortality of invasive aspergillosis and candidiasis, adequate diagnostic techniques permitting timely onset of treatment are of paramount importance. More than 90 % of all invasive fungal infections in immunocompromised individuals can be attributed to Candida and Aspergillus species. To date, standardized techniques permitting rapid, sensitive and, no less importantly, economic screening for the clinically most relevant fungi are lacking. In the present report, a real-time quantitative PCR assay, developed for the detection of the most common pathogenic Candida and Aspergillus species, is described. The single-reaction PCR assay targets a judiciously selected region of the 28S subunit of the fungal rDNA gene. The unique design of the universal primer/probe system, including a pan-Aspergillus and pan-Candida (Pan-AC) hydrolysis probe, facilitates the detection of numerous Aspergillus species (e.g. Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor and Aspergillus nidulans) and Candida species (e.g. Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Candida kefyr, Candida guilliermondii, Candida lusitaniae and Candida dubliniensis). The assay permits highly reproducible detection of 10 fg fungal DNA, which corresponds to a fraction of a fungal genome, and facilitates accurate quantification of fungal load across a range of at least five logs. Upon standardization of the technique using cultured fungal strains, the applicability in the clinical setting was assessed by investigating a series of clinical specimens from patients with documented fungal infections (n=17). The Pan-AC assay provides an attractive and economic approach to the screening and monitoring of invasive aspergillosis and candidiasis, which is readily applicable to routine clinical diagnosis.

Post-transplant monitoring of chimerism by lineage-specific analysis.

Molecular surveillance of hematopoietic chimerism is an important part of the routine diagnostic program in patients after allogeneic stem cell transplantation. Chimerism testing permits early prediction and documentation of successful engraftment and facilitates early risk assessment of impending graft rejection. In patients transplanted for treatment of malignant hematologic disorders, monitoring of chimerism can provide an early indication of incipient disease relapse. The investigation of chimerism has therefore become an indispensable tool for the management of patients during the post-transplant period. Increasing use of reduced-intensity conditioning, which is associated with prolonged duration of mixed hematopoietic chimerism, has further increased the clinical importance of chimerism analysis. At present, the most commonly used technical approach to the investigation of chimerism is microsatellite analysis by polymerase chain reaction. The investigation of chimerism within specific leukocyte subsets isolated from peripheral blood or bone marrow samples by flow sorting- or magnetic bead-based techniques provides more specific information on processes underlying the dynamics of donor/recipient chimerism. Moreover, cell subset-specific analysis permits the assessment of impending complications at a significantly higher sensitivity, thus providing a basis for earlier treatment decisions.

High-quality DNA from fingernails for genetic analysis.

The availability of high-quality germline DNA is an important prerequisite for a variety of genetic analyses. We have shown previously that fingernail clippings provide an optimal source of autologous, constitutional DNA for PCR-based applications. However, most existing protocols for nucleic acid purification from nails do not provide sufficiently high yields of pure and intact DNA for more demanding downstream analyses such as next generation sequencing (NGS). We have extensively tested and systematically modified a number of different protocols for DNA purification from nail material to optimize the yield and quality. The integrity of DNA was determined by PCR amplification of short (<300 bp), mid-range (>400 bp), and long-range (>2 kb) sequences using different target genes. Among the methods tested, the Prepfiler Forensic DNA Extraction kit was identified as the most appropriate approach to isolation of high-quality DNA from nail clippings. A standardized input of 20 mg nail material (1 to 10 pieces of fingernail clippings) yielded a mean of 1 µg DNA (range, 0.5 to 2.3 µg). Subsequent PCR-analysis revealed efficient amplifiability of short and mid-range targets in 93% and 90%, and long-range fragments in 60% of the samples tested. The adequacy for next generation sequencing applications was demonstrated by successful high-resolution HLA-typing in ten transplant recipients. Hence, the protocol presented facilitates the exploitation of fingernail material even for demanding genomic analyses both in research and diagnostics.

A multilocus technique for risk evaluation of patients with neuroblastoma.

PURPOSE: Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma. EXPERIMENTAL DESIGN: A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n = 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n = 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level. RESULTS: The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (κ = 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%. CONCLUSIONS: The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance.

Typing of human adenoviruses in specimens from immunosuppressed patients by PCR-fragment length analysis and real-time quantitative PCR.

Currently, 51 human adenovirus (AdV) serotypes, which are divided into six species (A to F), are known. AdV infections are a major cause of morbidity and mortality in immunosuppressed individuals, particularly in allogeneic stem cell transplant (SCT) recipients. Any AdV species may cause life-threatening disease, but little information is available on the clinical relevance of individual serotypes. The use of serological testing for serotype identification is limited due to the impaired immune response during the posttransplant period. A new molecular approach to serotype identification is presented here that exploits variable regions within the hexon gene. All serotypes belonging to the species A, B, C, E, and F can be determined by fragment length analysis of a single PCR product. For species C, which is the most prevalent in many geographic regions, an alternative technique based on serotype-specific real-time quantitative PCR was established. Of 135 consecutive pediatric patients screened for AdV infections after allogeneic SCT, 40 tested positive. Detailed analysis revealed the presence of 10 different serotypes; serotypes 1 and 2 from species C (C01 and C02) showed the highest prevalence, accounting for 77% of the AdV-positive cases. Representatives of other species were observed less commonly: serotype A12 in 6.5%; serotype A31 in 4.5%; and B03, B16, C05, C06, D19, and F41 in 2%. The approach to rapid molecular serotype analysis presented here provides a basis for detailed studies on adenovirus epidemiology and on the transmission of nosocomial infections. Moreover, in view of the increasing importance of tailored therapy approaches, serotype identification may in the future have implications for the selection of the most appropriate antiviral treatment.

Molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease.

Adenovirus (AdV) infection in the course of allogeneic stem cell transplantation (SCT) is associated with high transplant-related morbidity and mortality. Disseminated AdV disease is lethal in most instances. Early detection of AdV infection and identification of patients carrying a high risk of disseminated disease therefore remain a major challenge. In view of the large number of existing AdV types, we have established real-time polymerase chain reaction (PCR) assays permitting sensitive detection and quantification of all 51 currently known human AdV serotypes. In a series of 132 consecutive pediatric patients undergoing SCT, more than 5000 samples derived from peripheral blood (PB), stool, urine, and throat were screened for adenovirus infection by PCR during the posttransplantation period. Thirty-six patients (27%) tested positive by PCR, revealing AdV types of the subgenera A, B, C, D, and F. Except for enteritis in some patients with AdV positivity in stool, detection of the virus at sites other than PB was not associated with clinical signs of virus disease, and transplant-related mortality was not significantly different from AdV-negative patients. By contrast, 82% of patients who had detectable AdV in PB died from infectious complications (P <.001). Monitoring of PB specimens by real-time PCR permitted early diagnosis of invasive AdV infection in all instances. In patients who developed disseminated AdV disease, detection of the virus in PB preceded onset of clinical symptoms by a median of more than 3 weeks. The observation of AdV in peripheral blood may therefore serve as a basis for early initiation of preemptive antiviral treatment.