Rapid identification of compound mutations in patients with Philadelphia-positive leukaemias by long-range next generation sequencing.

An emerging problem in patients with Philadelphia (Ph)-positive leukaemias is the occurrence of cells with multiple mutations in the BCR-ABL1 tyrosine kinase domain (TKD) associated with high resistance to different tyrosine kinase inhibitors. Rapid and sensitive detection of leukaemic subclones carrying such changes, referred to as compound mutations, is therefore of increasing clinical relevance. However, current diagnostic methods including next generation sequencing (NGS) of short fragments do not optimally meet these requirements. We have therefore established a long-range (LR) NGS approach permitting massively parallel sequencing of the entire TKD length of 933bp in a single read using 454 sequencing with the GS FLX+ instrument (454 Life Sciences). By testing a series of individual and consecutive specimens derived from six patients with chronic myeloid leukaemia, we demonstrate that long-range NGS analysis permits sensitive identification of mutations and their assignment to the same or to separate subclones. This approach also facilitates readily interpretable documentation of insertions and deletions in the entire BCR-ABL1 TKD. The long-range NGS findings were reevaluated by an independent technical approach in select cases. Polymerase chain reaction (PCR) amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®-T plasmids, and >100 individual clones were subjected to analysis by Sanger sequencing. The NGS results were confirmed, thus documenting the reliability of the new technology. Long-range NGS analysis therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, and represents an important advancement of the diagnostic armamentarium for rapid assessment of impending resistant disease.

Species-specific identification of a wide range of clinically relevant fungal pathogens by the Luminex(®) xMAP technology.

Invasive fungal infections (IFI) are a common cause of life-threatening events in immunocompromised patients. Early detection and identification of the fungal pathogen is an important prerequisite for timely onset of the most appropriate treatment. Methods based on fungal culture are often too slow to be clinically useful. Other approaches to the identification of fungal species, including molecular techniques, are often restricted to a small number of the most commonly occurring pathogens and are therefore of limited use in the clinical setting. The development of assays for the detection and identification of a broad-range of clinically relevant fungal species is therefore an urgently needed step towards optimized diagnostics of IFI.The Luminex(®) xMAP technology offers a platform for the establishment of multiplex assays permitting high-throughput analysis of up to 100 different target molecules in a single test. Here we describe a Luminex(®)-based multiplex assay permitting rapid detection and identification of 10 fungal genera and 29 different species, including both commonly occurring and emerging fungal pathogens.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation.

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

Quantitative monitoring of BCR/ABL1 mutants for surveillance of subclone-evolution, -expansion, and -depletion in chronic myeloid leukaemia.

BACKGROUND: In chronic myeloid leukaemia (CML), clonal evolution with resistance to tyrosine kinase inhibitors (TKIs) is often triggered by BCR/ABL1 mutations. However, in the context of the complex pro-oncogenic signalling networks which ultimately lead to clonal expansion and disease progression, the exact contribution of BCR/ABL1 mutants remains uncertain. Recent data indicate that detection of BCR/ABL1 mutant subclones does not permit prediction of their expansion dynamics and their potential to become drivers of resistant disease. METHODS: To determine the patterns of clonal evolution and the distinct proliferation kinetics of individual BCR/ABL1 mutants during treatment, we employed ligase-dependent polymerase chain reaction (LD-PCR) analysis for quantitative surveillance of CML subclones with various tyrosine kinase domain (TKD) mutations including M244V, L248V, G250E, E255K, T315I, F317L-A/G, M351T and F359V. FINDINGS: Inadequate treatment responses were observed in 27 of 100 patients investigated and 16 were found to bear one or more BCR/ABL1 TKD mutations in separate subclones. Rapid subclone expansion upon onset or switch of TKI treatment was common and sometimes preceded corresponding changes in BCR/ABL1 transcript levels. Mutant subclones were found to respond differentially and sometimes unexpectedly to various treatment modalities. Decline and persistent depletion of specific mutation-bearing subclones in response to treatment could be documented by LD-PCR surveillance. INTERPRETATION: The observations show that quantitative monitoring of mutant BCR/ABL1 subclones by LD-PCR is a powerful tool for detection of clonal evolution, subclone-expansion and subclone-depletion and can contribute to optimised management of patients with CML.

Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem cell transplantation.

Timely diagnosis of impending graft rejection is crucial for effective therapeutic intervention after allogeneic hematopoietic stem cell transplantation (SCT). We have investigated the predictive potential of early leukocyte subset-specific chimerism for graft loss in children undergoing SCT. In total, 192 pediatric patients transplanted for the treatment of malignant and non-malignant diseases after reduced-intensity or myeloablative conditioning were investigated. Surveillance of lineage-specific chimerism was initiated upon first appearance of leukocyte counts amenable to cell sorting. Graft rejection occurred in 23 patients between 24 and 492 days post-transplant (median 63 days). The first chimerism analysis of T and NK cells performed at a median of 20 days after SCT identified three different risk groups that were independent from the conditioning regimen: recipient chimerism (RC) levels in T cells below 50% indicated a very low risk of rejection (1.4%), whereas high levels of RC (>90%) both in T and NK cells heralded graft loss in the majority of patients (90%) despite therapeutic interventions. RC >50% in T cells and ≤90% in NK cells defined an intermediate-risk group in which timely immunotherapy frequently prevented rejection. Early assessment of T- and NK-cell chimerism can therefore be instrumental in the risk assessment and therapeutic management of imminent graft rejection.

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats.

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30,000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients.

Invasive fungal disease (IFD) is a life-threatening event in immunocompromised patients, and there is an urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes. To assess the clinical potential of the assay, more than 600 consecutive specimens from 125 pediatric patients carrying a high risk of IFD were analyzed. An excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the European Organization for Research and Treatment of Cancer criteria was demonstrated, as revealed by the sensitivity of the assay of 96% (95% CI: 82-99%). The negative predictive value of the panfungal PCR assay presented was 98% (95% CI: 90-100%), while the specificity and the positive predictive value were 77% (95% CI: 66-85%) and 62% (95% CI: 47-75%), respectively. The results indicate that molecular screening of patients during febrile neutropenic episodes by the assay presented could help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. Our observations raise the possibility that rapid species identification may be required to increase the positive predictive value for impending fungus-related disease.

Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation.

Invasive adenovirus (AdV) infections are associated with high morbidity and mortality in allogeneic stem cell transplant recipients. We observed that molecular detection of the virus in stool specimens commonly precedes AdV viremia, suggesting that intestinal infections may represent a common source of virus dissemination. To address this notion, we have investigated 153 consecutive allogeneic transplantations in 138 pediatric patients by quantitative monitoring of AdV in stool specimens and peripheral blood by a pan-adenovirus real-time (RQ)-PCR approach. AdV was detectable in serial stool specimens in all cases of AdV viremia during the post-transplant course (P<0.0001). The incidence of AdV viremia in individuals with peak virus levels in stool specimens above 1 x 10E6 copies per gram (n=22) was 73% vs 0% in patients with AdV levels in stool specimens below this threshold (n=29; P<0.0001). Serial measurement of AdV levels in stool specimens by RQ-PCR permitted early diagnosis of impending invasive infection with a sensitivity and specificity of 100% (95% confidence interval (CI) 96-100%) and 83% (95% CI 67-92%), respectively. The median time span between detection of AdV loads in stool specimens above 1 x 10E6 copies per gram and first observation of viremia was 11 days (range 0-192). Quantitative monitoring of the AdV load in stool specimens therefore provides a rationale for early initiation of antiviral treatment with the aim of preventing progression to life-threatening invasive infection.

Species-specific identification of a wide range of clinically relevant fungal pathogens by use of Luminex xMAP technology.

In immunocompromised patients suffering from invasive fungal infection, rapid identification of the fungal species is a prerequisite for selection of the most appropriate antifungal treatment. We present an assay permitting reliable identification of a wide range of clinically relevant fungal pathogens based on the high-throughput Luminex microbead hybridization technology. The internal transcribed spacer (ITS2) region, which is highly variable among genomes of individual fungal species, was used to generate oligonucleotide hybridization probes for specific identification. The spectrum of pathogenic fungi covered by the assay includes the most commonly occurring species of the genera Aspergillus and Candida and a number of important emerging fungi, such as Cryptococcus, Fusarium, Trichosporon, Mucor, Rhizopus, Penicillium, Absidia, and Acremonium. Up to three different probes are employed for the detection of each fungal species. The redundancy in the design of the assay should ensure unambiguous fungus identification even in the presence of mutations in individual target regions. The current set of hybridization oligonucleotides includes 75 species- and genus-specific probes which had been carefully tested for specificity by repeated analysis of multiple reference strains. To provide adequate sensitivity for clinical application, the assay includes amplification of the ITS2 region by a seminested PCR approach prior to hybridization of the amplicons to the probe panel using the Luminex technology. A variety of fungal pathogens were successfully identified in clinical specimens that included peripheral blood, samples from biopsies of pulmonary infiltrations, and bronchotracheal secretions derived from patients with documented invasive fungal infections. Our observations demonstrate that the Luminex-based technology presented permits rapid and reliable identification of fungal species and may therefore be instrumental in routine clinical diagnostics.

Identification of fungal species by fragment length analysis of the internally transcribed spacer 2 region.

The rapid identification of fungal pathogens in clinical specimens is a prerequisite for timely onset of the most appropriate treatment. The aim of the present study was to develop a sensitive and rapid method for the species-specific identification of clinically relevant fungi. We employed fluorescent polymerase chain reaction (PCR)-fragment length analysis of the highly variable internally transcribed spacer 2 (ITS2) region to identify individual fungal species by their specific amplicon sizes. The specificity of the technique was ascertained by the detailed analysis of 96 strains derived from 60 different human-pathogenic fungal species. To achieve adequate sensitivity for species identification in patients with invasive fungal infection, who often display very low pathogen loads in peripheral blood, the ITS2 region was amplified by semi-nested PCR prior to amplicon-length analysis. Serial specimens from 26 patients with documented fungal infections were investigated. The fungal pathogens identified included different Aspergillus and Candida species, Rhizopus oryzae and Fusarium oxysporum. Fragment length analysis of the ITS2 region upon amplification by semi-nested PCR permits the sensitive identification of fungal species. The technique can be readily implemented in routine diagnostics.

Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.

A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.