Development of novel methodological approaches to detection and quantitative monitoring of point-mutated clones

Bei Patienten mit chronisch myeloischer Leukämie (CML) ist das Auftreten von Punktmutationen in der BCR-ABL1 Tyrosinkinasedomäne (TK) die am häufigsten beschriebene Ursache für Resistenzen gegen Tyrosinkinase-Inhibitoren (TKI). Für die Detektion von Punktmutationen ist eine Auswahl an Methoden verfügbar. Diese sind jedoch oft wenig sensitiv, wie zum Beispiel die Standardmethode der bidirektionalen Sequenzierung, mit einer Sensitivität von circa 20%. Das Auftreten von TKI resistenten Zellklonen muss jedoch nicht notwendigerweise mit einer bevorstehenden Progression der Krankheit assoziiert sein. Die Analyse von mutanten Zellklonen mit qualitativen Methoden könnte daher unzureichend sein, um klinisch relevante Klone zu identifizieren. Um diese Problematik bearbeiten zu können, wäre eine Methode von Vorteil, die die Detektion von Punktmutationen in einem klinisch relevanten Bereich mit der Möglichkeit zur Quantifizierung vereint. Solch eine Methode galt es zu entwickeln. Die neu etablierte LD-PCR Methode basiert auf der Hybridisierung spezifischer Sonden an die Wildtyp (WT) bzw. mutierte Sequenz gefolgt von einer ligations-abhängigen kompetitiven PCR. Die generierten Amplikons werden mittels Kapillarelektrophorese detektiert und quantifiziert. LD-PCR Assays wurden für folgende 21 häufig vorkommenden BCR-ABL1 TK Punktmutationen etabliert: M244V, L248V, V299L-C/T, G250E, Q252H-C/T, Y253F, Y253H, E255K, T315A, T315I, F317C, F317I, F317L-A/G, F317V, M351T, F359V, H396P and H396R. Das Konzept der LD-PCR ist auf jegliche Punktmutation umlegbar, wie am Beispiel der Punktmutation V617V im JAK2 Gen, die bei myeloproliferativen Erkrankungen prognostisch bedeutend ist, gezeigt werden konnte. Die LD-PCR Assays haben eine Sensitivität von 1-5% und ermöglichen eine quantitative Überwachung während des Behandlungsverlaufs. Die Anwendbarkeit dieser Methode bei CML Patienten unter TKI Behandlung konnte ebenso gezeigt werden wie das Vorhandensein einer Proliferationskinetik mutierter Zellklone. Die Dokumentation der Größe und der Expansion eines mutierten Zellklons könnte die Vorhersage einer bevorstehenden Resistenz ermöglichen und bei der klinischen Überwachung sowie bei der Therapiesteuerung von CML Patienten hilfreich sein.In patients with chronic myeloid leukemia (CML), the occurrence of point mutations within the BCR-ABL1 tyrosine kinase (TK) domain is currently the most common mechanism of resistance to therapy with TK inhibitors. To date a variety of different techniques to the detection of relevant point mutations is available. This mainly includes methods displaying a limited level of sensitivity e.g. bidirectional sequencing, the standard method of choice, providing a sensitivity of ~ 20%. However, the presence of cells carrying resistant mutations does not necessarily imply imminent disease progression. Mutation analysis by qualitative methods may therefore be insufficient to reliably identify clinically relevant resistant clones. To address this problem, an application permitting the detection of point mutated subclones of clinically relevant size and the additional option for quantitative analysis might be of great benefit. Thus the aim was to establish a technique combining both requirements. The newly developed LD-PCR relies on specific probe hybridization to mutant and wild-type sequences followed by ligation-dependent competitive PCR. Amplicons are detected and quantified via fluorescence-based capillary electrophoresis. Assays have been established for 21 common BCR-ABL1 TK point mutations including M244V, L248V, V299L-C/T, G250E, Q252H-C/T, Y253F, Y253H, E255K, T315A, T315I, F317C, F317I, F317L-A/G, F317V, M351T, F359V, H396P and H396R. Moreover the technique is adaptive to any kind of point mutation as shown for the detection of V617F point mutation within the JAK2 gene, relevant for several myeloproliferative disorders. The LD-PCR assays display a detection limit of 1-5% and permit quantitative monitoring of mutant clones during the course of treatment. The applicability in CML patients undergoing treatment with TK inhibitors and the existence of kinetic proliferation of mutated cells clones could be demonstrated. Assessment of the size and proliferation kinetics of clones carrying specific mutations could therefore be instrumental in the clinical surveillance and therapeutic management of CML patients

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

Additional file 2: of cFinder: definition and quantification of multiple haplotypes in a mixed sample

Archive containing test data. Include annotations and exemplary alignment files from AVA and CLC for sample 5, as well as simulated test reads for overlapping amplicons

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

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

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 (kappa = 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