The assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL). A broad spectrum of molecular techniques has been employed so far, including both direct Sanger sequencing and next generation sequencing. Oxford Nanopore Technologies recently released the MinION an USB-interfaced sequencer. In this paper we report our experience, with the MinION technology for the detection of the TP53 gene mutation in CLL patients. Twelve CLL patients at diagnosis were included in this study. All except one patient showed the TP53 gene deletion in Fluorescence in situ hybridization experiments. Patients were investigated for TP53 mutation by Sanger and by MinION sequencing. Analysis by Sanger was performed according with the IARC protocol. Analysis by MinION was performed adopting a strategy based on long template PCR, read error correction, and post variant calling filtering. Due to the high error rate of nanopore technology, sequence data were both used directly and before correction with two different in silico methods: ALEC and nanocorrect. A mean error rate of 15 % was detected before correction that was reduced to 4-5 % after correction. Analysis by Sanger sequencing was able to detect four patients mutated for TP53. MinION analysis detected one more mutated patient previously not detected from Sanger. In our hands, the Nanopore technology shows correlation with Sanger sequencing but more sensitive, manageable and less expensive, and therefore has proven to be a useful tool for TP53 gene mutation detection.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the BCR-ABL1 fusion gene generation as a consequence of the t(9;22)(q34;q11) rearrangement. The identification of the BCR-ABL1 transcript was of critical importance for both CML diagnosis and minimal residual disease (MRD) monitoring. In this review, we report the recent advances in the CML MRD monitoring based on RNA, DNA and protein analysis. The detection of the BCR-ABL1 transcript by the quantitative reverse-transcriptase polymerase chain reaction is the gold standard method, but other systems based on digital PCR or on GeneXpert technology have been developed. In the last years, DNA-based assays showed high sensitivity and specificity, and flow cytometric approaches for the detection of the BCR–ABL1 fusion protein have also been tested. Recently, new MRD monitoring systems based on the detection of molecular markers other than the BCR-ABL1 fusion were proposed. These approaches, such as the identification of CD26+ leukemic stem cells, microRNAs and mitochondrial DNA mutations, just remain preliminary and need to be implemented. In the precision medicine era, the constant improvement of the CML MRD monitoring practice could allow clinicians to choose the best therapeutic algorithm and a more accurate selection of CML patients eligible for the tyrosine kinase inhibitors discontinuation.

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future.

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

The molecular pathogenesis of hematological diseases is often driven by genetic and epigenetic alterations. Next-generation sequencing has considerably increased our genomic knowledge of these disorders becoming ever more widespread in clinical practice. In 2012 Oxford Nanopore Technologies (ONT) released the MinION, the first long-read nanopore-based sequencer, overcoming the main limits of short-reads sequences generation. In the last years, several nanopore sequencing approaches have been performed in various “-omic” sciences; this review focuses on the challenge to introduce ONT devices in the hematological field, showing advantages, disadvantages and future perspectives of this technology in the precision medicine era.