International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Protracted Clonal Trajectory of a JAK2 V617F-Positive Myeloproliferative Neoplasm Developing during Long-Term Remission from Acute Myeloid Leukemia.

Although transformation of the myeloproliferative neoplasms (MPNs) to acute myeloid leukemia (AML) is well documented, development of an MPN in patients previously treated for, and in remission from, AML is exceedingly rare. A case is described in which a patient was successfully treated for AML and in whom a JAK2 V617F-positive MPN was diagnosed after seven years in remission. Retrospective evaluation of the JAK2 V617F detected a low allele burden at AML diagnosis and following one course of induction chemotherapy. This putative chemoresistant clone subsequently expanded over the intervening seven years, resulting in a hematologically overt MPN. As AML relapse has not occurred, the MPN may have arose in a separate initiating cell from that of the AML. Alternatively, both malignancies possibly evolved from a common precursor defined by a predisposition mutation with divergent evolution into MPN through acquisition of the JAK2 V617F and AML through acquisition of different mutations. This case emphasizes the protracted time frame from acquisition of a disease-driving mutation to overt MPN and further underscores the clonal complexity in MPN evolution.

Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

BACKGROUND: The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. METHODS: Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. RESULTS: One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. CONCLUSION: Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.

Chronic Myeloid Leukemia with an e6a2 BCR-ABL1 Fusion Transcript: Cooperating Mutations at Blast Crisis and Molecular Monitoring.

A minority of chronic myeloid leukemia patients (CML) express a variety of atypical BCR-ABL1 fusion variants and, of these, the e6a2 BCR-ABL1 fusion is generally associated with an aggressive disease course. Progression of CML to blast crisis is associated with acquisition of additional somatic mutations yet these events have not been elucidated in patients with the e6a2 BCR-ABL1 genotype. Moreover, molecular monitoring is only sporadically performed in CML patients with atypical BCR-ABL1 fusion transcripts due to lack of consensus approaches or standardization. A case of CML is described in which comprehensive molecular analysis, including targeted next-generation sequencing, revealed a single ASXL1 mutation cooperating with an e6a2 BCR-ABL1 fusion transcript at blast crisis. A quantitative molecular monitoring approach was devised and adopted that reflected the disease response from initial treatment through allogeneic stem cell transplantation which resulted in undetectable e6a2 BCR-ABL1 transcripts. This case emphasizes the requirement for molecular monitoring in CML patients with atypical BCR-ABL1 fusion transcripts and emphasizes that comprehensive sequencing has the potential to identify targets for novel therapies in CML patients with advanced disease.

A novel molecular assay using hybridisation probes and melt curve analysis for CALR exon 9 mutation detection in myeloproliferative neoplasms.

AIMS: Somatic insertions/deletions in exon 9 of the calreticulin gene have been identified in patients with essential thrombocythemia and primary myelofibrosis. Over 55 mutations have been discovered, 80% of which consist of either type 1 52-bp deletion or type 2 5-bp insertion. Other mutations (types 3-5) in conjunction with types 1 and 2 account for >87% of identified mutations. The aim of this study was development of a rapid PCR-based assay using LightCycler Hybridisation Probes for the detection of type 1-5 CALR mutations. METHOD: A real-time PCR assay using a novel HybProbe set was developed for use on the LightCycler 480 Instrument II. The acceptor probe was labelled with LC640 and Faststart DNA Master HybProbe kit was used for PCR reactions. RESULTS: Assay limit of detection was determined to be seven target copies with a probability of 95%. The specificity of the assay was determined by using synthetic constructs of CALR wild-type and CALR mutation types 1-5 with no non-specific detection observed. Samples from 21 patients with essential thrombocythemia (ET) and 12 patients with primary myelofibrosis (PMF), together with 29 control samples from patients diagnosed with various conditions, were screened using the assay. Of these, 24 were found to have mutations in CALR exon 9, with the assay detecting 8 type 1 mutations, 12 type 2 mutations, 2 type 24 mutations, 1 type 20 mutation and 1 31-bp deletion. CONCLUSIONS: The novel assay described has potential for application as a rapid, sensitive, high-throughput screening method in the clinical diagnostics setting.

Characterization of a novel variant BCR-ABL1 fusion transcript in a patient with chronic myeloid leukemia: Implications for molecular monitoring.

Molecular monitoring of BCR-ABL1 transcript levels using quantitative polymerase chain reaction is an essential part of the modern management of chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Establishing the diagnostic BCR-ABL1 fusion transcript is necessary in order to select appropriate primers and probes for such monitoring. A case is described in which quantitative polymerase chain reaction failed to detect the presence of BCR-ABL1 fusion transcript in a Philadelphia chromosome-positive chronic myeloid leukemia patient. Further investigation demonstrated a novel in-frame BCR-ABL1 fusion transcript with a breakpoint in BCR exon 13 and insertion of a sequence of ABL1 intron 1, therefore enabling subsequent molecular monitoring. This case highlights the requirement for characterization of the BCR-ABL1 transcript type at chronic myeloid leukemia diagnosis. Issues concerning standardized methodological approaches and interpretation of transcript levels in such rare cases are discussed.

Monitoring Minimal Residual Disease in the Myeloproliferative Neoplasms: Current Applications and Emerging Approaches.

The presence of acquired mutations within the JAK2, CALR, and MPL genes in the majority of patients with myeloproliferative neoplasms (MPN) affords the opportunity to utilise these mutations as markers of minimal residual disease (MRD). Reduction of the mutated allele burden has been reported in response to a number of therapeutic modalities including interferon, JAK inhibitors, and allogeneic stem cell transplantation; novel therapies in development will also require assessment of efficacy. Real-time quantitative PCR has been widely adopted for recurrent point mutations with assays demonstrating the specificity, sensitivity, and reproducibility required for clinical utility. More recently, approaches such as digital PCR have demonstrated comparable, if not improved, assay characteristics and are likely to play an increasing role in MRD monitoring. While next-generation sequencing is increasingly valuable as a tool for diagnosis of MPN, its role in the assessment of MRD requires further evaluation.

CALR mutation profile in Irish patients with myeloproliferative neoplasms.

Insertion and/or deletion mutations of the CALR gene have recently been demonstrated to be the second most common driver mutations in the myeloproliferative neoplasms (MPNs) of essential thrombocythemia (ET) and primary myelofibrosis (PMF). Given the diagnostic and emerging prognostic significance of these mutations, in addition to the geographical heterogeneity reported, the incidence of CALR mutations was determined in an Irish cohort of patients with MPNs with a view to incorporate this analysis into a prospective screening program. A series of 202 patients with known or suspected ET and PMF were screened for the presence of CALR mutations. CALR mutations were detected in 58 patients. Type 1 and Type 1-like deletion mutations were the most common (n=40) followed by Type 2 and Type 2-like insertion mutations (n=17). The CALR mutation profile in Irish ET and PMF patients appears similar to that in other European populations. Establishment of this mutational profile allows the introduction of a rational, molecular diagnostic algorithm in cases of suspected ET and PMF that will improve clinical management.

Inter-Laboratory Evaluation of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed. METHODS: DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance. RESULTS: An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected. CONCLUSION: In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.

Acute Lymphoblastic Leukemia Arising in CALR Mutated Essential Thrombocythemia.

The development of acute lymphoblastic leukemia in an existing myeloproliferative neoplasm is rare with historical cases unable to differentiate between concomitant malignancies or leukemic transformation. Molecular studies of coexisting JAK2 V617F-positive myeloproliferative neoplasms and mature B cell malignancies indicate distinct disease entities arising in myeloid and lymphoid committed hematopoietic progenitor cells, respectively. Mutations of CALR in essential thrombocythemia appear to be associated with a distinct phenotype and a lower risk of thrombosis yet their impact on disease progression is less well defined. The as yet undescribed scenario of pro-B cell acute lymphoblastic leukemia arising in CALR mutated essential thrombocythemia is presented. Intensive treatment for the leukemia allowed for expansion of the original CALR mutated clone. Whether CALR mutations in myeloproliferative neoplasms predispose to the acquisition of additional malignancies, particularly lymphoproliferative disorders, is not yet known.