Droplet digital PCR assay for quantifying of CALR mutant allelic burden in myeloproliferative neoplasms

Abstract

Dear Editor,Calreticulin (CALR) gene mutations (CALR mut ) have recently been discovered in about 20-35 % of patients affected by essential thrombocythemia (ET) and primary myelofibrosis (PMF) [1,2]. Several molecular assays have been developed to detect the most frequent CALR mut (type 1 consisting of a 52-bp deletion, and type 2 of a 5-bp insertion) [3,4]. All these techniques are useful for identifying CALR mut at the diagnosis, but they are not suitable for minimal residual disease (MRD) monitoring, since the maximum sensitivity is 1 %. The droplet digital PCR (ddPCR) technology is a third-generation PCR method that started to be used in hematological malignancies [5][6][7]. We describe a ddPCR assay with a sensitivity of 0.01 % developed for the absolute quantification of CALR type 1 and 2 mutations and analyze a cohort of 57 JAK2V617F-negative myeloproliferative neoplasm patients. ddPCR experiments were performed using the QX-200 instrument (BioRad) and specific primers and probes were designed for both type 1 and type 2 mutations (see Supplementary Files). CALR mut load in each sample was expressed as fractional abundance (FA, mutant allele/mutant allele + wild-type allele). The CALR mut allelic burden resulted heterogeneous in both ET (min.13.8 %-max. 51 %) and PMF (min. 34.5 %-max. 51.3 %) patients. We show that the median CALR mut allelic burden at diagnosis was significantly higher in PMF patients as compared to ET case (47.9 vs 43.8 %, p = 0.008) whereas no significant difference was observed between the type 1 and 2 mutations (Fig. 1a). Moreover, no relationship between the gene mutation type and the CALR mut amount was observed within each group of ET and PMF patients. In our ET series, there were 14 (29.7 %) patients with a very low FA, <30 %; this group was not statistically different in terms of hemoglobin, white blood cells and platelet counts, age, sex, thrombosis and/or hemorrhage, and CALR mut type compared to those with FA >30 %. The PMF group included ten patients, too few for any type of statistical considerations.Sequential evaluations by ddPCR experiments were performed in three patients to monitor the CALR mut load during treatment. CALR mut load at diagnosis was 15.8 and 48 % in two ET patients. The former patient was treated with interferon-α (IFN-α) and after 5 years from diagnosis the FA was 7.7 %. The latter was also treated with IFN-α and after 2 years from diagnosis, the CALR mut load was 14.7 %. Both patients had stable disease and a well-controlled platelet count. A 44-year-old man at PMF diagnosis showed a FA of 49.7 %; 8 years later, we observed a leukemic transformation. At the time of the AML evolution, the CALR mut load was 0 %; this finding was also confirmed by PCR qualitative analysis. The patient underwent induction chemotherapy, achieving complete remission, then allogeneic bone marrow transplantation (ABMT) from a HLA-matched related donor. Two months later, the FA observed by ddPCR analysis was 0.01 %; 7 months after, the ABMT and AML relapsed and at this time, the ...

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia

“…In fact, in comparison with quantitative analog PCR, ddPCR has the potential to make quantitative analyses more reproducible, and precise quantitation might facilitate a variety of clinical tests. However, there are only few studies focused on the use of ddPCR in hematological malignancies [21–26]. In a recent report, NOTCH1 mut were detected by NGS in 11% of monoclonal B cell lymphocytosis (MBL) and 13.4% of CLL Binet stage A patients [27]; moreover, this mutation was frequently observed at a low clonal level, particularly in MBL patients, and sequential analyses demonstrated that the NOTCH1 mut generally did not appear during the disease course, and that the mutational burden in positive cases remained stable over time.…”

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study

“…Quantitative real-time PCR (qPCR) has been widely applied to nucleic acid-based diagnostic tests, but lack of standardization and a relatively poor accuracy have hindered its usefulness in some clinical applications. Droplet digital PCR (ddPCR) is a novel PCR-based technology that has already been successfully used for sensitive and reproducible detection of different pathogenetic variants [11,12].…”

Monitoring minimal residual disease by ddPCR in acute lymphoblastic leukemia associated with the FGFR1 gene rearrangement

“…On the contrary, the ddPCR technique can estimate the absolute quantitation of a molecular target without the need for calibrators or standard samples; moreover, it is especially powerful in MRD monitoring, as previously demonstrated. [10][11][12] We report for the first time the absolute quantitation of the BCR- ALL with t(8;22)/BCR-FGFR1 fusion is a very aggressive tumor characterized by rapid evolution, for which HSCT is strongly recommended; however, the recent use of tyrosine kinase inhibitors has expanded the treatment opportunities for these patients. 5 Our results demonstrate that ddPCR can not only provide quantitative data about the residual tumor burden, but also, as demonstrated for other hematologic malignancies, 11,12 it has adequate sensitivity to provide the chance of following disease response and modulating treatment, maybe preventing disease progression.…”