Digital-PCR assay for screening and quantitative monitoring of calreticulin (CALR) type-2 positive patients with myelofibrosis following allogeneic stem cell transplantation

Badbaran, Anita; Christopeit, Maximilian; Aranyossy, Tim; Ayuk, Francis; Wolschke, Christine

doi:10.1038/bmt.2016.14

Cited by 15 publications

(15 citation statements)

References 13 publications

(13 reference statements)

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“…Implementation of next generation sequencing (NGS) into the clinical diagnostic laboratory setting is complex with specific technical training of staff and informatics expertise for result interpretation required, although advances have been made in an effort to make the technique more user-friendly. Digital PCR (dPCR) has recently been described for accurate and highly sensitive quantification of type 1 and/or type 2 CALR mutant allele burden 22 23. However, the quantitative detection of all identified CALR mutations would require the development of a large number of individual or multiplex assays using dPCR.…”

Section: Discussionmentioning

confidence: 99%

A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

Keaney¹,

O’Connor

Krawczyk

et al. 2017

J Clin Pathol

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Section: Discussionmentioning

confidence: 99%

A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

Keaney¹,

O’Connor

Krawczyk

et al. 2017

J Clin Pathol

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“…Although the importance of the CALR allelic burden determination has not yet been defined at the disease onset, the utility of and need for a sensitive method, like our ddPCR assay, are unquestionable for the purposes of MRD monitoring [8][9][10].…”

Section: Dear Editormentioning

confidence: 99%

“…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 CALR mut load was 13.5 % (Fig. 1b, c).Although the importance of the CALR allelic burden determination has not yet been defined at the disease onset, the utility of and need for a sensitive method, like our ddPCR assay, are unquestionable for the purposes of MRD monitoring [8][9][10].Electronic supplementary material The online version of this article ( …”

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confidence: 98%

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

et al. 2016

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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 ...

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“…As compared to FLA or qPCR, dPCR was capable of detecting delayed clearance of MRD after ASCT and earlier detection of increasing MRD levels prior to clinical relapse [59, 60]. Sequential dPCR evaluations in a limited number MPN patients treated with interferon have shown reductions in the CALR mutant allele burden and, in one case, disappearance of the mutant clone at the time of transformation to acute myeloid leukemia [61].…”

Section: Emerging Approachesmentioning

confidence: 99%

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

Haslam

Langabeer

2016

BioMed Research International

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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.

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Digital-PCR assay for screening and quantitative monitoring of calreticulin (CALR) type-2 positive patients with myelofibrosis following allogeneic stem cell transplantation

Cited by 15 publications

References 13 publications

A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

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

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

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