Quantification of the Mutant CALR Allelic Burden by Digital PCR

Mansier, Olivier; Migeon, Marina; Saint-Lézer, A.; James, Chloé; Verger, Emmanuelle; Robin, Marie; Socié, Gérard; Bidet, Audrey; Mahon, François‐Xavier; Cassinat, Bruno; Lippert, Éric

doi:10.1016/j.jmoldx.2015.07.007

Cited by 30 publications

(12 citation statements)

References 26 publications

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“…The ddPCR was performed on a QX-100 Droplet Digital System (Bio-Rad, Hercules, CA), and the mutated allele burden was analysed by multiplex PCR assays for the CALR type 1 and type 2 mutations on 10–17.000 droplets. The assays and reactions were performed as previously described [ 29 ], with the following changes for the type 2 assay: primer concentrations were 300 nM and probe concentration was 200 nM while the annealing temperature was set to 60°C for both assays.…”

Section: Methodsmentioning

confidence: 99%

“…For routine diagnostic purposes, screening for CALR mutations by fragment analysis is sufficiently sensitive as the majority of CALR mutation positive samples appear to have above 15% mutant alleles [ 27 ]. However, post-transplantation relapse monitoring of CALR mutation positive AML patients and evaluation of treatment response in ET patients during IFN treatment have established the value of sensitive and quantitative determination of CALR mutations [ 22 , 23 , 28 , 29 ]. Sensitive quantitative polymerase chain reaction (qPCR) assays are currently the method of choice for monitoring mutant allele burden including deep IFN-induced remissions as seen in a subset of JAK2- V617F positive MPN patients [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

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Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment

et al. 2016

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Discovery of somatic mutations in the calreticulin gene (CALR) has identified a subgroup of Philadelphia-negative chronic myeloproliferative neoplasms (MPN) with separate haematological characteristics and prognosis. CALR mutations serve as novel markers both of diagnostic value and as targets for monitoring molecular responses during therapy. Interferon-α (IFN) selectively targets the malignant clone in a subset of MPN patients and can induce both haematological and molecular remissions in CALR mutated essential thrombocythemia (ET) patients. We investigated the response to IFN in a cohort of 21 CALR mutated MPN patients including ET, prefibrotic primary myelofibrosis (pre-PMF), and primary myelofibrosis (PMF) with a median follow-up of 31 months. For evaluation of a molecular response, we developed highly sensitive quantitative PCR (qPCR) assays for monitoring the mutant allele burden of the two most prevalent CALR mutations (type 1 and type 2). Thirteen patients (62%) experienced a decrease in the mutant allele burden with a median decline of 29% from baseline. However, only four patients, including patients with ET, pre-PMF, and PMF diagnosis, achieved molecular responder (MR) status with >50% reduction in mutant allele burden according to European LeukemiaNet (ELN) guidelines. MR patients displayed significant differences in the dynamics of the CALR mutant load with regard to time to response and dynamics in mutant allele burden after discontinuation of IFN treatment. Furthermore, we highlight the prognostic value of the CALR mutant allele burden by showing a close association with leucocyte- and platelet counts, hemoglobin concentration, in addition to plasma lactate dehydrogenase (LDH) irrespective of molecular response and treatment status.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment

et al. 2016

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“…However, only in the last few years dPCR has been broadly introduced in molecular diagnostics in different areas of medicine [ 11 , 12 , 13 ]. In hematology, several studies have indicated applicability of dPCR for the detection of minimal residual disease using a variety of molecular markers [ 14 , 15 , 16 , 17 ]. As noted above, principal suitability of dPCR for analysis of hematopoietic chimerism after allogeneic SCT has also been shown [ 7 , 8 ].…”

Section: Introductionmentioning

confidence: 99%

Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

Stahl

Rothe²,

Böhme³

et al. 2016

IJMS

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Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current "gold standard" for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.

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“…Total RNA was extracted using Trizol reagent (Life technologies, Courtaboeuf, France), treated by DNAse (Ambion) and reverse-transcribed with First Strand cDNA synthesis kit (Roche Diagnostics, Meylan, France). CALR mRNA expression was assessed by digital droplets PCR (ddPCR) as previously described [49]. UPR target gene expression was evaluated by RT-qPCR as previously described [50].…”

Section: Rt-qpcr and Ddpcrmentioning

confidence: 99%

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

Mansier

Prouzet‐Mauléon

Jégou

et al. 2019

Cancers

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Background: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase – Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. Methods: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. Results: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. Conclusions: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.

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Quantification of the Mutant CALR Allelic Burden by Digital PCR

Cited by 30 publications

References 26 publications

Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment

Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment

Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

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