title: Quantification of the Mutant CALR Allelic Burden by Digital PCR: Application to Minimal Residual Disease Evaluation after Bone Marrow Transplantation.
abstract: With the recent discovery of CALR mutations, >80% of patients with myeloproliferative neoplasms carry a phenotype-driving mutation. For JAK2 V617F, the most frequent mutation in myeloproliferative neoplasms, accurate determination of mutational loads is of interest at diagnosis, for phenotypic and prognostic purposes, and during follow-up for minimal residual disease assessment. We developed a digital PCR technique that allowed the accurate determination of CALR allelic burdens for the main mutations (types 1 and 2). Compared with the commonly used fluorescent PCR product analysis, digital PCR is more precise, reproducible, and accurate. Furthermore, this method reached a very high sensitivity. We detected at least 0.025% CALR mutants. It can thus be used for patient characterization at diagnosis and for minimal residual disease monitoring. When applied to patients with primary myelofibrosis who underwent hematopoietic stem cell transplant, the digital PCR detected low levels of minimal residual disease. After negativation of the mutational load in all patients, the disease reappeared at a low level in one patient, preceding hematologic relapse. In conclusion, digital PCR adapted to type 1 and 2 CALR mutations is an inexpensive, highly precise, and sensitive technique suitable for evaluation of myeloproliferative neoplasm patients during follow-up.
authors.affiliation: Hematology Laboratory, CHU de Bordeaux, Bordeaux, France; Leukemic Hematopoiesis and Therapeutic Targets Laboratory, Biothérapie des maladies génétiques et cancers, Université de Bordeaux, Bordeaux, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Mansier
authors.affiliation: Hematology Laboratory, CHU de Bordeaux, Bordeaux, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Migeon
authors.affiliation: Service of Blood Diseases, CHU de Bordeaux, Bordeaux, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Saint-Lézer
authors.affiliation: Hematology Laboratory, CHU de Bordeaux, Bordeaux, France; Cardiovascular Adaptation to Ischemia, Université de Bordeaux, Bordeaux, France.
authors.forename: Chloé
authors.initials: C
authors.lastname: James
authors.affiliation: Cell Biology Service, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Verger
authors.affiliation: Hematology Transplants Service, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Robin
authors.affiliation: Hematology Transplants Service, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: Gérard
authors.initials: G
authors.lastname: Socié
authors.affiliation: Hematology Laboratory, CHU de Bordeaux, Bordeaux, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Bidet
authors.affiliation: Hematology Laboratory, CHU de Bordeaux, Bordeaux, France; Leukemic Hematopoiesis and Therapeutic Targets Laboratory, Biothérapie des maladies génétiques et cancers, Université de Bordeaux, Bordeaux, France.
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Mahon
authors.affiliation: Cell Biology Service, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Cassinat
authors.affiliation: Hematology Laboratory, CHU de Bordeaux, Bordeaux, France; Leukemic Hematopoiesis and Therapeutic Targets Laboratory, Biothérapie des maladies génétiques et cancers, Université de Bordeaux, Bordeaux, France. Electronic address: eric.lippert@univ-brest.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Lippert
mesh_terms: D000483:Alleles; D016026:Bone Marrow Transplantation; D037282:Calreticulin / Q000235:genetics*; D005091:Exons; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D009154:Mutation; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D055728:Primary Myelofibrosis / Q000175:diagnosis* / Q000235:genetics
chemical_list: D037282:Calreticulin