Filomena Daraio

Genetic and clinical heterogeneity of ferroportin disease

Ferroportin is encoded by the SLC40A1 gene and mediates iron export from cells by interacting with hepcidin. SLC40A1 gene mutations are associated with an autosomal type of genetic iron overload described as haemochromatosis type 4, or HFE4 (Online Mendelian Inheritance in Man number 606069), or ferroportin disease. We report three families with this condition caused by novel SLC40A1 mutations. Denaturing high‐performance liquid chromatography was employed to scan for the SLC40A1 gene. A D181V (A846T) mutation in exon 6 of the ferroportin gene was detected in the affected members of an Italian family and shown to have a de novo origin in a maternal germinal line. This mutation was associated with both parenchymal and reticuloendothelial iron overload in the liver, and with reduced urinary hepcidin excretion. A G80V (G543T) mutation in exon 3 was found in the affected members of an Italian family with autosomal hyperferritinaemia,. Finally, a G267D (G1104A) mutation was identified in exon 7 in a family of Chinese descent whose members presented with isolated hyperferritinaemia. Ferroportin disease represents a protean genetic condition in which the different SLC40A1 mutations appear to be responsible for phenotypic variability. This condition should be considered not only in families with autosomal iron overload or hyperferritinaemia, but also in cases of unexplained hyperferritinaemia.

A certified plasmid reference material for the standardisation of BCR–ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Growth hormone (GH)-induced reconstitution of CD8+ CD28+ T lymphocytes in a rare case of severe lymphopenia associated with Juvenile Haemochromatosis and Turner's syndrome

This paper describes a rare case of Turners syndrome associated with Juvenile Haemochromatosis and severe lymphopenia, followed‐up for a period of 5 years. Because of the indication for treatment with growth hormone (GH), this case was observed as a model to analyse the effects of GH on growth, iron mobilization and lymphocyte reconstitution. For this purpose, a serial study of the T lymphocyte subpopulations CD4+, CD8+, CD8+ CD28+ and CD8+ CD28‐ was performed by immunophenotyping during the follow‐up period. Besides the impact of both phlebotomy treatment and GH on the rapid growth and mobilization of 20·8 g of iron in 136 weeks, the most relevant observation was the finding of a significant expansion of CD8+ T lymphocytes expressing the costimulatory marker CD28 in the setting of the severe lymphopenia. These findings constitute new clinical evidence supporting the notion that the GH/IGF‐1 system has an important role on the maintenance of T cell homeostasis in vivo, and that GH may be regarded as a putative therapeutic agent in T lymphocyte reconstitution.

Second-Generation Tyrosine Kinase Inhibitors Can Induce Complete Molecular Response in Ph-Positive Acute Lymphoblastic Leukemia After Allogeneic Stem Cell Transplant

Allogeneic stem cell transplant (SCT) is thought to be the only curative therapy of Phþ acute lymphoblastic leukemia (ALL). Recent results showed that Phþ ALL with minimal residual disease (MRD) after SCT benefitted from the use of imatinib. Three patients with Phþ ALL fromour institution showed different degrees of MRD increase after transplant and were treated with second-generation tyrosine kinase inhibitors, obtaining a complete molecular remission. These cases suggest a posttransplant application of TKIs to traet or prevent the relapse of Phþ ALL.

CALR-positive myeloproliferative disorder in a patient with Ph-positive chronic myeloid leukemia in durable treatment-free remission: a case report

Current diagnostic criteria for Philadelphia-negative myeloproliferative neoplasia (MPN) have been redefined by the discovery of Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL) and calreticulin (CALR) genetic alterations. Only few cases of coexistence of CALR-mutated MPN and Philadelphia-positive chronic myeloid leukemia (CML) have been described so far. Here we report the case of a patient with CML diagnosed in 2001, treated with imatinib and pegylated interferon (IFN) frontline. She reached complete molecular remission (CMR) and discontinued imatinib, maintaining treatment free remission. Due to persistent thrombocytosis, we repeated bone marrow (BM) analysis and diagnosed CARL-mutated essential thrombocythemia (ET). A CALR-positive clone was found to be present since 2001, and was unaffected by imatinib treatment, possibly representing a molecular abnormality arising at stem cell level.

Sensitive Replicate Real-Time Quantitative PCR of BCR-ABL Shows Deep Molecular Responses in Long-Term Post–Allogeneic Stem Cell Transplantation Chronic Myeloid Leukemia Patients

Real-time quantitative PCR (RT-qPCR) is commonly used for follow-up of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors, but its current sensitivity does not allow detection of very low BCR-ABL levels. Therefore RT-qPCR negativity is not synonymous with complete molecular response. Replicate RT-qPCR had shown increased sensitivity in tyrosine kinase inhibitor-treated patients and was, therefore, used here to evaluate whether RT-qPCR-negative post-allogeneic stem cell transplantation (SCT) patients harbor detectable disease. Samples from 12 patients were tested at 2 time points using 82 replicates of BCR-ABL RT-qPCR. One patient (38 months after SCT) had detectable transcripts at baseline and none at the follow-up test, done at a median of 107 months after SCT. This suggests cure from CML in the majority of allogeneic SCT patients who have no transcripts detectable by replicate RT-qPCR for BCR-ABL.