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 (MR(1)-MR(4)), 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 MR(4.5) 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 MR(4.5) 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.

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 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) 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).

Lupus anticoagulant and thrombosis in splenic marginal zone lymphoma.

INTRODUCTION: Splenic marginal zone lymphoma (SMZL) is a rare low-malignant Non-Hodgkin lymphoma (NHL), in which immune mediated paraneoplastic phenomena such as autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia (ITP) and C1 esterase inhibitor deficiency are relatively common. MATERIALS AND METHODS: We performed a multicenter retrospective study in 70 patients on the prevalence and clinical features of antiphospholipid antibodies (aPLA) in SMZL. RESULTS AND CONCLUSIONS: Nine patients (13%) had the diagnosis of a lupus anticoagulant (LA). The occurrence of venous thromboembolic events was significantly higher in LA positive patients compared to LA negative patients (4/9 [44%] vs 5/61 [8%], p = 0.002), especially within 12 months after splenectomy (3/6 [50%] vs 2/28 [7%], p = 0.007). None of the patients with LA had a persistent complete remission of LA after splenectomy, but complete remission of LA was achieved in 2/2 patients after rituximab-bendamustine immuno-chemotherapy. In conclusion, our data show a relatively high prevalence of aPLA in SMZL and an increased risk of postsplenectomy thrombosis in these patients. The fact that rituximab-bendamustine was effective for eradicating LA may be considered as an argument for using immuno-chemotherapy as first line therapy in SMZL patients with LA.

The biological and clinical significance of MLL abnormalities in haematological malignancies.

The MLL (Mixed Lineage Leukaemia or Myeloid/Lymphoid Leukaemia) gene on chromosome 11q23 is frequently involved in chromosomal translocations associated with human acute leukaemias. These translocations lead to fusion genes generally resulting in novel chimeric proteins containing the amino terminus of MLL fused in-frame to one of about 30 distinct partner proteins. Abnormalities involving the MLL gene are observed in leukaemias of either lymphoid or myeloid lineage derivation, as well as in poorly differentiated or biphenotypic leukaemias. They are frequently seen in infant patients, and patients with therapy-related secondary AML following treatment with inhibitors of topoisomerase II (epipodophyllotoxins). In the majority of cases, abnormalities involving the MLL gene are associated with a very poor prognostic outcome. In this review, we will discuss some of the recent advances in MLL research resulting from biological as well as clinical studies.

Prognostic significance of molecular staging by PCR-amplification of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma (DLBCL).

The prognostic value of the detection of peripheral blood (PB) and/or bone marrow (BM) involvement by polymerase chain reaction (PCR) amplification of rearranged immunoglobulin heavy chain (IgH) and immunoglobulin kappa light chain (Igkappa) genes was evaluated in 155 patients with diffuse large B-cell lymphomas (DLBCL). Immunoglobulin gene rearrangements (IgR) were detected in 35/155 (23%) patients. The presence of IgR in PB/BM was related to clinical stage (CS I-III vs CS IV; P<0.001), histopathological detection of BM involvement (P<0.001), and the International Prognostic Index (P<0.001). IgR-positive cases had a significantly lower complete remission (CR) rate (18/35, 51%) than IgR-negative patients (85/120, 71%; P=0.042), and a significantly poorer overall survival (OAS) at 5 years (25 vs 66%; P<0.001). There was a significant difference in the estimated OAS at 5 years between patients with negative BM histology and negative PCR results (66%), patients with negative BM histology but positive IgR (37%), and patients with positive BM histology (12%). Our results indicate that molecular methods improve the accuracy of staging in patients with DLBCL and define a group of patients with normal bone marrow histology who have a significantly poorer OAS due to molecular detection of PB/BM involvement.