M. E. Sarasquete

MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenström's macroglobulinemia.

We evaluated the MYD88 L265P mutation in Waldenström’s macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10−3). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23−CD27++FMC7++), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3–23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.

Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry

We have analyzed the applicability, sensitivity and prognostic value of allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) as a method for minimal residual disease (MRD) assessment in patients with multiple myeloma (MM), comparing the results with those of multiparameter flow cytometry (MFC). A total of 170 patients enrolled in three consecutive Spanish trials achieving at least partial response after treatment were included. Lack of clonality detection (n=31), unsuccessful sequencing (n=17) and suboptimal ASO performance (n=51) limited the applicability of PCR to 42% of cases. MRD was finally investigated in 103 patients (including 32 previously studied) with persistent disease identified by PCR and MFC in 54% and 46% of cases, respectively. A significant correlation in MRD quantitation by both the techniques was noted (r=0.881, P<0.001), being reflective of treatment intensity. Patients with <10−4 residual tumor cells showed longer progression-free survival (PFS) compared with the rest (not reached (NR) vs 31 months, P=0.002), with similar results observed with MFC. Among complete responders (n=62), PCR discriminated two risk groups with different PFS (49 vs 26 months, P=0.001) and overall survival (NR vs 60 months, P=0.008). Thus, although less applicable than MFC, ASO RQ-PCR is a powerful technique to assess treatment efficacy and risk stratification in MM.

SNP-based mapping arrays reveal high genomic complexity in monoclonal gammopathies, from MGUS to myeloma status

Genetic events mediating transformation from premalignant monoclonal gammopathies (MG) to multiple myeloma (MM) are unknown. To obtain a comprehensive genomic profile of MG from the early to late stages, we performed high-resolution analysis of purified plasma cells from 20 MGUS, 20 smoldering MM (SMM) and 34 MM by high-density 6.0 SNP array. A progressive increase in the incidence of copy number abnormalities (CNA) from MGUS to SMM and to MM (median 5, 7.5 and 12 per case, respectively) was observed (P=0.006). Gains on 1q, 3p, 6p, 9p, 11q, 19p, 19q and 21q along with 1p, 16q and 22q deletions were significantly less frequent in MGUS than in MM. Although 11q and 21q gains together with 16q and 22q deletions were apparently exclusive of MM status, we observed that these abnormalities were also present in minor subclones in MGUS. Overall, a total of 65 copy number-neutral LOH (CNN-LOH) were detected. Their frequency was higher in active MM than in the asymptomatic entities (P=0.047). A strong association between genetic lesions and fragile sites was also detected. In summary, our study shows an increasing genomic complexity from MGUS to MM and identifies new chromosomal regions involved in CNA and CNN-LOH.

Bisphosphonate‐related osteonecrosis: genetic and acquired risk factors

The objectives of this study were to review epidemiological, clinical and biological aspects associated with the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ) in multiple myeloma (MM) patients, with special emphasis on the genetic aspects. A detailed review of previously described risk factors as well as recent genetic findings mostly comprises this work. The most recent meeting abstracts and relevant articles published in journals covered by the Science Citation Index and Medline are also examined. The review pays special attention to the genetic component of BRONJ. A total of 15 series and 14 guidelines or revisions were selected to fit the aims of the review. Gene variability was reviewed in depth to give a clinical illustration on the genetic aspects of BRONJ. Crude prevalence and 5-year cumulative incidence were considered as the most important end points for predictive purposes. Several acquired factors were recognized as predictors for BRONJ in MM, especially intravenous bisphosphonates, dental trauma and advanced age. Among genetic factors, polymorphisms on CYP2C8 gene arise as a promising risk factor. Bisphosphonate-related osteonecrosis of the jaw can be predicted with a conjunction of genetic and environmental risk factors.

Phenotypic identification of subclones in multiple myeloma with different chemoresistant, cytogenetic and clonogenic potential

Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia.

There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

Low expression of ZHX2, but not RCBTB2 or RAN, is associated with poor outcome in multiple myeloma

RAN, ZHX2 and RCBTB2 (CHC1L) expression was evaluated by quantitative real time reverse transcription polymerase chain reaction in plasma cells from 85 monoclonal gammopathies: 58 symptomatic multiple myeloma (MM) (52 untreated, six relapsed), eight smouldering MM, five monoclonal gammopathy of undetermined significance, four plasma cell leukaemias and 10 myeloid cell lines. ZHX2 was weakly expressed in high‐risk/proliferative disease compared to low‐risk or indolent disease. High ZHX2 expression was associated with better response and longer survival after high‐dose therapy. RCBTB2 expression was weaker in hyperdiploid versus non‐hyperdiploid cases while RAN was more expressed in symptomatic MM and cell lines.

Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders

Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype–phenotype correlations are important for predicting the clinical course of the disease and to allow tailor‐made follow‐up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next‐generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost.

Detection of MYD88 L265P mutation by real-time allele-specific oligonucleotide polymerase chain reaction.

MYD88 L265P mutation has been reported in ∼90% of Waldenström’s Macroglobulinemia (WM) patients and immunoglobulin M (IgM) monoclonal gammopathies of uncertain significance (MGUS), as well as in some cases of lymphoma and chronic lymphocytic leukemia. The present study aimed to develop a real-time allele-specific oligonucleotide PCR (ASO-RQ-PCR) to detect the MYD88 L265P mutation. We first evaluated the reproducibility and sensitivity of the technique with a diluting experiment of a previously known positive sample. Then, we evaluated the applicability of the methodology by analyzing 30 selected patients (10 asymptomatic WM, 10 symptomatic WM, and 10 IgM MGUS) as well as 10 healthy donors. The quantitative ASO-PCR assay could detect the MYD88 L265P mutation at a dilution of 0.25%, showing an inverse correlation between the tumor cell percentage and the cycle threshold (CT) value, thus allowing for tumor burden quantitation. In addition, mutated cases were distinguished from the unmutated by >10 cycles of difference between CTs. To sum up, ASO-RQ-PCR is an inexpensive, robust, and optimized method for the detection of MYD88 L265P mutation, which could be considered as a useful molecular tool during the diagnostic work-up of B-cell lymphoproliferative disorders.