Quantitative PCR: an alternative approach to detect common copy number alterations in multiple myeloma.

Chromosome 1q gains and 13q deletions are common cytogenetic aberrations in multiple myeloma (MM) that confer a poor prognosis. There are several techniques for the targeted study of these alterations, but interphase fluorescence in situ hybridization (FISH) is the current gold standard. The aim of the present study was to validate quantitative PCR (qPCR) as an alternative to FISH studies in CD138+-enriched plasma cells (PCs) from MM patients at diagnosis. We analyzed 1q gains and 13q deletions by qPCR in 57 and 60 MM patients, respectively. qPCR applicability was 84 and 88% for 1q and 13q, respectively. The qPCR and FISH methods had a sensitivity and specificity of 88 and 71% for 1q gains, and 79 and 100% for 13q deletions. A second qPCR assay for each region was carried out to confirm the previous results. Paired qPCR (two assays) and FISH results were available from 53 MM patients: 26 for 1q amplification and 27 for 13q deletion. qPCR assays gave concordant results (qPCR-consistent) in 20 of the 26 (77%) 1q gains and 25 of the 27 (93%) 13q deletions. Considering only the consistent data, the overall concordance among qPCR and FISH was 85 and 100% for 1q gains and 13q deletions, respectively. Our results show a substantial agreement between qPCR and the gold standard FISH technique, indicating the potential of qPCR as an alternative approach, particularly when the starting material is too scarce or cells are too damaged to obtain accurate results from FISH studies.

From Waldenström's macroglobulinemia to aggressive diffuse large B-cell lymphoma: a whole-exome analysis of abnormalities leading to transformation.

Transformation of Waldenström's macroglobulinemia (WM) to diffuse large B-cell lymphoma (DLBCL) occurs in up to 10% of patients and is associated with an adverse outcome. Here we performed the first whole-exome sequencing study of WM patients who evolved to DLBCL and report the genetic alterations that may drive this process. Our results demonstrate that transformation depends on the frequency and specificity of acquired variants, rather than on the duration of its evolution. We did not find a common pattern of mutations at diagnosis or transformation; however, there were certain abnormalities that were present in a high proportion of clonal tumor cells and conserved during this transition, suggesting that they have a key role as early drivers. In addition, recurrent mutations gained in some genes at transformation (for example, PIM1, FRYL and HNF1B) represent cooperating events in the selection of the clones responsible for disease progression. Detailed comparison reveals the gene abnormalities at diagnosis and transformation to be consistent with a branching model of evolution. Finally, the frequent mutation observed in the CD79B gene in this specific subset of patients implies that it is a potential biomarker predicting transformation in WM.

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

INTRODUCTION: 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. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.

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.

Mutation status and immunoglobulin gene rearrangements in patients from northwest and central region of Spain with chronic lymphocytic leukemia.

The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.

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.

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.

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.

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.

Frequency of HLA-A, -B and -DRB1 specificities and haplotypic associations in the population of Castilla y León (northwest-central Spain).

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y León and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y León is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.

Deregulation of microRNA expression in the different genetic subtypes of multiple myeloma and correlation with gene expression profiling.

Specific microRNA (miRNA) signatures have been associated with different cytogenetic subtypes in acute leukemias. This finding prompted us to investigate potential associations between genetic abnormalities in multiple myeloma (MM) and singular miRNA expression profiles. Moreover, global gene expression profiling was also analyzed to find correlated miRNA gene expression and select miRNA target genes that show such correlation. For this purpose, we analyzed the expression level of 365 miRNAs and the gene expression profiling in 60 newly diagnosed MM patients, selected to represent the most relevant recurrent genetic abnormalities. Supervised analysis showed significantly deregulated miRNAs in the different cytogenetic subtypes as compared with normal PC. It is interesting to note that miR-1 and miR-133a clustered on the same chromosomal loci, were specifically overexpressed in the cases with t(14;16). The analysis of the relationship between miRNA expression and their respective target genes showed a conserved inverse correlation between several miRNAs deregulated in MM cells and CCND2 expression level. These results illustrate, for the first time, that miRNA expression pattern in MM is associated with genetic abnormalities, and that the correlation of the expression profile of miRNA and their putative mRNA targets is useful to find statistically significant protein-coding genes in MM pathogenesis associated with changes in specific miRNAs.

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.

The presence of DRB1*01 allele in multiple myeloma patients is associated with an indolent disease.

The human leukocyte antigen (HLA) system could play an essential role in multiple myeloma (MM) disease control. This report describes the results comparing HLA-DRB1 phenotypic frequencies in 181 MM patients (53 smoldering/indolent MM and 128 symptomatic MM patients) and healthy individuals. Higher DRB1*01 phenotypic frequencies were found in the smoldering patients compared with symptomatic MM patients (38% vs 14%, P = 0.001) and with the healthy individuals (38% vs 22%, P = 0.01). Additionally, higher DRB1*07 phenotypic frequencies were found in symptomatic MM compared with control population (38% vs 28%, P = 0.01). The present data suggest that HLA-DRB1*01 individuals may have a better ability to efficiently present myeloma-related antigens to immunocompetent cells, which could favor a better immune response against the tumor. This would translate into a more appropriate disease control associated with more indolent disease and prolonged survival.

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.