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.

The genomic profiling of high-risk smoldering myeloma patients treated with an intensive strategy unveils potential markers of resistance and progression.

Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.

Efficacy of mouth rinses and toothpaste on tooth whitening.

OBJECTIVES: People increasingly desire tooth whitening. Considering the wide range of whitening products on the market, this study evaluated the efficacy of whitening toothpastes and mouth rinses compared with the 10% carbamide peroxide (CP) whitening gel. METHODS: We obtained 120 cylindrical specimens from bovine teeth, which were darkened for 24 hours in a coffee solution. The color measurement was performed by a spectrophotometer using the CIE L*a*b* system, and specimens were divided into six groups according to the use of the following agents: group 1, conventional fluoridated toothpaste; group 2, Close Up White Now; group 3, Listerine Whitening; group 4, Colgate Plax Whitening; group 5, experimental mouth rinse with Plasdone; and group 6, 10% CP Whiteness Perfect. After the simulation of 12 weeks of treatment for groups 1 to 5 and 14 days of treatment for group 6, the specimens were subjected to a new color reading. RESULTS: Data were subjected to one-way analysis of variance (α=0.05), which showed significant differences among groups after 12 weeks for ΔE (p=0.001). Results of the Tukey test revealed that groups 3, 4, and 6 presented significantly higher color alteration than groups 1, 2, and 5. CONCLUSIONS: The whitening toothpaste Close Up White Now and the experimental mouth rinse with Plasdone showed similar color alteration as conventional toothpaste after a 12-week treatment simulation. These groups presented significantly lower color alteration compared with whitening mouth rinses Listerine and Colgate Plax Whitening, which showed similar results to those observed after 14 days of bleaching with 10% CP treatment.

Identification of a novel recurrent gain on 20q13 in chronic lymphocytic leukemia by array CGH and gene expression profiling.

BACKGROUND: The presence of genetic changes is a hallmark of chronic lymphocytic leukemia (CLL). The most common cytogenetic abnormalities with independent prognostic significance in CLL are 13q14, ATM and TP53 deletions and trisomy 12. However, CLL displays a great genetic and biological heterogeneity. The aim of this study was to analyze the genomic imbalances in CLL cytogenetic subsets from both genomic and gene expression perspectives to identify new recurrent alterations. PATIENTS AND METHODS: The genomic imbalances and expression levels of 67 patients were analyzed. The novel recurrent abnormalities detected with bacterial artificial chromosome array were confirmed by FISH and oligonucleotide microarrays. In all cases, gene expression profiling was assessed. RESULTS: Copy number alterations were identified in 75% of cases. Overall, the results confirmed FISH studies for the regions frequently involved in CLL and also defined a new recurrent gain on chromosome 20q13.12, in 19% (13/67) of the CLL patients. Oligonucleotide expression correlated with the regions of loss or gain of genomic material, suggesting that the changes in gene expression are related to alterations in copy number. CONCLUSION: Our study demonstrates the presence of a recurrent gain in 20q13.12 associated with overexpression of the genes located in this region, in CLL cytogenetic subgroups.

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.

Gene expression profiling of B lymphocytes and plasma cells from Waldenström's macroglobulinemia: comparison with expression patterns of the same cell counterparts from chronic lymphocytic leukemia, multiple myeloma and normal individuals.

The tumoral clone of Waldenström's macroglobulinemia (WM) shows a wide morphological heterogeneity, which ranges from B lymphocytes (BL) to plasma cells (PC). By means of genome-wide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell counterparts from chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes downregulated in WM-BL were IL4R, which plays a relevant role in CLL B-cell survival, and BACH2, which participates in the development of class-switched PC. Interestingly, one of the upregulated genes in WM-BL was IL6. A set of four genes was able to discriminate clonal BL from WM and CLL: LEF1 (WNT/beta-catenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5, which was overexpressed in WM-PC, and IRF4 and BLIMP1, which were underexpressed. In addition, three of the target genes activated by PAX5 - CD79, BLNK and SYK - were upregulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell counterpart.

Prognostic and biological implications of genetic abnormalities in multiple myeloma undergoing autologous stem cell transplantation: t(4;14) is the most relevant adverse prognostic factor, whereas RB deletion as a unique abnormality is not associated with adverse prognosis.

Fluorescence in situ hybridization (FISH) has become a powerful technique for prognostic assessment in multiple myeloma (MM). However, the existence of associations between cytogenetic abnormalities compels us to re-assess the value of each abnormality. A total of 260 patients with MM at the time of diagnosis, enrolled in the GEM-2000 Spanish transplant protocol, have been analyzed by FISH in order to ascertain the independent influence on myeloma prognosis of IGH translocations, as well as RB and P53 deletions. Survival analyses showed that patients with t(4;14), RB or P53 deletions had a significantly shorter survival than patients without these abnormalities. However, patients with RB deletions without other abnormalities in FISH analysis, displayed a similar outcome to those patients without genetic changes by FISH (46 vs 54 months, P=0.3). In the multivariate analysis the presence of t(4;14), RB deletion associated with other abnormalities, age >60 years, high proportion of S-phase cells and advanced stage of the disease according to the International Staging System retained their independent prognostic influence. In summary, RB deletion as a sole abnormality does not lead to a shortening in the survival of MM patients, whereas t(4;14) confers the worst prognosis in MM patients treated with high-dose chemotherapy.

Prognostic value of antigen expression in multiple myeloma: a PETHEMA/GEM study on 1265 patients enrolled in four consecutive clinical trials.

Persistence of minimal residual disease (MRD) after treatment for myeloma predicts inferior outcomes, but within MRD-positive patients there is great heterogeneity with both early and very late relapses. Among different MRD techniques, flow cytometry provides additional information about antigen expression on tumor cells, which could potentially contribute to stratify MRD-positive patients. We investigated the prognostic value of those antigens required to monitor MRD in 1265 newly diagnosed patients enrolled in the GEM2000, GEM2005MENOS65, GEM2005MAS65 and GEM2010MAS65 protocols. Overall, CD19pos, CD27neg, CD38lo, CD45pos, CD81pos, CD117neg and CD138lo expression predicted inferior outcomes. Through principal component analysis, we found that simultaneous CD38lowCD81posCD117neg expression emerged as the most powerful combination with independent prognostic value for progression-free survival (HR:1.69; P=0.002). This unique phenotypic profile retained prognostic value among MRD-positive patients. We then used next-generation flow to determine antigen stability throughout the course of the disease, and found that the expression of antigens required to monitor MRD is mostly stable from diagnosis to MRD stages, except for CD81 whose expression progressively increased from baseline to chemoresistant tumor cells (14 vs 28%). Altogether, we showed that the phenotypic profile of tumor cells provides additional prognostic information, and could be used to further predict risk of relapse among MRD-positive patients.

Molecular biology of myeloma.

Multiple myeloma (MM) is a B-cell malignancy characterised by the accumulation of clonal plasma cells (PC) in the bone marrow (BM). The molecular bases for this incurable disease have been widely investigated in the last years, and the development of modern genomic technologies has contributed to the understanding of the pathogenesis of MM. The molecular mechanisms that explain the cellular origin of myeloma cells, the cytogenetic abnormalities and their clinical implications, and the biological information provided by gene expression profiling analysis are reviewed in this paper. In addition, a molecular classification of MM in seven groups based on the relationship between gene expression profiling, chromosomal translocations and prognostic outcome is also presented. And finally, the recent hypothesis of a potential unifying event in the pathogenesis of MM, supported by cyclin D deregulation in virtually all MM tumours, will be summarised.

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.

Differentiation stage of myeloma plasma cells: biological and clinical significance.

The notion that plasma cells (PCs) are terminally differentiated has prevented intensive research in multiple myeloma (MM) about their phenotypic plasticity and differentiation. Here, we demonstrated in healthy individuals (n=20) that the CD19-CD81 expression axis identifies three bone marrow (BM)PC subsets with distinct age-prevalence, proliferation, replication-history, immunoglobulin-production, and phenotype, consistent with progressively increased differentiation from CD19+CD81+ into CD19-CD81+ and CD19-CD81- BMPCs. Afterwards, we demonstrated in 225 newly diagnosed MM patients that, comparing to normal BMPC counterparts, 59% had fully differentiated (CD19-CD81-) clones, 38% intermediate-differentiated (CD19-CD81+) and 3% less-differentiated (CD19+CD81+) clones. The latter patients had dismal outcome, and PC differentiation emerged as an independent prognostic marker for progression-free (HR: 1.7; P=0.005) and overall survival (HR: 2.1; P=0.006). Longitudinal comparison of diagnostic vs minimal-residual-disease samples (n=40) unraveled that in 20% of patients, less-differentiated PCs subclones become enriched after therapy-induced pressure. We also revealed that CD81 expression is epigenetically regulated, that less-differentiated clonal PCs retain high expression of genes related to preceding B-cell stages (for example: PAX5), and show distinct mutation profile vs fully differentiated PC clones within individual patients. Together, we shed new light into PC plasticity and demonstrated that MM patients harbouring less-differentiated PCs have dismal survival, which might be related to higher chemoresistant potential plus different molecular and genomic profiles.

Gene expression profile reveals deregulation of genes with relevant functions in the different subclasses of acute myeloid leukemia.

Bone marrow samples from 43 adult patients with de novo diagnosed acute myeloid leukemia (AML)--10 acute promyelocytic leukemias (APL) with t(15;17), four AML with inv(16), seven monocytic leukemias and 22 nonmonocytic leukemias--were analyzed using high-density oligonucleotide microarrays. Hierarchical clustering analysis segregated APL, AML with inv(16), monocytic leukemias and the remaining AML into separate groups. A set of only 21 genes was able to assign AML to one of these three classes: APL, inv(16) and other AML subtype without a specific translocation. Quantitative RT-PCR performed for 18 out of these predictor genes confirmed microarray results. APL expressed high levels of FGF13 and FGFR1 as well as two potent angiogenic factors, HGF and VEGF. AML with inv(16) showed an upregulation of MYH11 and a downregulation of a gene encoding a core-binding factor protein, RUNX3. Genes involved in cell adhesion represented the most altered functional category in monocytic leukemias. Two major groups emerged from the remaining 22 AML: cluster A with 10 samples and cluster B with 12. All the eight leukemias that were either refractory to treatment or that relapsed afterwards were assigned to cluster B. In the latter cluster, CD34 upregulation and serine proteases downregulation is consistent with a maturation arrest and lack of granulocytic differentiation.

Genetic heterogeneity of BCR/ABL+ adult B-cell precursor acute lymphoblastic leukemia: impact on the clinical, biological and immunophenotypical disease characteristics.

Philadelphia-positive (Ph(+)) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous disease with a very poor prognosis. In this study, we analyzed the frequency of supernumerary Ph, trisomy 8, monosomy 7, and del(9p21) by FISH and its relationship with the characteristics of the disease, in 46 BCR/ABL(+) adult BCP-ALL patients. The frequency of supernumerary Ph, trisomy 8, monosomy 7 and del(9p21) was 30%, 20%, 15%, and 24%, respectively. Although all patients displayed a BII/common phenotype, supernumerary Ph and trisomy 8 were associated with higher expression of CD19 and CD22 and of CD19, CD34, CD45, and HLA-DR, respectively; in turn, cases with monosomy 7 showed lower CD19, CD22, CD34, and cCD79a and del(9p21)(+) blasts were CD13(-) and CD33(-). Overall, similar clinical and hematological features were observed at presentation, independently of the underlying genetic abnormalities. However, relapse-free survival (RFS) was significantly shorter in cases with supernumerary Ph, trisomy 8, and del(9p21), the latter being the most powerful independent prognostic factor for RFS.

In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4ß1 cytoplasmic-binding proteins.

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4ß1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are ß1-integrin-binding partners that regulate ß1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4ß1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4ß1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4ß1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4ß1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4ß1 activity.

Bence Jones proteinuria in smoldering multiple myeloma as a predictor marker of progression to symptomatic multiple myeloma.

The diagnosis of smoldering multiple myeloma (SMM) includes patients with a heterogeneous risk of progression to active multiple myeloma (MM): some patients will never progress, whereas others will have a high risk of progression within the first 2 years. Therefore, it is important to improve risk assessment at diagnosis. We conducted a retrospective study in a large cohort of SMM patients, in order to investigate the role of Bence Jones (BJ) proteinuria at diagnosis in the progression to active MM. We found that SMM patients presenting with BJ proteinuria had a significantly shorter median time to progression (TTP) to MM compared with patients without BJ proteinuria (22 vs 88 months, respectively; hazard ratio=2.3, 95% confidence interval=1.4-3.9, P=0.002). We also identified risk subgroups based on the amount of BJ proteinuria: ⩾500 mg/24 h, <500 mg/24 h and without it, with a significantly different median TTP (13, 37 and 88 months, P<0.001). Thus, BJ proteinuria at diagnosis is an independent variable of progression to MM that identifies a subgroup of high-risk SMM patients (51% risk of progression at 2 years) and ⩾500 mg of BJ proteinuria may allow, if validated in another series, to reclassify these patients to MM requiring therapy before the end-organ damage development.

Differences in genetic changes between multiple myeloma and plasma cell leukemia demonstrated by comparative genomic hybridization.

To analyze the genomic differences between multiple myeloma (MM) and plasma cell leukemia (PCL), a total of 30 cases were studied by comparative genomic hybridization (CGH). In five cases with a low proportion of plasma cells (PC) in bone marrow, an enrichment of PC was performed by using immunomagnetic beads conjugated with the monoclonal antibody B-B4. In 24 out of the 25 MM (96%) and in all five PCL (100%) patients DNA copy number changes were identified by CGH analysis; in the MM case without chromosomal imbalances, the immunomagnetic enrichment of PC had failed. The most recurrent changes in MM patients were gains at chromosomes 15q (48%), 11q (44%), 3q (40%), 9q (40%) and 1q (36%). By contrast, all PCL patients showed gains in 1q. Losses of chromosomal material were significantly more frequent in PCL than in MM patients (P = 0.03): losses on 13q in 80% of PCL vs 28% of MM; and on chromosome 16 in 80% vs 12%, respectively. In addition, PCL patients showed losses of 2q and 6p that were not present in MM. The CGH data show differences in chromosomal imbalances between MM and PCL.

Abnormalities on 1q and 7q are associated with poor outcome in sporadic Burkitt's lymphoma. A cytogenetic and comparative genomic hybridization study.

Comparative genomic hybridization (CGH) studies have demonstrated a high incidence of chromosomal imbalances in non-Hodgkin's lymphoma. However, the information on the genomic imbalances in Burkitt's Lymphoma (BL) is scanty. Conventional cytogenetics was performed in 34 cases, and long-distance PCR for t(8;14) was performed in 18 cases. A total of 170 changes were present with a median of four changes per case (range 1-22). Gains of chromosomal material (143) were more frequent than amplifications (5) or losses (22). The most frequent aberrations were gains on chromosomes 12q (26%), Xq (22%), 22q (20%), 20q (17%) and 9q (15%). Losses predominantly involved chromosomes 13q (17%) and 4q (9%). High-level amplifications were present in the regions 1q23-31 (three cases), 6p12-p25 and 8p22-p23. Upon comparing BL vs Burkitt's cell leukemia (BCL), the latter had more changes (mean 4.3 +/- 2.2) than BL (mean 2.7 +/- 3.2). In addition, BCL cases showed more frequently gains on 8q, 9q, 14q, 20q, and 20q, 9q, 8q and 14q, as well as losses on 13q and 4q. Concerning outcome, the presence of abnormalities on 1q (ascertained either by cytogenetics or by CGH), and imbalances on 7q (P=0.01) were associated with a short survival.

In vivo murine model of acquired resistance in myeloma reveals differential mechanisms for lenalidomide and pomalidomide in combination with dexamethasone.

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.

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.

Genome-wide profiling of methylation identifies novel targets with aberrant hypermethylation and reduced expression in low-risk myelodysplastic syndromes.

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.