Antonino Neri

International Myeloma Working Group molecular classification of multiple myeloma: spotlight review

Myeloma is a malignant proliferation of monoclonal plasma cells. Although morphologically similar, several subtypes of the disease have been identified at the genetic and molecular level. These genetic subtypes are associated with unique clinicopathological features and dissimilar outcome. At the top hierarchical level, myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. The latter is mainly composed of cases harboring IgH translocations, generally associated with more aggressive clinical features and shorter survival. The three main IgH translocations in myeloma are the t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Trisomies and a more indolent form of the disease characterize hyperdiploid myeloma. A number of genetic progression factors have been identified including deletions of chromosomes 13 and 17 and abnormalities of chromosome 1 (1p deletion and 1q amplification). Other key drivers of cell survival and proliferation have also been identified such as nuclear factor- B-activating mutations and other deregulation factors for the cyclin-dependent pathways regulators. Further understanding of the biological subtypes of the disease has come from the application of novel techniques such as gene expression profiling and array-based comparative genomic hybridization. The combination of data arising from these studies and that previously elucidated through other mechanisms allows for most myeloma cases to be classified under one of several genetic subtypes. This paper proposes a framework for the classification of myeloma subtypes and provides recommendations for genetic testing. This group proposes that genetic testing needs to be incorporated into daily clinical practice and also as an essential component of all ongoing and future clinical trials.

Identification of microRNA expression patterns and definition of a microRNA/mRNA regulatory network in distinct molecular groups of multiple myeloma

To date, little evidence of miRNA expression/deregulation in multiple myeloma has been reported. To characterize miRNA in the context of the major multiple myeloma molecular types, we generated miRNA expression profiles of highly purified malignant plasma cells from 40 primary tumors. Furthermore, transcriptional profiles, available for all patients, were used to investigate the occurrence of miRNA/predicted target mRNA pair anticorrelations, and the miRNA and genome-wide DNA data were integrated in a subset of patients to evaluate the influence of allelic imbalances on miRNA expression. Differential miRNA expression patterns were identified, which were mainly associated with the major IGH translocations; particularly, t(4;14) patients showed specific overexpression of let-7e, miR-125a-5p, and miR-99b belonging to a cluster at 19q13.33. The occurrence of other lesions (ie, 1q gain, 13q and 17p deletions, and hyperdiploidy) was slightly characterized by specific miRNA signatures. Furthermore, the occurrence of several allelic imbalances or loss of heterozygosity was found significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 or miR-140-3p at 16q22. Finally, the integrative analysis based on computational target prediction and miRNA/mRNA profiling defined a network of putative functional miRNA-target regulatory relations supported by expression data.

IRTA1 and IRTA2, novel immunoglobulin superfamily receptors expressed in B cells and involved in chromosome 1q21 abnormalities in B cell malignancy.

Abnormalities of chromosome 1q21 are common in B cell malignancies, but their target genes are largely unknown. By cloning the breakpoints of a (1;14) (q21;q32) chromosomal translocation in a myeloma cell line, we have identified two novel genes, IRTA1 and IRTA2, encoding cell surface receptors homologous to the Fc and inhibitory receptor families. Both genes are selectively expressed in mature B cells: IRTA1 in marginal zone B cells and IRTA2 in centrocytes, marginal zone B cells, and immunoblasts. As a result of the t(1;14), IRTA1 is fused to the immunoglobulin Calpha domain to produce a chimeric IRTA1/Calpha fusion protein. In tumor cell lines with 1q21 abnormalities, IRTA2 expression is deregulated. Thus, IRTA1 and IRTA2 are novel immunoreceptors implicated in B cell development and lymphomagenesis.

Oct-4 Expression in Adult Human Differentiated Cells Challenges Its Role as a Pure Stem Cell Marker

The Oct‐4 transcription factor, a member of the POU family that is also known as Oct‐3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct‐4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct‐4 expression. On the contrary, we demonstrated, using reverse transcription‐polymerase chain reaction (PCR) (total RNA, Poly A+), real‐time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct‐4. These observations raise the question as to whether the role of Oct‐4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct‐4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic‐specific gene with a large number of pseudogenes that may contribute to false identification of Oct‐4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct‐4 in fully differentiated cells.

Aberrant global methylation patterns affect the molecular pathogenesis and prognosis of multiple myeloma

We used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal gammopathy of undetermined significance [MGUS], presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell-cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.

Synthetic miR-34a Mimics as a Novel Therapeutic Agent for Multiple Myeloma: In Vitro and In Vivo Evidence

Purpose: Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. Experimental Design: Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. Results: Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. Conclusions: Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a–based treatment strategies in MM patients. Clin Cancer Res; 18(22); 6260–70. ©2012 AACR.

Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and Bcl2A1 as critical target genes

Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPbeta and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.

Thalidomide Downregulates Angiogenic Genes in Bone Marrow Endothelial Cells of Patients With Active Multiple Myeloma

PURPOSE To study the antiangiogenic effect of thalidomide. PATIENTS AND METHODS The expression of key angiogenic genes was studied in bone marrow endothelial cells (ECs) of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies unattributed/unassociated (MG[u]), diffuse large B-cell non-Hodgkins lymphoma, in a Kaposis sarcoma (KS) cell line, and in healthy human umbilical vein ECs (HUVECs) following exposure to therapeutic doses of thalidomide. RESULTS Thalidomide markedly downregulates the genes in a dose-dependent fashion in active MMECs and KS cell line, but upregulates them or is ineffective in nonactive MMECs, MG(u)ECs, NHL-ECs, and in HUVECs. Secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor also diminishes according to the dose in culture conditioned media (CM) of active MMECs and KS, whereas it does not change in the other CM. CONCLUSION Inhibition by thalidomide is probably confined to the genes of active MMECs and KS. This would account for its higher efficacy in these diseases.

Gene Expression Profiling Uncovers Molecular Classifiers for the Recognition of Anaplastic Large-Cell Lymphoma Within Peripheral T-Cell Neoplasms

PURPOSE To unravel the regulatory network underlying nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) -mediated lymphomagenesis of anaplastic large-cell lymphoma (ALCL) and to discover diagnostic genomic classifiers for the recognition of patients with ALK-positive and ALK-negative ALCL among T-cell non-Hodgkins lymphoma (T-NHL). PATIENTS AND METHODS The transcriptome of NPM-ALK-positive ALCL cell lines was characterized by silencing the expression of ALK or STAT3, a major effector of ALK oncogenic activity. Gene expression profiling (GEP) was performed in a series of systemic primary T-NHL (n = 70), including a set of ALK-positive and ALK-negative ALCL (n = 36). Genomic classifiers for ALK-positive and ALK-negative ALCL were generated by prediction analyses and validated by quantitative reverse-transcriptase polymerase chain reaction and/or immunohistochemistry. RESULTS In ALCL cell lines, two thirds of ALK-regulated genes were concordantly dependent on STAT3 expression. GEP of systemic primary T-NHL significantly clustered ALK-positive ALCL samples in a separate subgroup, underscoring the relevance of in vitro ALK/STAT3 signatures. A set of genomic classifiers for ALK-positive ALCL and for ALCL were identified by prediction analyses. These gene clusters were instrumental for the distinction of ALK-negative ALCL from peripheral T-cell lymphomas not otherwise specified (PTCLs-NOS) and angioimmunoblastic lymphomas. CONCLUSION We proved that experimentally controlled GEP in ALCL cell lines represents a powerful tool to identify meaningful signaling networks for the recognition of systemic primary T-NHL. The identification of a molecular signature specific for ALCL suggests that these T-NHLs may represent a unique entity discernible from other PTCLs, and that a restricted number of genes can be instrumental for clinical stratification and, possibly, therapy of T-NHL.

Molecular Classification of Multiple Myeloma: A Distinct Transcriptional Profile Characterizes Patients Expressing CCND1 and Negative for 14q32 Translocations

PURPOSE The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups. MATERIALS AND METHODS The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. RESULTS Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures. CONCLUSION Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.