Daniela Intini

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.

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.

Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma.

Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the α-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

Analysis of FGFR3 gene mutations in multiple myeloma patients with t(4;14)

The t(4;14)(p16.3;q32) in multiple myeloma (MM) leads to an apparent deregulation of the FGFR3 and WHSC1/MMSET genes. FGFR3 mutations, known to be associated with genetic skeletal disorders, have also been identified in a few cases of MM (mainly cell lines) with t(4;14). We investigated FGFR3 mutations in a series of 53 MM cases; 11 cases with t(4;14) and FGFR3 overexpression were analysed using reverse transcription polymerase chain reaction, while the remaining cases were studied at DNA level. The Arg248Cys mutation, which is associated with some lethal forms of skeletal disorders, was found in one case with t(4;14). Our results indicate that FGFR3 mutations occur in only a small fraction of MM cases with t(4;14).

Molecular and Transcriptional Characterization of 17p Loss in B-Cell Chronic Lymphocytic Leukemia

Distinct genetic abnormalities, such as TP53 deletion at 17p13.1, have been identified as having adverse prognostic relevance in B‐cell chronic lymphocytic leukemia (B‐CLL), and conventional cytogenetic studies have shown that TP53 deletion in B‐CLL is mainly associated with the loss of 17p due to complex chromosomal rearrangements. We used an integrative genomic approach to investigate the significance of 17p loss in 18 B‐CLLs in Binet stage A, carrying a TP53 monoallelic deletion detected by means of fluorescence in situ hybridization (FISH). Genome‐wide DNA analysis using single nucleotide polymorphism (SNP) arrays of 12 of 18 samples showed 17p loss in 11 cases, with breakpoints scattered along the 17p11.2 region. FISH analysis confirmed these findings and revealed 17p loss in a small fraction of leukemic cells in the remaining TP53‐deleted case, and it also indicated 17p loss in the six cases not investigated by means of SNP arrays. Mutations in exons 2–11 of the remaining TP53 allele were found in 9 of 12 deleted samples. Gene‐expression profiling of 60 B‐CLLs, including seven patients with 17p loss, identified 40 differentially expressed genes in 17p‐ versus 17p normal samples, 35 of which were downregulated in 17p‐tumors. The majority (30 of 35) of these transcripts, including putative tumor suppressor genes, mapped to 17p, thus indicating a remarkable gene‐dosage effect. Our data provide evidence that 17p loss may play an additional pathogenetic role in B‐CLL and suggest that the concomitant loss of multiple tumor suppressor genes could be responsible for the highly adverse prognostic relevance associated with TP53 loss.

Characterization of oncogene dysregulation in multiple myeloma by combined FISH and DNA microarray analyses

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF, and MAFB genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23), and t(14;20)(q32;q12), respectively, in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) by DNA microarray analysis and compared the results with the presence of translocations as assessed by dual‐color FISH or RT‐PCR. A t(4;14) was found in 6 MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in 1 MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in 1 PCL. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased MMSET expression was found in 1 MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.

BCL10 gene mutations rarely occur in lymphoid malignancies

BCL10, a gene involved in apoptosis signalling, has recently been identified through the cloning of chromosomal breakpoints in extranodal (MALT-type) marginal zone lymphomas carrying the t(1;14)(p22;q32) translocation. BCL10 was also found mutated in these cases as well as in other types of lymphoid and solid tumors, suggesting that its inactivation may play an important pathogenetic role; however, this has been questioned by recent studies showing a lack of somatic mutations in human cancers. We report the mutation analysis of exons 1–3 of the BCL10 gene in DNAs from 228 cases of lymphoid malignancies (30 B cell chronic lymphocytic leukemias, 123 B and 45 T non-Hodgkins lymphomas and 30 multiple myelomas). Somatic mutations were detected in four cases (2%): one small lymphocytic, one follicular and two diffuse large cell lymphomas. The mutations were all within exon 3 and have not been previously reported. Our data suggest that BCL10 mutations may play only a limited role in the pathogenesis of lymphoid neoplasms.

Diagnostic role and prognostic significance of a simplified immunophenotypic classification of mature B cell chronic lymphoid leukemias

We verified the diagnostic and prognostic role of a simplified immunophenotypic classification (IC) in a series of 258 patients (M/F: 1.4; median age: 64 years; median follow-up: 64 months; 75 deaths) with mature B cell lymphoid leukemias (MBC-LL) for whom no histopathological diagnosis was available because of minimal or no lymph node involvement. The IC was based on the reactivity of three pivotal immunophenotypic markers: CD5, CD23 and SIg intensity. On the basis of different expression patterns, we identified four diagnostic clusters (C) characterized by distinct clinico-biological features and different prognoses: C1 (149 patients) identified most classical B cell chronic lymphocytic leukemias (CLL-type cluster; SIgdim/CD5+/CD23+); C2, 38 patients whose clinico-hematological characteristics were intermediate between C1 and C3 (CLL-variant cluster; SIgbright/CD5+/CD23+/−or SIgdim/CD5−/−/CD23 indifferent); C3 (16 patients) most situations consistent with mantle cell lymphoma in leukemic phase (MCL-type cluster; SIgbright/CD5+/CD23−); and C4, 55 cases, most of whom were consistent with leukemic phase lymphoplasmacytic/splenic marginal zone lymphomas (LP/S-type cluster; SIgbright/CD5−/+/CD23 indifferent). At univariate survival analysis, prognosis worsened from C1 to C4, C2 and C3 (P = 0.0001), and this was maintained at multivariate analysis (P = 0.006), together with CD11c expression (P = 0.0043), age at diagnosis (cut-off 70 years; P = 0.0008) and platelet count (cut-off 140 × 109/l; P = 0.0034). Besides recognising the two well-known situations of classic B-CLL and MCL, our IC identified situations with distinct prognostic and/or clinical behaviors.

Molecular targeting of the PKC‐β inhibitor enzastaurin (LY317615) in multiple myeloma involves a coordinated downregulation of MYC and IRF4 expression

The protein kinase C (PKC) pathway has been shown to play a role in the regulation of cell proliferation in several haematological malignancies, including multiple myeloma (MM). Recent data have shown that a PKC inhibitor, enzastaurin, has antiproliferative and proapoptotic activity in a large panel of human myeloma cell lines (HMCLs). In order to further characterise the effect of enzastaurin in MM, we performed gene expression profiling of enzastaurin‐treated KMS‐26 cell line. We identified 62 upregulated and 32 downregulated genes that are mainly involved in cellular adhesion (CXCL12, CXCR4), apoptosis (CTSB, TRAF5, BCL2L1), cell proliferation (IGF1, GADD45A, BCMA (B‐cell maturation antigen), CDC20), transcription regulation (MYC, MX11, IRF4), immune and defence responses. Subsequent validation by Western blotting of selected genes in four enzastaurin‐treated HMCLs was consistent with our microarray analysis. Our data indicate that enzastaurin may affect important processes involved in the proliferation and survival of malignant plasma cells as well as in their interactions with the bone marrow microenvironment and provide a preclinical rationale for the potential role of this drug in the treatment of MM. Copyright

Molecular and transcriptional characterization of the novel 17p11.2‐p12 amplicon in multiple myeloma

Multiple myeloma (MM) is a malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2‐p12, an unstable chromosomal region characterized by a large number of low‐copy repeats, which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An ∼5 Mb 17p11.2‐p12 amplified region was detected in the KMS‐26 myeloma cell line by SNP microarray analysis. Further fluorescence in situ hybridization mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary database of myeloma cell lines identified 12 significantly overexpressed genes in the KMS‐26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a database including 141 plasma cell dyscrasia primary tumors showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in the KMS‐26, whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, similarly to solid tumors, amplification/gain of the 17p11.2‐p12 region in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma‐associated genes.