Acute stroke after intravitreal bevacizumab to treat choroidal neovascularization due to angioid streaks in pseudoxanthoma elasticum : a severe systemic adverse event after an off-label procedure.

To report the occurrence of acute stroke after intravitreal bevacizumab administration to treat choroidal neovascularization due to angioid streaks in a patient affected by pseudoxanthoma elasticum. A 54-year-old man with pseudoxanthoma elasticum had vision loss because of choroidal neovascularization due to angioid streaks. He underwent two intravitreal bevacizumab injections. Three days after the second procedure the patient was afflicted by acute stroke. Intravitreal injection of bevacizumab to treat choroidal neovascularization due to angioid streaks in pseudoxanthoma elasticum could lead to severe systemic adverse events.

Molecular targeting of the PKC-beta 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.

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.

Integrative genomic analysis reveals distinct transcriptional and genetic features associated with chromosome 13 deletion in multiple myeloma.

BACKGROUND AND OBJECTIVES: The chromosome 13 deletion (Delta13) is one of the most frequent chromosomal alterations in multiple myeloma (MM). Delta13 is associated with an unfavorable prognosis, although there is increasing agreement that its prognostic relevance must be related to the ploidy status and the presence of different chromosomal translocations. The aim of this study was to provide a comprehensive analysis of the transcriptional features of Delta13 in MM. DESIGN AND METHODS: Highly purified plasma cells from 80 newly diagnosed MM patients were characterized by means of fluorescence in situ hybridization (FISH) and high-density oligonucleotide microarray for gene expression profiling and chromosomal alterations. RESULTS: We identified 67 differentially expressed genes in the patients with and without the chromosome 13 deletion, all of which were downregulated in the cases with Delta13: 44 mapped along the whole chromosome 13, seven on chromosome 11 and three on chromosome 19. Functional analyses of the selected genes indicated their involvement in protein biosynthesis, ubiquitination and transcriptional regulation. An integrative genomic approach based on regional analyses of the gene expression data identified distinct chromosomal regions whose global expression modulation could differentiate Delta13-positive cases, in particular the upregulation of 1q21-1q42 and the downregulation of 19p and almost the entire chromosome 11. FISH analyses confirmed the close relationship between Delta13-positivity and the presence of extra copies of 1q21-1q42 (p=6 x 10(-4)) or the absence of chromosome 11 and 19 trisomy (p=5 x 10(-4)). INTERPRETATION AND CONCLUSIONS: Our results indicate that distinct types of chromosomal aberrations are closely related to the transcriptional profiles of Delta13-positive cases, suggesting that the contribution of Delta13 to the malignancy should be considered together with associated abnormalities.

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 alpha-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.

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.

Molecular and biological characterization of three novel interleukin-6-dependent human myeloma cell lines.

BACKGROUND AND OBJECTIVES: Established human myeloma cell lines (HMCL) have significantly contributed to the investigation of the biological aspects of multiple myeloma. Our study reports the molecular and biological characterization of three novel interleukin-6 (IL-6)-dependent HMCL (CMA-01, CMA-02, CMA-03) established from the malignant plasma cells of myeloma patients with extramedullary disease. DESIGN AND METHODS: The immunophenotype, cell growth characteristics, IL-6 pathway, chromosomal alterations and gene expression profiles of the three HMCL were investigated. RESULTS: The plasma cell origin of the three Epstein-Barr virus-negative HMCL was confirmed by immunophenotypic analysis. Cytogenetic and fluorescence in situ hybridization analyses revealed the presence of complex karyotypes with many numerical and structural chromosomal abnormalities. All three HMCL are positive for the t(8;14); CMA-01 and CMA-02 showed t(11;14) and t(14;16) translocations, respectively. The three HMCL grow slowly at a relatively low saturation density and depend on exogenous IL-6 for their survival and proliferation. The comparison of the gene expression profiles of the three HMCL versus those of the purified tumor plasma cells from which the cell lines were derived identified a set of differentially expressed genes mainly involved in the cell proliferation pathway. INTERPRETATION AND CONCLUSIONS: Extensively characterized large HMCL panels that reflect the heterogeneity of the disease may improve our understanding of the pathogenetic events and clinical progression of multiple myeloma.

Cell cycle regulators in multiple myeloma: prognostic implications of p53 nuclear accumulation.

Multiple myeloma (MM) is characterized by a multistep process of tumorigenesis involving genes that control cell cycle progression. The prevalence and clinical implications of p53, p21, HDM-2, p27, and cyclin E immunoreactivity in MM patients, however, have not been fully elucidated. We evaluated the immunoreactivity (IR) for p53, p21, HDM-2, p27, cyclin E, and Ki-67 in bone marrow biopsies from 48 patients. In 34 (70.8%) cases, TP53 gene mutations and HDM-2 gene amplification were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and Southern blot densitometric analyses in the corresponding bone marrow aspirates. Nineteen (39.6%) biopsy specimens exhibited > or =10% neoplastic cells immunoreactive for p53, 23 (47.9%) for p21, 28 (58.3%) for HDM-2, 29 (60.4%) for cyclin E, and 16 (33.3%) for Ki-67; 23 (47.9%) tumors had > or =50% neoplastic cells immunoreactive for p27. TP53 gene mutations in exons 5 through 8 were detected in 3 (8.8%) cases, whereas none exhibited HDM-2 gene amplification. In the cases bearing a wild-type TP53 gene, no association was found between p53 accumulation and HDM-2 or p21 IR. The same cases had been previously investigated for the presence of the t(11;14) translocation and cyclin D1 IR; interestingly, a significant inverse correlation between cyclin D1 and p27 or cyclin E IR was noted. In addition to clinical stage and Bartl's histologic stage and grade, p53 accumulation was significantly associated with survival, and it maintained its prognostic significance in a multivariate analysis adjusted for age, clinical stage, and relapse. Our data suggest that the immunohistochemical evaluation of p53 IR in bone marrow biopsies may represent an adjunct in MM patient prognostication.

Lack of Bcl10 gene mutations in laryngeal squamous cell carcinoma.

The Bcl10 gene encodes a protein probably involved in some apoptotic regulatory pathways. Bcl10 mutations lead to the translation of truncated proteins that show gain-of-function transforming activity; it has been suggested that Bcl10 may represent a major target gene for inactivation in many human cancers. To define the frequency of Bcl10 mutations in laryngeal squamous cell carcinoma and their possible association with tumour progression, we investigated a large panel of tumours representative of all grades and stages of malignancy. To detect pathogenic mutations in exons 1, 2 and 3 of the Bcl10 gene, we performed a silver-staining polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis followed by direct DNA sequencing. We revealed the presence of SSCP variants in 18 out of 91 laryngeal tumours. Direct DNA sequencing showed previously described polymorphisms but no pathogenic mutations. We have strong evidence that the Bcl10 gene is not involved in laryngeal carcinogenesis.

Supportive transfusion therapy in cancer patients with acquired defects of hemostasis.

Bleeding occurs in approximately 10% of patients with cancer: supportive transfusion therapy with Platelets Concentrates (PC), Fresh Frozen Plasma (FFP) and plasma-derived or recombinant concentrates is often required for the cessation and prevention of the bleeding episodes. The most frequent causes of bleeding in cancer is thrombocytopenia followed by liver insufficiency with or without vitamin K deficiency, disseminated intravascular coagulation (DIC) and the inappropriate or excessive use of anticoagulants. Other acquired hemostatic defects such as acquired hemophilia (AHA) and acquired von Willebrand syndrome (AVWS) are rare but they can be life-threatening. Thrombocytopenia in cancer patients may be the consequence of marrow invasion, chemotherapy or platelet auto-antibodies; patients with severe hypoproliferative thrombocytopenia, must be treated with PC and carefully followed to assess refractoriness to PC. The management of the other acquired defects of hemostasis usually requires the use of FFP and specific plasma-derived or recombinant concentrates. PC, FFP and plasma-derived concentrates can induce complications and/or adverse events in cancer patients: these include mainly allergic (ALR) or anaphylactic reactions (ANR), Transfusion-Associated Graft-Versus-Host Disease (TA-GVHD), Trasfusion-transmitted bacteriemia (TTB), Transfusion-Related Acute Lung Injury (TRALI), Acute Hemolytic Transfusion Reactions (AHTR), Febrile Non Hemolytic Transfusion Reactions (FNHTR). Therefore, modifications such as leukocyte-reduction and irradiation of the blood components to be transfused in cancer patients are recommended to reduce the risk of these complications.

Relevance of Ras gene mutations in the context of the molecular heterogeneity of multiple myeloma.

Ras gene mutations are a recurrent genetic lesion in multiple myeloma (MM). Here, we report a mutation analysis of N- and K-Ras genes in purified plasma cell populations from a panel of 81 newly diagnosed MM patients stratified according to the most frequent genetic and molecular features associated with the neoplasia. Ras gene mutations, mostly involving the N-Ras gene, were detected in 20% of the patients. Ras mutations did not correlate with the presence of chromosome 13q deletion, trisomy of chromosome 11, 1q amplification or hyperdiploidy. In addition, despite an appreciable association with tumours overexpressing Cyclin D1, Ras mutations did not correlate at significant levels with any of the proposed groups in the TC classification, based on the presence of the major IgH chromosomal translocations and expression of Cyclin D genes. Finally, transcription analyses revealed the presence of differentially expressed transcripts in human multiple myeloma cell lines carrying the Ras gene mutations but not in primary tumours. Overall, these data suggest that Ras gene mutations are not likely to represent a master lesion in MM but its relevance needs to be considered in the context of other genetic abnormalities.

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 approximately 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.

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. Disclosure of potential conflicts of interest is found at the end of this article.

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.