ODZ1 allows glioblastoma to sustain invasiveness through a Myc-dependent transcriptional upregulation of RhoA.

Long-term survival remains low for most patients with glioblastoma (GBM), which reveals the need for markers of disease outcome and novel therapeutic targets. We describe that ODZ1 (also known as TENM1), a type II transmembrane protein involved in fetal brain development, plays a crucial role in the invasion of GBM cells. Differentiation of glioblastoma stem-like cells drives the nuclear translocation of an intracellular fragment of ODZ1 through proteolytic cleavage by signal peptide peptidase-like 2a. The intracellular fragment of ODZ1 promotes cytoskeletal remodelling of GBM cells and invasion of the surrounding environment both in vitro and in vivo. Absence of ODZ1 by gene deletion or downregulation of ODZ1 by small interfering RNAs drastically reduces the invasive capacity of GBM cells. This activity is mediated by an ODZ1-triggered transcriptional pathway, through the E-box binding Myc protein, that promotes the expression and activation of Ras homolog family member A (RhoA) and subsequent activation of Rho-associated, coiled-coil containing protein kinase (ROCK). Overexpression of ODZ1 in GBM cells reduced survival of xenografted mice. Consistently, analysis of 122 GBM tumour samples revealed that the number of ODZ1-positive cells inversely correlated with overall and progression-free survival. Our findings establish a novel marker of invading GBM cells and consequently a potential marker of disease progression and a therapeutic target in GBM.

Molecular cytogenetics of childhood hematological malignancies.

Cytogenetic and molecular analyses are essential for the classification of childhood hematologic malignancies. Nearly all children with leukemia should have an adequate cytogenetic analysis which in 80-90% is expected to show clonal chromosomal abnormalities. Moreover, with the availability of appropriate gene probes and sophisticated molecular techniques, genetic rearrangements become detectable in the majority of leukemia patients. Genetic abnormalities often associate with particular clinical-biological characteristics of the disease. In ALL, for example, genetic alterations together with distinct immunologic and clinical features, define various subgroups. In AML, unique cytogenetic rearrangements have been identified and associated with distinct morphological subgroups. Apart from the diagnostic assessment, cytogenetic studies provide valuable prognostic information which may influence treatment choices. Molecular analysis has also become of important value in the management of children with leukemias, as it serves, for example, to identify genetic abnormalities not detected by chromosomal analysis and to monitor residual disease during treatment and follow-up. Thus, genetic analyses of leukemic cells provide information of clinical relevance as well as contributing to our understanding of leukemogenesis. Alternative classifications of acute leukemias which take into account genetic information are being proposed. The available cytogenetic and molecular data have then to be included in clinical protocols, either by selecting individualized therapies in certain leukemia subtypes or by modifying treatment according to quantification of residual disease. We are closer to our main goal which is not only to classify 100% of childhood leukemias accurately, but to cure all of them.

Detection of 11q23/MLL rearrangements in infant leukemias with fluorescence in situ hybridization and molecular analysis.

Cytogenetic abnormalities of band 11q23 have been found in more than 50% of infant leukemias regardless of the phenotype. Using probes for the MLL gene at 11q23, MLL rearrangements have been identified in 70-80% of all infant leukemias including virtually all of the cases with 11q23 translocations, as well as cases with apparently normal karyotypes. We reviewed the chromosomal pattern of 26 cases of infant leukemias (12 ALL, 12 AML, two AUL). Eleven had 11q23 translocations, five had other abnormalities, and 10 had a normal karyotype. To determine whether 11q23/MLL rearrangements were present in the leukemia cells of patients with a normal karyotype, we performed FISH and molecular studies of eight of these patients who had adequate material. Three were found to have 11q23/MLL abnormalities, two of them detected by FISH; one ALL case had a t(11;19) (q23;p13.3), and one AML case had a t(11;19) (q23;p13.1). Retrospective review confirmed the presence of the t(11;19) in a small percentage of poor quality metaphase cells in both cases. A rearrangement of the MLL gene was detected by Southern blot analysis of leukemic cells from a third patient with ALL; one cell with a deletion of 11q23 was found on karyotypic review. Therefore, in our series the actual incidence of 11q23 abnormalities in infant leukemias was 54% (14/26): 67% in ALL (8/12) and 50% in AML (6/12). Our findings suggest that most infant leukemias with apparently normal karyotypes that have a molecular rearrangement of the MLL gene are undetected subtle translocations.(ABSTRACT TRUNCATED AT 250 WORDS)

The BCL6 gene in B-cell lymphomas with 3q27 translocations is expressed mainly from the rearranged allele irrespective of the partner gene.

The BCL6 gene, which functions as a transcription repressor, is the target of multiple chromosomal translocations in non-Hodgkin's lymphomas (NHL). These translocations occur in the nontranslated region of the BCL6 gene, juxtaposing regulatory sequences of the diverse partner genes to the open reading frame of the BCL6 gene and thus are thought to deregulate BCL6 gene expression. The levels of expression of the BCL6 gene and protein have been demonstrated to predict the clinical outcome of diffuse large B-cell lymphomas. By contrast, the prognostic significance of BCL6 gene translocations is unclear. In this study we have sought an explanation for this apparent discrepancy. We examined tumors with a variety of different BCL6 translocations and therefore with a variety of potentially substituted promoters. We found no increase in total BCL6 mRNA levels in the NHL specimens harboring BCL6 gene translocation. Indeed, some of these tumors expressed relatively low quantities of the BCL6 mRNA. We also sought to determine whether BCL6 transcription occurs from the rearranged or from the normal untranslocated allele in these tumors. We demonstrate that lymphoma cell lines and majority of NHL tumor specimens expressed BCL6 mRNA predominantly from the rearranged allele that may come under the control of various partner gene promoters. However, few NHL tumors with BCL6 gene translocations expressed BCL6 mRNA equally from the rearranged and the nonrearranged alleles. Neither the nature of the substituted promoters nor the presence of activating mutations in the BCL6 regulatory sequences correlated with the allelic expression of the BCL6 gene in these tumors.

Bcl-6 mutation status provides clinically valuable information in early-stage B-cell chronic lymphocytic leukemia.

In B-cell chronic lymphocytic leukemia (B-CLL), somatic mutation of IgVH genes defines a subgroup with favorable prognosis, whereas the absence of IgVH mutations is correlated with a worse outcome. Mutations of the BCL-6 gene are also observed in a subset of B-CLL, but the clinical significance of this molecular alteration remains uncertain. We examined the distribution of IgVH and BCL-6 gene mutations in 95 well-characterized patients with Binet stage A B-CLL, and correlated them with clinical, laboratory, cytogenetic findings and disease progression. Mutations of the BCL-6 gene were observed only in cases harboring mutated IgVH. Unexpectedly, coexistence of IgVH and BCL-6 mutations was correlated with shorter treatment-free interval (TFI) compared to cases harboring only IgVH mutation (median, 55 months vs not reached; P=0.01), resembling the clinical course of unmutated IgVH cases (median TFI, 44 months). As expected, deletions of 17p13 (P53 locus) and 11q22 (ATM locus) were observed in cases with unmutated IgVH, except one patient who showed mutations of both IgVH and BCL-6. No other statistically significant differences were observed among the genetic subgroups. Our data indicate that BCL-6 mutations identify a subgroup of Binet stage A B-CLL patients with a high risk of progression despite the presence of mutated IgVH gene.

Identification and molecular characterization of CALM/AF10fusion products in T cell acute lymphoblastic leukemia and acute myeloid leukemia.

The t(10;11)(p12-p13;q14-q21) observed in a subset of patients with either acute lymphoblastic leukemia or acute myeloid leukemia has been shown to result in the fusion of AF10 on chromosome 10 with CALM (also named CLTH) on chromosome 11. AF10 was originally identified as a fusion partner of MLL in the t(10;11)(p12-p13;q23) observed in myeloid leukemia. CALM is a newly isolated gene, cloned as the fusion partner of AF10 in the monocytoid cell line, U937. In order to understand the relationship between MLL, AF10, CALM and the leukemic process, fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction were used to study a series of nine leukemia patients with a t(10;11). Six had myeloid leukemia (AML-M0, AML-M1, AML-M4 and AML-M5) and three had T cell lymphoblastic leukemia. We identified four different CALM/AF10 fusion products in five patients and AF10/CALM reciprocal message in one. We conclude that fusion of CALM and AF10 is a recurring abnormality in both lymphoid and myeloid leukemias of various types including AML-M5, and that the breakpoints in the two types of leukemia do not differ. Our data indicate that the CALM/AF10 fusion product on the der(10) chromosome is critical to leukemogenesis. Leukemia (2000) 14, 100-104.

Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo.

We report on the chromosomal pattern of 120 patients with childhood AML de novo. One hundred and fifteen patients (96%) had adequate samples for analysis; 98 (85%) of these showed clonal karyotypic abnormalities. They were classified into cytogenetic subgroups which were closely correlated with FAB subtypes: t(8;21) and M2 (n = 9); t(15;17) and M3 (n = 12); inv(16) and M4Eo (n = 9); t(9;11) and M5a (n = 10); t(11q23) other than t(9;11) and M4-M5 (n = 11); and t(1;22) and M7 (n = 4). In patients with -7/del(7q) (n = 6), leukemia was preceded by MDS in half of the cases, although they had diverse FAB subtypes. Thirty-seven patients had miscellaneous abnormalities. Despite a high CR rate, patients with t(8;21) had a very poor survival: only one child was event-free at 3 years from diagnosis. One third of patients with t(15;17) died during induction. Those eight who achieved CR fared well: only two relapsed, and six were event-free survivors. Patients with inv(16) had a high remission rate and a long survival: five children were in CR 20 to 136 months. Both groups with t(9;11) and t(11q23) had a high remission rate: however, outcome was superior for the t(9;11) group when compared to either the t(11q23) group (EFS at 3 years +/- SE, 56 +/- 17% vs. 11 +/- 10%, p = 0.07) or to the remaining patients (p = 0.06). Both -7/del(7q) and t(1;22) groups had low CR rates (50%) and poor survival. Cytogenetic analysis identifies clinically distinct subsets of childhood AML and is useful in tailoring treatment for these patients. Favorable cytogenetic groups (t(15;17), inv(16), and t(9;11)) may do well with current therapy protocols, whereas unfavorable groups (t(11q23), t(8;21), -7/del(7q), and t(1;22)) require more effective therapies.

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.

Detection of translocations affecting the BCL6 locus in B cell non-Hodgkin's lymphoma by interphase fluorescence in situ hybridization.

Structural alterations in 3q27 affecting the BCL6 locus are among the most frequent changes in B-NHL. The aim of the present study was to establish an interphase-FISH assay for the detection of all diverse BCL6 translocations in B-NHL. Two different approaches were tested, one using a PAC-clone spanning the major breakpoint region (MBR) of BCL6 (span-assay), and another using two BAC clones flanking the MBR (flank-assay). Interphase FISH with the span-assay detected the various BCL6 translocations in seven B-NHL cell lines. The dual-color flank-assay was evaluated in two laboratories independently: in normal controls, the cutoff level for false-positive signals was 2.6%, whereas the cutoff level for false-negatives in the seven cell lines was 7.5%. To test the feasibility of the FISH strategies, 30 samples from patients with B-NHL with cytogenetic abnormalities of 3q27 were evaluated with both assays. In 21 cases, the span-assay indicated a BCL6 rearrangement. In 18 of the 21 cases, the dual-color flank-assay confirmed the translocation including 12 different partner chromosomal loci. The three false-positive cases detected with the span-assay showed trisomy of chromosome 3 by cytogenetic analyses, and they were correctly classified as non-rearranged with the flank-assay. In summary, our FISH strategy using two differently labeled flanking BCL6 BAC probes provides a robust, sensitive, and reproducible method for the detection of common and uncommon abnormalities of BCL6 gene in interphase nuclei. The routine application of this assay to patients with B-NHL will allow the assessment of the diagnostic and prognostic significance of BCL6 rearrangements.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.

Chromosomal abnormalities in women with breast cancer after autologous stem cell transplantation are infrequent and may not predict development of therapy-related leukemia or myelodysplastic syndrome.

We determined prospectively the incidence of chromosomal abnormalities in patients with high-risk breast cancer (HRBC) after high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT), and correlated the cytogenetic abnormalities with the development of post-transplant myelodysplastic syndrome or acute myeloid leukemia (MDS/AML). From 1990 to 1999, 229 women with HRBC underwent ASCT. Cytogenetic analysis of bone marrow (BM) cells was performed 12-59 months after ASCT in 60 consecutive women uniformly treated with six courses of FAC/FEC followed by HDCT and ASCT. With a median follow-up of 36 months after ASCT, there were no cases of MDS/AML among the 229 patients. In the selected cohort of 60 patients, three (5%) showed clonal chromosomal abnormalities (two single trisomy X and one t(1;6)), whereas two additional patients showed non-clonal reciprocal translocations. Two of the patients with clonal aberrations had blood cytopenias as well as subtle dysplastic pictures in BM which were not classifiable as MDS according to the FAB criteria. Similar dysplastic features were also observed in four patients with normal karyotypes. All cytogenetic aberrations were transient and disappeared, except a +X detected by FISH in a residual cell population in one of the patients. Retrospective cytogenetic and FISH studies of samples obtained after six cycles of FAC/FEC and before transplant demonstrated no chromosomal abnormalities in any of the five patients with post-ASCT karyotypic changes. Early changes in karyotype detected in breast cancer patients following ASCT are transient and do not correlate with or predict development of MDS/AML. As these aberrations were not present before ASCT, they may be related to the HDCT regimen or transplant procedure rather than to the prior adjuvant therapy. Our results suggest that ASCT may be less likely to cause MDS or AML in breast cancer patients as compared to other malignancies. Bone Marrow Transplantation (2000) 25, 1203-1208.

Identification of two subgroups of mantle cell leukemia with distinct clinical and biological features.

INTRODUCTION: Mantle cell leukemia (MCLeu) has been considered as a leukemic form of mantle cell lymphoma (MCL). However, the presence of certain features rarely observed in MCL, such as transformation to prolymphocytic leukemia (PLL) or indolent clinical course, suggests that MCLeu may represent a distinct disorder. METHODS: Seven cases of MCLeu with t(11;14)(q13;q32) and BCL1-IGH gene rearrangement were ascertained among 140 newly diagnosed chronic B-cell lymphoproliferative disorders with leukemic expression. Comparative genomic hybridization, FISH for specific gene loci, and immunological studies were preformed in them. RESULTS: In comparison with CLL, MCLeu cases had low immunological scores < or =2 with respect to B-CLL (P<0.0001). Expression of CD38 was absent in 43% of MCLeu and in 44% of B-CLL. Comparative genomic hybridization analysis identified genomic imbalances in 86% of MCLeu with a similar pattern than in MCL: gains of 3q, 8q involving MYC gene and 15q, and losses of 6q, 9p, 13q and 17p affecting P53 gene. Differently from MCL and CLL, genomic loss of 8p was frequently detected in MCLeu (83%). Although clinical presentation of MCLeu was indistinguishable from CLL, all patients but one had disease progression within three years. According to the immunologic and genomic profiles, two distinct subgroups of MCLeu were defined: one related to PLL, showing CD38-, deletion of P53, and MYC amplification and another which corresponds to a leukemic form of classical MCL, presenting with CD38+ and normal P53 and MYC status. CONCLUSION: MCLeu and MCL are closely related disorders, as they show similar genomic and molecular patterns. However, the deletion of the short arm of chromosome 8 may represent a specific marker for MCLeu. Two distinct subgroups of MCLeu may also be distinguished according to the immunologic and genomic cell profiles.

Variant three-way translocation of inversion 16 in AML-M4Eo confirmed by fluorescence in situ hybridization analysis.

The inv(16) and t(16;16) characterize a subgroup of acute myelomonocytic leukemia (AML) with distinct morphological features and a favorable prognosis. Both cytogenetic abnormalities result in a fusion of CBF beta at 16q22 and MYH11 gene at 16p13, whose detection by PCR and fluorescence in situ hybridization (FISH) is useful for diagnosis and monitoring of the disease. Variant translocations of inv(16)/t(16;16) are very rare and whether they are also associated with a favorable prognosis is unknown. We report a patient presenting with typical AML-M4Eo and a three-way translocation of inv(16) involving 16p13, 16q22, and 3q22. FISH studies on bone marrow (BM) chromosomes using CBFB and MYH11 DNA probes revealed a fusion of CBFB and MYH11 on 16q of the der(16), as well as a signal from MYH11 on 16p but not from CBFB; normal signals for both probes were present on the normal 16. Neither of these labeled probes was on the der(3), but the translocation between the der(3) and der(16) was confirmed by using a chromosome 16 painting probe. Molecular analysis of BM cells using RT-PCR identified a CBFB-MYH11 fusion transcript type D. After achieving complete remission, the patient relapsed. We conclude that FISH and PCR are feasible tools to distinguish cases with variant abnormalities of inv(16) from cases with other chromosome 16 abnormalities. Variant abnormalities of inv(16) may be not associated with favorable prognosis.

Epigenetic regulation of miRNA genes in acute leukemia.

MicroRNAs (miRNAs) are small non-coding RNA molecules that can negatively regulate gene expression at the post-transcriptional level. miRNA expression patterns are regulated during development and differentiation of the hematopoietic system and have an important role in cell processes such as proliferation, apoptosis, differentiation or even in tumorigenesis of human tumors and in particular of hematological malignancies such as acute leukemias. Various miRNAs and their functions have been intensively studied in acute leukemias but the mechanisms that control their expression are largely unknown for the majority of aberrantly expressed miRNAs. miRNA expression can be regulated by the same genetic mechanism that modulate protein coding genes such as mutation, deletion, amplification, loss of heterozygosity and translocations. In this review we focus on the regulation of miRNAs in acute leukemias mediated by alterations in epigenetic mechanisms such as DNA methylation and histone code, describing the role of these alterations in the pathogenesis, diagnosis and prognosis of acute leukemias and their possible use as new therapeutic targets and biomarkers.

Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia.

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.

Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo.

Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy because of its aggressive behavior. Cancer-initiating or progenitor cells have been described to be the only cell population with tumorigenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells or the early precursors may be beneficial. We have established different cultures of glioblastoma-initiating cells (GICs) derived from surgical specimens and found that, after induction of differentiation, the NFκB transcriptional pathway was activated, as determined by analyzing key proteins such as p65 and IκB and the upregulation of a number of target genes. We also showed that blockade of nuclear factor (NF)κB signaling in differentiating GICs by different genetic strategies or treatment with small-molecule inhibitors, promoted replication arrest and senescence. This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1, because its downregulation by RNA interference reproduced a similar phenotype. Furthermore, these results were confirmed in a xenograft model. Intravenous treatment of immunodeficient mice bearing human GIC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultrastructural alterations of the brain parenchyma were detected. These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed to promote premature senescence of differentiating GICs by blocking key factors within the NFκB pathway.