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.

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.

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.

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.

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.

Co-amplified genes at 8p12 and 11q13 in breast tumors cooperate with two major pathways in oncogenesis.

Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high-resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate oncogenes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. These studies also suggested that CCND1 at 11q13 induced expression of ZNF703 mapping at 8p12, which was subsequently shown to be mediated by the Rb/E2F pathway. Nine candidate oncogenes from 8p12 and four from 11q13 were further evaluated for oncogenic function. None of the genes individually promoted colony formation in soft agar or collaborated with each other functionally. On the other hand, FGFR1 and DDHD2 at 8p12 cooperated functionally with MYC, whereas CCND1 and ZNF703 cooperated with a dominant negative form of TP53. These observations highlight the complexity and functional consequences of the genomic rearrangements that occur in these breast cancer amplicons, including transcriptional cross-talk between genes in the 8p and 11q amplicons, as well as their cooperation with major pathways of tumorigenesis.

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.

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.

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.

Soluble intercellular adhesion molecule-1 (s-ICAM-1/s-CD54) in diffuse large B-cell lymphoma: association with clinical characteristics and outcome.

BACKGROUND: High serum levels of soluble intercellular adhesion molecule-1(s-ICAM-1/s-CD54) have been associated with adverse clinical features and poor outcome in chronic lymphocytic leukemia, Hodgkin's disease and non-Hodgkin's lymphoma, but their value in the different subtypes of non-Hodgkin's lymphoma has not been well addressed. PATIENTS AND METHODS: Our aim was to study the serum levels of s-ICAM-1 in diffuse large B-cell lymphoma (DLBCL) and to correlate them with clinical characteristics and outcome. We analyzed the serum levels of s-ICAM-1 in a series of 55 patients with DLBCL diagnosed in a single institution. s-ICAM-1 levels were quantified by an immunoenzymatic assay. Median age was 62 years (range 22-96); 29 (53%) were male. Twenty-eight (51%) presented with advanced clinical stage (III/IV), 32 (58%) had extranodal involvement, 28 (51%) had high serum lactate dehydrogenase (LDH) and 23 (43%) had high beta2-microglobulin levels. All patients received anthracycline-containing regimens. Correlation between clinical variables and s-ICAM-1 levels were tested with the Mann-Whitney U-test and survival was plotted by the Kaplan-Meier method, and curves compared with the log-rank test. RESULTS: Serum levels of s-ICAM-1 were significantly increased in patients with DLBCL compared with normal controls (589 +/- 487 versus 279 +/- 65 ng/ml, respectively; P <0.001). Higher levels of s-ICAM-1 were present in patients with B symptoms, advanced stage and increased LDH and beta2-microglobulin. s-ICAM-1 levels also correlated with achievement of a complete response. Patients with s-ICAM-1 over 668 ng/ml had a shorter time to treatment failure (TTF) (3-year TTF, 59% versus 20%, respectively; P = 0.01) and overall survival (OS) (3-year OS, 58% versus 22%, respectively; P = 0.04) than the remainders. When only low and low-intermediate risk patients in the international prognostic index score were considered, those with s-ICAM-1 over 668 ng/ml also had worse TTF and OS. CONCLUSIONS: In DLBCL, s-ICAM-1 levels correlated with high tumor burden and lymphoma dissemination and may contribute to assessment of prognosis.

Loss of a novel tumor suppressor gene locus at chromosome 8p is associated with leukemic mantle cell lymphoma.

Patients with mantle cell lymphoma (MCL) may present with either nodal or leukemic disease. The molecular determinants underlying this different biologic behavior are not known. This study compared the pattern of genetic abnormalities in patients with nodal and leukemic phases of MCL using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) for specific gene loci. Although both leukemic and nodal MCL showed similar genomic patterns of losses (involving 6q, 11q22-q23, 13q14, and 17p13) and gains (affecting 3q and 8q), genomic loss of chromosome 8p occurred more frequently in patients with leukemic disease (79% versus 11%, P <.001). Subsequent CGH analysis confirmed the genomic loss of 8p21-p23 in 6 of 8 MCL cell lines. Interestingly, MYC gene amplification was restricted to cases with 8p deletion. These data indicate the presence of a novel tumor suppressor gene locus on 8p, whose deletion may be associated with leukemic dissemination and poor prognosis in patients with MCL.

Additional cytogenetic changes do not influence the outcome of patients with newly diagnosed acute promyelocytic leukemia treated with an ATRA plus anthracyclin based protocol. A report of the Spanish group PETHEMA.

BACKGROUND AND OBJECTIVES: To analyze in patients with de novo acute promyelocytic leukemia (APL) treated with an ATRA plus anthracyclin-based protocol if the presence of additional cytogenetic aberrations to the t(15;17) influences: 1. clinical and biological presenting features; 2. disease outcome. DESIGN AND METHODS: One hundred and thirteen patients with newly diagnosed APL enrolled in the APL-96 protocol of the Spanish PETHEMA group were studied by conventional karyotyping, FISH and RT-PCR for the PML-RARa fusion. Treatment was homogeneous in all cases and consisted of anthracyclines and ATRA. RESULTS: Additional chromosome aberrations were observed in 30% of cases. The most frequent secondary changes were +8 (14 cases), and abnormalities of chromosomes 9 or 3 (4 patients each), and of chromosomes 1 and 8 (3 cases each). No clinical, biological, morphological, immunophenotypic or molecular differences were observed between the group of APLs with t(15;17) alone and the group of patients with additional changes. Patients with additional changes had a higher rates of complete remission (CR) and 4-year disease-free survival (DFS) (97%, and 97%, respectively) than patients with t(15;17) alone (CR, 70% and DFS, 84%) but these differences were not statistically significant. INTERPRETATION AND CONCLUSIONS: Patients with APL and additional cytogenetic abnormalities do not show different clinical, biological, morphological or molecular features as compared to patients with t(15;17) alone. The prognosis of patients with APL and t(15;17) alone and those with additional changes is similar in both groups. This study indicates that there is no rationale for administering more intensive treatment in APL patients with additional cytogenetic abnormalities receiving ATRA plus anthracycline-based chemotherapy.

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.

Novel genomic imbalances in B-cell splenic marginal zone lymphomas revealed by comparative genomic hybridization and cytogenetics.

Splenic marginal zone lymphoma (SMZL) has recently been recognized in the World Health Organization classification of hematological diseases as distinct type of non-Hodgkin's lymphoma. In contrast to the well-established chromosomal changes associated with other B-cell non-Hodgkin's lymphoma, few genetic alterations have been found associated with SMZL. The aim of our study was to analyze by comparative genomic hybridization (CGH) the chromosomal imbalances in 29 patients with SMZL and to correlate these findings with clinical and biological characteristics and patient outcome. In 21 cases, cytogenetic studies were simultaneously performed. Most of the patients (83%) displayed genomic imbalances. A total of 111 DNA copy number changes were detected with a median of four abnormalities per case (range, 1 to 12). Gains (n = 92) were more frequent than losses (n = 16), while three high-level amplifications (3q26-q29, 5p11-p15, and 17q22-q25) were observed. The most frequent gains involved 3q (31%), 5q (28%), 12q and 20q (24% each), 9q (21%), and 4q (17%). Losses were observed in 7q (14%) and 17p (10%). SMZL patients with genetic losses had a shorter survival than the remaining SMZL patients (P < 0.05). In summary, chromosomal imbalances in regions 3q, 4q, 5q, 7q, 9q, 12q, and 20q have been detected by CGH in SMZL. Patients with SMZL displaying genetic losses by CGH had a short survival.

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.

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.

Incidence, characterization and prognostic significance of chromosomal abnormalities in 640 patients with primary myelodysplastic syndromes. Grupo Cooperativo Español de Citogenética Hematológica.

Recently, a consensus International Prognostic Scoring System (IPSS) for predicting outcome and planning therapy in the myelodysplastic syndromes (MDS) has been developed. However, the intermediate-risk cytogenetic subgroup defined by the IPSS includes a miscellaneous number of different single abnormalities for which real prognosis at present is uncertain. The main aims of this study were to evaluate in an independent series the prognostic value of the IPSS and to identify chromosomal abnormalities with a previously unrecognized good or poor prognosis in 640 patients. In univariate analyses, cases with single 1q abnormalities experienced poor survival, whereas those with trisomy 8 had a higher risk of acute leukaemic transformation than the remaining patients (P = 0.004 and P = 0.009 respectively). Patients with single del(12p) had a similar survival to patients with a normal karyotype and showed some trend for a better survival than other cases belonging to the IPSS intermediate-risk cytogenetic subgroup (P = 0.045). Multivariate analyses demonstrated that IPSS cytogenetic prognostic subgroup, proportion of bone marrow blasts and haemoglobin level were the main prognostic factors for survival, and the first two characteristics and platelet count were the best predictors of acute leukaemic transformation risk. A large international co-operative study should be carried out to clarify these findings.