Hypermethylation of the calcitonin gene in acute lymphoblastic leukaemia is associated with unfavourable clinical outcome

We analysed calcitonin (CALC1) gene hypermethylation using semiquantitative differential polymerase chain reaction in 105 patients with adult (n = 49) and childhood (n = 56) acute lymphoblastic leukaemia (ALL), and studied the association of CALC1 hypermethylation with clinical presentation features and disease outcome. We also investigated the possible relationship between CALC1 methylation status and expression of the cell cycle inhibitor gene p57KIP2. We observed CALC1 hypermethylation in bone marrow cells from 43% (45 out of 105) of ALL patients. Clinical, molecular and laboratory features did not differ significantly between hypermethylated and hypomethylated patients, only T-cell lineage was associated with hypermethylation (14% vs. 47%, P = 0025). Complete remission rate was similar in both groups although hypermethylated patients had a higher relapse rate (68% vs. 19%, P < 0.00001) and mortality rate (55% vs. 36%, P = 0.06) than hypomethylated patients. Estimated disease-free survival (DFS) at 6 years was 66.1% for hypomethylated patients and 5.3% for hypermethylated patients (P < 0,00001). Multivariate analysis from potential prognostic factors demonstrated that CALC1 methylation status was an independent prognostic factor in predicting DFS (P = 0.0001). Separate analysis of adult and childhood ALL patients showed similar results to the whole series. In addition, hypermethylated patients showed downregulation of p57KIP2 expression. Our results suggest that CALC1 gene hypermethylation is associated with an enhanced risk of relapse independently of known poor-prognostic factors and we describe, for the first time, a possible implication of the p57KIP2 gene in the genesis and prognosis of ALL.     

Npm1 is a haploinsufficient suppressor of myeloid and lymphoid malignancies in the mouse

Nucleophosmin (NPM1) gene has been heavily implicated in cancer pathogenesis both as a putative proto-oncogene and tumor suppressor gene. NPM1 is the most frequently mutated gene in acute myeloid leukemia (AML), while deletion of 5q, where NPM1 maps, is frequent in patients with myelodysplastic syndromes (MDS). We have previously shown that mice heterozygous for Npm1 (Npm1+/-) develop a hematologic syndrome with features of human MDS. Here we analyzed Npm1+/- mutants to determine their susceptibility to cancer. Npm1+/- mice displayed a greater propensity to develop malignancies compared with Npm1+/+ mice. The Npm1+/- cohort frequently developed hematologic malignancies of both myeloid and lymphoid origin with myeloid malignancies displaying the highest incidence. Malignant cells retained the wild-type allele with normal localization and expression of Npm1 at the protein level, suggesting that complete Npm1 loss is not a prerequisite for tumorigenesis. Our results conclusively demonstrate that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment.     

Genomic organization of the 5' region of the human thyroglobulin gene

OBJECTIVE: The purpose of the present work is to establish the intron-exon organization from exon 12 to exon 23 of the human thyroglobulin gene and to construct a physical map of the 5' terminal half of the gene. DESIGN: Screening of a genomic library and subsequent restriction map, hybridization and sequencing methods have been employed to characterize the recombinant positive phages. METHODS: A human genomic DNA library was screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. Intron/exon junction sequences were determined by the Taq polymerase-based chain terminator method. Finally, the thyroglobulin gene was mapped using the Gene Bridge 4 radiation hybrid clone panel. RESULTS: We isolated and characterized four lambda phage clones that include nucleotides 3002 to 4816 of the thyroglobulin mRNA, encompassing exons 12 to 23 of the gene. The exon sizes range between 78 and 219 nucleotides. We found that the GT-AG splicing sequences rule was perfectly respected in all the introns. A total of 7302 intronic bases was analyzed. Hormogenic tyrosine 5 and 1291 are encoded by exons 2 and 18. Also, seven alternative spliced variants are associated with the 5' region. Thyroglobulin gene maps to 5,5 centiRays from the AFMA053XF1 marker, in chromosome 8. CONCLUSIONS: The present study shows that the first 4857 bases of thyroglobulin mRNA are divided into 23 exons and the four phages isolated include 32.6 kb genomic DNA, covering 1815 nucleotides of exonic sequence distributed in 12 exons, from exon 12 to 23.     

Hypermethylation of calcitonin gene in adult acute leukemia at diagnosis and during complete remission

Hypermethylation of the calcitonin gene has been described in various hematologic malignancies. In order to assess its frequency and potential usefulness as a marker for leukemic cells and to detect potential clinical correlations, 180 adult patients (aged > 15 years) with newly diagnosed acute leukemia including 133 cases of acute myeloid leukemia (AML) and 47 cases of acute lymphoblastic leukemia (ALL) were tested for its presence in leukemic blasts at diagnosis by Southern blot technique and polymerase chain reaction (PCR) using 3 sets of primers (P550, P566, P1400), amplifying the most frequent sites of hypermethylation upstream or within the gene. In AML, 92 patients (69%) had hypermethylation detected by Southern blot at diagnosis. This hypermethylation could be confirmed by PCR in 18 of 36 tested cases (50%). Hypermethylation was not significantly associated to any clinical or hematological characteristic of the disease. In ALL, 44 patients (94%) had hypermethylation detected by Southern blot at diagnosis. This hypermethylation could be confirmed by PCR in 33 of the 43 tested cases (77%). Sensitivity of PCR assessed by dilution was 1 to 0.1%. Hypermethylation was not either significantly related to any clinical or hematologic characteristics of the disease. Seven ALL cases which were positive by PCR at diagnosis and achieved cytological CR could be tested during CR. Five cases were negative and did not relapse after 3 to 27 months in CR. One case was positive at the beginning of CR and became negative after autologous transplant. However, he relapsed after 9 months in CR, 3 months after the last negative test. PCR for Bcr/Abl was also negative at this time. We conclude that hypermethylation of the calcitonine gene is frequent at diagnosis in adult acute leukemia, particularly in ALL.     

Alterations of the retinoic acid receptor alpha (RAR alpha) gene in myeloid and lymphoid malignancies

The retinoic acid receptor alpha (RAR alpha) protein plays a central role in myeloid differentiation, and chromosomal translocations disrupting the RAR alpha gene are implicated in the development of acute myeloid leukaemia (AML). To identify haemopoietic malignant disorders which may also be linked to RAR alpha abnormalities, Southern blot analysis was performed in DNA from 153 patients with haematological malignancies other than AML FAB type M3 using RAR alpha cDNA probes. Alterations of RAR alpha were detected in 1/42 myelodysplastic syndromes (MDS), 2/24 AML, 3/47 B-chronic lymphocytic leukaemias (B-CLL), 0/40 lymphomas and 0/60 normal individuals. These data strongly suggest that alterations of RAR alpha might predispose for myeloid and lymphoid disorders.     

Hypermethylation of the promoter region is associated with the loss of MEG3 gene expression in human pituitary tumors

MEG3 is a human homolog of the mouse maternal imprinted gene, Gtl2. Gtl2 has been suggested to be involved in fetal and postnatal development and to function as an RNA. Recently our laboratory demonstrated that a cDNA isoform of MEG3, MEG3a, inhibits cell growth in vitro. Interestingly, MEG3 is highly expressed in the normal human pituitary. In striking contrast, no MEG3 expression was detected in human clinically nonfunctioning pituitary tumors. These data indicate that this imprinted gene may be involved in pituitary tumorigenesis. In the present study we investigated the mechanism underlying the absence of MEG3 expression in human clinically nonfunctioning pituitary tumors. No genomic abnormality was detected in the tumors examined. Instead, we found that two 5'-flanking regions, immediately in front of and approximately 1.6-2.1 kb upstream of the first exon, respectively, were hypermethylated in tumors without MEG3 expression compared with the normal pituitary. Reporter assays demonstrated that these two regions are functionally important in gene expression activation. Furthermore, treatment of human cancer cell lines with a methylation inhibitor resulted in MEG3 expression. We conclude that hypermethylation of the MEG3 regulatory region is an important mechanism associated with the loss of MEG3 expression in clinically nonfunctioning pituitary tumors.     

Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for gamma interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5' CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell line, was treated with gamma interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma cell line, only did so when treated with 5-aza-2'-deoxycytidine followed by gamma interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of gamma interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell-lineage lymphomas.     

The vent-like homeobox gene VENTX promotes human myeloid differentiation and is highly expressed in acute myeloid leukemia

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34⁺ leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT–PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33⁺ myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34⁺/CD38⁻ or CD34⁺ human progenitor cells. In contrast to this, leukemic CD34⁺/CD38⁻ cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34⁺ cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34⁺ human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.     

Tabulation of myeloid, lymphoid and intestinal malignancies in Crohn's disease.

A variety of malignant complications occur in Crohn's disease, and previous studies have recorded an increased intestinal cancer risk. The present investigation tabulated myeloid and lymphoid malignancies compared with intestinal cancers in 1000 consecutively evaluated patients with Crohn's disease who were followed over an extended period by a single clinician. Myeloid and lymphoid neoplasms were present in 0.5% of patients, while cancer in the intestinal tract was detected in 1%. Most of these patients with a malignancy had Crohn's disease for a prolonged period of more than 20 years and had negative outcomes, including death or presentations with advanced disease. In this cohort, lymphoma was not detected in a single patient after definition of Crohn's disease, possibly reflecting the limited use of immunosuppressives or infused biological agents in this clinical practice. Bypassed rectal 'stumps' were associated with subsequent colorectal cancer in half of all males with colon cancer in this series, suggesting an important risk factor following colectomy in Crohn's disease. Epithelial dysplasia was detected in only a single male patient before colorectal cancer, implying that this histopathological marker may be a poor predictor of subsequent colon cancer development in Crohn's disease, an inflammatory bowel disease process that is typically patchy or focal in distribution in the intestinal tract.     

The incidence of lymphoid and myeloid malignancies among hospitalized Crohn's disease patients

An association may exist between Crohn's disease (CD) and lymphoid/myeloid malignancies. We aimed to evaluate the 2-year cumulative incidence rate of lymphoid/myeloid malignancy among hospitalized CD patients. This is a retrospective cohort study using hospital discharge data from California and Virginia. Cohorts were defined by the presence or absence of a CD diagnosis in all patients discharged during a single calendar year (Year-2). The presence or absence of lymphoid/myeloid malignancy was determined for all hospitalizations during a 4-year period (Year-1 to Year-4) for each member of both cohorts. To obtain a 2-year cumulative incidence rate, patients with lymphoid/myeloid malignancy prior to or at the time of their first admission in Year-2 were excluded. Patients were followed for 8 quarters after this admission for the incidence of lymphoid/myeloid malignancy. Cumulative incidence rates and odds ratios were calculated. The crude 2-year incidence rate of lymphoid/myeloid malignancy among hospitalized CD patients was 3.87/1.000 CD patients (21/5,426; 95% CI = 2.40-5.92). The odds ratio adjusted for age, gender, and race was 2.04 (95% CI = 1.33-3.14, p < 0.001). The 2-year cumulative incidence of lymphoid/myeloid malignancies among hospitalized CD patients is greater than that seen in hospitalized patients without CD. This finding supports the need for further prospective population-based studies.     

The interleukin 3 gene is located on human chromosome 5 and is deleted in myeloid leukemias with a deletion of 5q.

The gene IL-3 encodes interleukin 3, a hematopoietic colony-stimulating factor (CSF) that is capable of supporting the proliferation of a broad range of hematopoietic cell types. By using somatic cell hybrids and in situ chromosomal hybridization, we localized this gene to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, IL-3 was found to be deleted in the 5q-chromosome of one patient with refractory anemia who had a del(5)(q15q33.3), of three patients with refractory anemia (two patients) or acute nonlymphocytic leukemia (ANLL) de novo who had a similar distal breakpoint [del(5)(q13q33.3)], and of a fifth patient, with therapy-related ANLL, who had a similar distal breakpoint in band q33 [del(5)(q14q33.3)]. Southern blot analysis of somatic cell hybrids retaining the normal or the deleted chromosome 5 from two patients with the refractory anemia 5q- syndrome indicated that IL-3 sequences were absent form the hybrids retaining the deleted chromosome 5 but not from hybrids that had a cytologically normal chromosome 5. Thus, a small segment of chromosome 5 contains IL-3, GM-CSF (the gene encoding granulocyte-macrophage-CSF), CSF-1 (the gene encoding macrophage-CSF), and FMS (the human c-fms protooncogene, which encodes the CSF-1 receptor). Our findings and earlier results indicating that GM-CSF, CSF-1, and FMS were deleted in the 5q-chromosome, suggest that loss of IL-3 or of other CSF genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).     

c-fms expression is a molecular marker of human acute myeloid leukemias

The c-fms protooncogene product was identified as the CSF-1 or M-CSF receptor, a polypeptide growth factor that plays a major role in myelomonocytic differentiation. This led us to look for expression of c-fms in fresh acute myeloid leukemia (AML) cells, using Northern blot analysis. c-fms expression was found in the leukemic cells of 28 AML patients, regardless of their stage of differentiation, which was assessed in the French-American-British (FAB) classification. However, the level of c-fms expression was especially high in AML of the M5 stage. High levels of expression were not accompanied by either amplification or rearrangements of the c-fms gene in AML cell DNAs. In contrast, c-fms expression was not found in acute lymphoid leukemias, whether of T or B origin. Thus, c-fms expression appears as a specific molecular marker of leukemogenesis in the myeloid lineage.     

High level of endothelial cell-specific gene expression by a combination of the 5' flanking region and the 5' half of the first intron of the VE-cadherin gene

To develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5' flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5' half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin(+) progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation.