Alternative mechanisms associated with silencing of CDKN1C in Beckwith-Wiedemann syndrome.

BACKGROUND: Mutations in the imprinted gene CDKN1C account for approximately 10% of Beckwith-Wiedemann syndrome (BWS) cases. Fibroblasts from BWS patients with loss of methylation (LOM) at the imprinting control region (ICR) KvDMR1 have reduced CDKN1C expression. Another group of BWS patients with downregulated CDKN1C expression but with normal methylation at KvDMR1 has been identified. OBJECTIVE: To investigate the mechanism of CDKN1C silencing in BWS in these two classes of patients. METHODS: The CDKN1C promoter region was analysed for changes in DNA methylation using bisulphite sequencing, and for alterations in chromatin structure using the chromatin immunoprecipitation (ChIP) assay. RESULTS: There was only spurious CpG methylation of the CDKN1C promoter in fibroblast DNA from both normal individuals and patients with BWS, irrespective of the methylation status of KvDMR1. There was no detectable change in chromatin structure at the CDKN1C promoter in patients with LOM at KvDMR1. BWS patients with downregulated CDKN1C and normal methylation at KvDMR1 had depletion of dimethylated H3-K4 and enrichment of dimethylated H3-K9 and HP1gamma at the CDKN1C promoter, suggesting that in these cases gene silencing is associated with repressive chromatin changes. CONCLUSIONS: CDKN1C may be downregulated by multiple mechanisms including some that do not involve promoter methylation. In BWS patients with normal methylation at KvDMR1 and reduced expression of CDKN1C, repressive chromatin may play a role, but the absence of methylation and repressive chromatin structure at the CDKN1C promoter in BWS patients with LOM at KvDMR1 argues for a direct role of this epimutation in silencing CDKN1C.

NUP98-HOXD13 gene fusion in therapy-related acute myelogenous leukemia.

A novel chromosomal translocation, t(2;11)(q31;p15), was identified in a patient with therapy-related acute myelogenous leukemia (t-AML). Fluorescence in situ hybridization experiments mapped the breakpoint near NUP98; Southern blot analysis demonstrated that the nucleoporin gene NUP98 was disrupted by this translocation. We used rapid amplification of cDNA ends to identify a chimeric mRNA. An in-frame, chimeric mRNA that fused NUP98 sequences to the homeobox gene HOXD13 was cloned; the predicted fusion protein contains both the GLFG repeats from NUP98 as well as the homeodomain from HOXD13. The NUP98-HOXD13 fusion is structurally similar to the NUP98-HOXA9 fusion previously identified in patients with AML, leading to the speculation that NUP98-homeobox gene fusions may be oncogenic. Moreover, this report, along with a recent study that demonstrated NUP98-DDX10 fusions in patients with t-AML, raises the possibility that NUP98 may be a previously unsuspected target for chromosomal translocations in patients with t-AML.

Simultaneous presentation of acute monoblastic leukemia and mantle cell lymphoma: case report and review of the literature.

This paper reports a 73-year old woman with simultaneous presentation of acute monoblastic leukemia (acute myeloid leukemia (AML), French-American-British (FAB) type M5a) and mantle cell lymphoma. The patient presented with wasting, generalized lymphadenopathy, an extensive infiltrative rash and pancytopenia. Bone marrow and lymph node histopatholology showed extensive infiltration by leukemic monoblasts. Marrow cytogenetics revealed a complex karyotype, including t(8;16)(p11;p13). Flow cytometric immunophenotyping of peripheral blood, lymph node and bone marrow demonstrated two populations, expressing CD5, CD19, CD20 and CD22 and CD45, HLA-DR, CD13, CD33, CD14 and CD38, respectively. A focus of abnormal lymphocytes in the lymph node biopsy demonstrated BCL1 expression and t(11;14)(p11;p13) by fluorescence in situ hybridization and immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction. The patient received infusional cytarabine, daunorubicin and etoposide chemotherapy, with complete remission of both the AML and the mantle cell leukemia. To the authors' knowledge, this is the first report of simultaneous presentations of AML, FAB M5a and mantle cell lymphoma. The case is discussed and the literature is reviewed.

Cytogenetic study of maturing granulocytes in bone marrow of patients with acute myelogenous leukemia.

To determine the cytogenetic origin of maturing granulocytes in the bone marrow of patients with acute myelogenous leukemia, bone marrow cells were studied using a modified cytogenetic technique, which does not disrupt the cell membrane, in conjunction with periodic acid-Schiff (PAS) staining. In four cases successfully studied, myeloblasts were PAS-negative and granulocytes were PAS-positive. In three cases successfully studied following 0-2 days of culture, metaphase spreads with abnormal karyotypes characteristic of the patients' leukemic clones were seen in five of five, six of nine, and four of four PAS-positive cells successfully studied. These patients' bone marrows were AN, AA, and AA, respectively, by standard cytogenetic study. Therefore, the cytogenetic status of PAS-positive cells did not necessarily correlate with presence or absence of normal metaphases determined by standard cytogenetic study. Bone marrow cells which underwent full and partial granulocytic maturation in suspension culture were studied following 2 weeks of culture. Abnormal karyotypes were seen in five of five and two of two metaphases in PAS-positive cells successfully studied in two patients. Therefore, we have demonstrated that when acute myelogenous leukemia cells undergo myeloid maturation in culture, the mature cells may be definitely proven to derive from leukemic progenitors rather than from normal stem cells.

Truncated STAT proteins are prevalent at relapse of acute myeloid leukemia.

Signal transducer and activator of transcription (STAT) proteins are implicated in the control of cell survival, proliferation and differentiation in response to hematopoietic cytokines. C-terminally truncated STAT isoforms (STATbeta), as opposed to the full length form (STATalpha), have a competitive or even transdominant negative effect on gene induction mediated by the STAT pathway. We have previously demonstrated that while constitutively active STAT proteins were detected in ten of 36 (28%) for STAT3 and eight of 36 (22%) for STAT5 in pretreatment samples from newly diagnosed acute myeloid leukemia (AML) patients, a significantly larger fraction of samples [21 of 27 (78%)] expressed STATbeta proteins. To determine whether STATbeta expression was maintained or increased after relapse in AML, we compared STAT activity and isoform expression at diagnosis and at relapse in 17 patients. In this selected group, constitutively active STAT3 was detected in 13 of 17 (76%) AML samples at diagnosis but was detected in only four of these patients at relapse. Constitutively active STAT5 was detected in three of 17 (18%) AML samples at diagnosis; but only two at relapse. In contrast, STATbeta protein expression was observed in 12 of the 17 pretreatment samples (71%) and in 16 of 17 samples at relapse. Only one patient did not express STATbeta at relapse. Our results suggest that STATbeta isoform expression, rather than level of constitutive activity, may be involved in disease progression in AML.

Continued cell cycling ability of HL-60 cells following 3HaraC incorporation: a combination of "double-labeling" and sister chromatid analysis.

This study was designed to determine if HL-60 cells could undergo one or more cycles of DNA synthesis despite containing 3H-cytosine arabinoside (3HaraC) in their genome. HL-60 cells were incubated with 3HaraC for 2 hours, washed and maintained in a medium containing bromodeoxyuridine (BrdU). At fixed time points, cells were arrested in metaphase and prepared for chromosomal analysis. Treatment of the sample by an immunofluorescent monoclonal anti-BrdU antibody allowed us to determine the differential fluorescent pattern of sister chromatids in metaphase cells that had undergone two or more rounds of DNA synthesis in the presence of BrdU. Processing the samples by autoradiography demonstrated the presence of black grains (3HaraC) overlying the chromosomes. Thus, we were able to examine each metaphase for the presence of 3HaraC as well as the number of cycles it had completed in the presence of BrdU. We showed that despite the presence of 3HaraC in their DNA, some HL-60 cells were able to undergo two or more complete rounds of DNA replication.

A t(1;11) in acute nonlymphocytic leukemia FAB type M4.

Abnormalities involving the long arm of chromosome #11 have been shown to be involved in a high proportion of acute nonlymphocytic leukemias, specifically FAB types M4 and M5. In particular, band 11q23 seems to be preferentially affected. Reported herein is a case of acute nonlymphocytic leukemia type M4 showing a t(1;11)(q21;q23).

t(Y;18) in acute myeloblastic leukemia FAB type M2.

Changes affecting the Y chromosome in neoplasia have been documented. However, this usually occurs as a loss of the Y chromosome in the affected cells accompanied by or preceding other karyotypic changes. Involvement of the Y chromosome in a translocation is extremely rare, there being only 14 cases reported in the literature. Presented here is a case of t(Y;18) in acute myeloblastic leukemia type M2 (FAB).

A novel t(1;8)(p13;p11) in a thymic carcinoma with unusual giant cell features and renal metastasis.

Cytogenetic analysis of a thymic carcinoma metastatic to the left kidney revealed the presence of a t(1;8)(p13;p11). In addition to a previously undescribed translocation, this tumor histologically showed unusual giant cell features.

Recurrent involvement of 11q13 in acute nonlymphocytic leukemia.

Rearrangements of 11q have been associated with acute nonlymphocytic leukemia (ANLL), particularly M4 and M5 (FAB classification). Though 11q23 is most often involved, 11q13 is also affected. We report two cases of ANLL with translocations involving band 11q13.

Tetrasomy of chromosome 8: an interesting and rare cytogenetic phenomenon in acute nonlymphocytic leukemia.

Trisomy of chromosome #8 is a change commonly associated with acute nonlymphocytic leukemia (ANLL). However, tetrasomy of chromosome #8 in ANLL as a single chromosome change without accompanying abnormalities is extremely rare and, to the best of our knowledge, has not been reported to date. We present here a case of acute monocytic leukemia (AMoL, FAB M5b) with four copies of chromosome #8.

Consistent chromosome changes in leiomyosarcoma.

Karyotypic analysis of a leiomyosarcoma of the retroperitoneum revealed some structural and numerical changes. Review of the literature showed that some of these changes, namely involvement of 1p13 and 11p13 and monosomy 9, 18 and 22 seemed to occur frequently. These changes could be characteristic of leiomyosarcoma and define a cytogenetic subgroup within this tumor entity.

A uterine leiomyoma showing both t(12;14) and del(7) abnormalities.

The involvement of chromosomes 12 and 14 in uterine leiomyomas has been well established. However, in a recent report of only a del(7)(q22.1q31.32) or (q11.2q22.3) in two cases of typical uterine leiomyoma, Boghosian et al. hypothesized that this could represent a cytogenetic subgroup of uterine leiomyomas. We report a case of uterine leiomyoma with both the t(12;14) and del(7) in all the cells examined and discuss the implications of this in terms of critical chromosomal rearrangements underlying the route to benign cellular proliferation.

Deletion of chromosome 22 without bcr rearrangement and without juxtaposition of c-abl in a case of acute myeloid leukemia.

We describe a patient with acute myeloid leukemia (AML) who had a deletion of chromosome 22 at q11 as a sole chromosomal abnormality, resulting in the karyotype 46,XY,del(22)(q11). Southern blot analysis showed no bcr rearrangement and fluorescence in situ hybridization indicated no juxtaposition of c-abl. This study indicates that molecular events other than bcr rearrangement and c-abl juxtaposition were involved in leukemogenesis in this patient. We hypothesize that a tumor suppressor candidate gene may be located on the long arm of chromosome 22; its loss may lead to malignant transformation.

Trisomy 4: an entity within acute nonlymphocytic leukemia.

Trisomy 4 is a newly recognized primary chromosome change in leukemia. Five cases of acute nonlymphocytic leukemia (ANLL) are described from the United States and France. As in cases from Belgium, the only chromosome abnormality detected in the leukemic cells was trisomy 4. This was associated preferentially with ANLL of the M4 type (by FAB classification): acute myelomonocytic leukemia.

A t (11;21) (13;q22) in Ph-positive chronic myelogenous leukemia.

Reciprocal translocations, in addition to that of the Ph chromosome, though rare, have been reported in chronic myelogenous leukemia (CML). We describe here a case of Ph-positive CML with a new translocation, t (11;21) (q13;q22), and missing Y, which were present both during transformation to the blastic crisis and in the subsequent reversion to the chronic phase. The possible significance of this abnormality is discussed.

Complex cytogenetic changes in Ph-negative chronic myelogenous leukemia.

A Ph-negative chronic myelogenous leukemia (CML) with t(3;7)(q21;q32), t(4;9)(q21;q34), and del(8)(q22) is reported. This case is rather unusual for Ph-negative CML in being associated with complex chromosome changes. The patient was diagnosed as in the accelerated phase of CML. It will be important to study this malignant disorder in detail cytogenetically and molecularly in order to ascertain its nature and place among the myeloproliferative disorders.

Translocation t(1;22) in congenital acute megakaryocytic leukemia.

Acquired chromosomal abnormalities have been reported in 80 patients with congenital acute leukemia, the commonest being t(4;11). We report here a case of acute megakaryocytic leukemia with a rare translocation of t(1;22)(p13.3;q13.3). The course of the disease was short, with the patient surviving less than a year after the initial diagnosis.

Chromosome changes in metastatic human melanoma.

Cytogenetic studies were performed on human malignant melanoma cells from eight metastatic lesions. Five tumors displayed near-triploid and three near-diploid chromosome numbers. Chromosomes #1, #6, #7, followed by #2 and #9, were found to be most frequently involved in structural aberrations. Aberrations involving chromosome #1, with deletions or translocations of 1p, involving region 1p12-1p22 in seven of eight breakpoints of the p arm were observed. Seven of nine breakpoints of 6q were located at region 6q15-6q21. Most of the breakpoints on chromosome #7 occurred near the centromeric region. All tumors had additional chromosome material involving 1q, chromosome #7 (7q in two tumors), and in five tumors an increased dose of chromosome #6 (6p in one tumor). The nonrandom breakpoints of these and other chromosomes involved diverse bands, including loci of oncogenes and fragile sites. The observation of nonrandom chromosomal changes in advanced malignant melanoma suggests that genes important in the progression of melanoma are located on chromosomes #1, #6, and #7.

Translocation t(6;9)(p22.3;q34) in myelodysplastic syndrome--refractory anemia with excess blasts.

A 69-year-old male patient with refractory anemia with excess blasts (RAEB) was found to have a consistent chromosomal abnormality, t(6;9)(p22.3;q34), in the bone marrow and unstimulated peripheral blood cells. Twenty patients with t(6;9) and leukemia have been reported; some of them had a myelodysplastic syndrome (MDS) before developing overt ANLL. Our patient was still in the MDS stage when the t(6;9) was found. This result suggests that t(6;9) represents one of the pathways from MDS to leukemia in patients with ANLL.