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M H Dreyling

Publications and source records attributed to M H Dreyling.

13 recordsLinked to original sources
PubMed1998

MLL and CALM are fused to AF10 in morphologically distinct subsets of acute leukemia with translocation t(10;11): both rearrangements are associated with a poor prognosis.

The translocation t(10;11)(p13;q14) has been observed in acute lymphoblastic leukemia (ALL) as well as acute myeloid leukemia (AML). A recent study showed a MLL/AF10 fusion in all cases of AML with t(10;11) and various breakpoints on chromosome 11 ranging from q13 to q23. We recently cloned CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), the fusion partner of AF10 at 11q14 in the monocytic cell line U937. To further define the role of these genes in acute leukemias, 10 cases (9 AML and 1 ALL) with cytogenetically proven t(10;11)(p12-14;q13-21) and well-characterized morphology, immunophenotype, and clinical course were analyzed. Interphase fluorescence in situ hybridization (FISH) was performed with 2 YACs flanking the CALM region, a YAC contig of the MLL region, and a YAC spanning the AF10 breakpoint. Rearrangement of at least one of these genes was detected in all cases with balanced t(10;11). In 4 cases, including 3 AML with immature morphology (1 AML-M0 and 2 AML-M1) and 1 ALL, the signals of the CALM YACS were separated in interphase cells, indicating a translocation breakpoint within the CALM region. MLL was rearranged in 3 AML with myelomonocytic differentiation (2 AML-M2 and 1 AML-M5), including 1 secondary AML. In all 3 cases, a characteristic immunophenotype was identified (CD4+, CD13-, CD33+, CD65s+). AF-10 was involved in 5 of 6 evaluable cases, including 1 case without detectable CALM or MLL rearrangement. In 2 complex translocations, none of the three genes was rearranged. All cases had a remarkably poor prognosis, with a mean survival of 9.6 +/- 6.6 months. For the 7 AML cases that were uniformly treated according to the AMLCG86/92 protocols, disease-free and overall survival was significantly worse than for the overall study group (P = .03 and P = .01, respectively). We conclude that the t(10;11)(p13;q14) indicates CALM and MLL rearrangements in morphologically distinct subsets of acute leukemia and may be associated with a poor prognosis.

Acute Disease↗
PubMed1998

Codeletion of CDKN2 and MTAP genes in a subset of non-Hodgkin's lymphoma may be associated with histologic transformation from low-grade to diffuse large-cell lymphoma.

Identifying the various genetic alterations that contribute to lymphomagenesis is key to our improved understanding of the biological behavior of the disease. Recently, we and others have defined a tumor suppressor region on the short arm of chromosome 9 harboring a cluster of genes, including MTAP, CDKN2A (p16INK4a), and CDKN2B (p15INK4B), which is frequently deleted in a variety of tumor types. To determine whether this region is involved in a particular subset of malignant lymphomas, we have examined 16 cases of diffuse large-cell lymphoma (DLCL) (including three cases that evolved from low-grade non-Hodgkin lymphoma (NHL) (transformed DLCL)), and nine cases of low-grade NHL that had subpopulations of large cells with a diffuse growth pattern (seven follicular NHL, one chronic lymphocytic leukemia, one mycosis fungoides). Interphase fluorescence in situ hybridization was performed on these samples using a 250-kb cosmid contig (COSp16), which encompasses MTAP, CDKN2A, and CDKN2B. Six of the 16 DLCLs and one of nine low-grade NHLs had deletions of COSp16. COSp16 was homozygously deleted in four cases; two cases had hemizygous deletions, and one case had a partial homozygous deletion of the cosmid contig. Three of 13 cases of de novo DLCL, all three transformed DLCLs, and one of nine low-grade NHL had COSp16 deletions. Although the numbers are small, COSp16 deletion was associated with transformed DLCL in contrast to de novo DLCL (P < 0.04, Fisher's exact test) or low-grade NHL (P < 0.02). The COSp16 deletion was mostly submicroscopic and was not observed in association with any specific recurring cytogenetic abnormalities. These results suggest that targeted deletion of the CDKN2A region occurs in a subset of non-Hodgkin's lymphomas, and may be associated with transformed lymphomas.

Adolescent↗
PubMed1997

Alterations of the cyclin D1/p16-pRB pathway in mantle cell lymphoma.

Mantle cell lymphoma (MCL) has recently become generally accepted as a subentity of malignant lymphomas that is characterized by the chromosomal translocation t(11;14)(q13;q32), resulting in the overexpression of cyclin D1. Cyclin D1 forms a complex with cell cycle-dependent kinase (cdk) 4, which inactivates the retinoblastoma protein (pRB) via phosphorylation. However, in transgenic mice, the overexpression of cyclin D1 alone is not sufficient for the development of malignant lymphoma. To determine whether other members of the pRB pathway contribute to the malignant transformation of MCL, we analyzed 37 cases of MCL that were well characterized by morphology, immunophenotype, and/or interphase cytogenetics [detection of t(11;14)(q13;q32)]. Interphase fluorescence in situ hybridization was performed using a cosmid contig (250 kb) of the CDKN2/p16 region (encoding an inhibitor of the cyclin D1/cdk4 complex) and a phage contig (200 kb) of the Rb region. CDKN2/p16 deletion was detected in 15 cases (41%), including 6 homozygous deletions; Rb was deleted in 15 cases (41%), all of which were hemizygous deletions. Nine cases (24%) had deletions of both CDKN2/p16 and Rb. Further analysis of a subset of 17 MCLs revealed a highly significant correlation between CDKN2/p16 deletion and proliferation index, determined by the rate of Ki67 expression (P = 0.014; t test). No significant correlation was found between CDKN2/p16 deletion and the blastoid variant of MCL (P = 0.23; Fisher's test) or between proliferation index and blastoid morphology (P = 0.51; t test). Deletion of Rb did not have any impact on cell proliferation in addition to CDKN2/p16 deletion (P = 0.76; t test). Additional analysis of 13q14 deletions suggests that these deletions may target another gene telomeric to Rb. We conclude that deletion of CDKN2/p16 occurs in approximately one-half of MCLs and is a more relevant indicator of the proliferative features as compared to morphological criteria. In contrast, although deletions of chromosomal band 13q14 are frequent in MCL, inactivation of Rb seems not to be involved in the pathogenesis of MCL.

Animals↗
PubMed1996

The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.

The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line.

Adaptor Proteins, Vesicular Transport↗
PubMed1996

Preferential loss of expression of p16(INK4a) rather than p19(ARF) in breast cancer.

The tumor suppressor p16(INK4a) has been shown to be inactivated in numerous cancer lines and primary tumors. Recently, we reported loss of heterozygosity of the region in which p16(INK4a) is located in more than one-half of primary breast tumors. However, mutational analysis of these same tumors revealed mutation of p16(INK4a) to be infrequent. Other possible modes of inactivation, such as de novo methylation and homozygous deletion, have since been shown to occur in numerous neoplasias. Furthering the complexity of this locus, a transcript overlapping the p16(INK4a) coding sequence and encoding a novel peptide with growth-suppressive activity, p19(ARF), has been described. To clearly elucidate the target of aberrations affecting this subchromosomal region and approximate frequency in breast cancer, we performed a comprehensive study including p16 deletion analysis by means of interphase chromosomal fluorescence in situ hybridization, methylation analysis of the first exon encoding p16(INK4a) (exon 1alpha), mutational analysis of exon 1beta by single-strand conformational polymorphism analysis of p19(ARF) transcripts, and expression of both alpha and beta transcripts by reverse transcription PCR. Homozygous deletion of p16, as determined by interphase chromosomal fluorescence in situ hybridization, was observed in 3 of 18 (17%) tumors analyzed, whereas de novo methylation of exon 1alpha was observed in an additional 17% (4 of 23). Reduced expression of p16(INK4a) was observed in 11 tumors (48%), including all those in which homozygous deletion or complete methylation was observed. No mutations of exon 1 beta were detected, and expression of its transcript was variable, with 13% demonstrating decreased expression and 17% demonstrating overexpression. These results further support p16(INK4a) as a target of inactivation in the 9p21 region for breast cancer.

Breast Neoplasms↗
PubMed1995

Refined mapping of genomic rearrangements involving the short arm of chromosome 9 in acute lymphoblastic leukemias and other hematologic malignancies.

Deletions of chromosomal band 9p21 have been detected in various tumor types as well as in more than 20% of acute lymphoblastic leukemia (ALL). These deletions frequently include the entire interferon (IFN) gene cluster as well as the methylthioadenosine phosphorylase (MTAP) gene. Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) was proposed as a candidate tumor-suppressor gene on 9p21 because it is frequently deleted in cell lines derived from multiple tumor types. To determine if CDKN2 or another closely related gene on 9p is the target of 9p deletions in ALL and other hematologic malignancies, we analyzed 20 primary patient samples (13 ALL, 2 acute myeloid leukemias [AML], and 5 non-Hodgkin's lymphomas [NHL]) with 9p rearrangements using Southern blot analysis, fluorescence in situ hybridization (FISH), and single-strand conformation polymorphism (SSCP) for alterations of CDKN2. Homozygous deletions of the CDKN2/CDKN2B (p15) region were detected in 10 cases (50%; 6 ALL, 2 AML, and 2 NHL). In 1 additional case, the intensity of the Southern blot band was significantly reduced, suggesting a CDKN2 deletion in a subpopulation of the malignant cells. No CDKN2 or CDKN2B rearrangements were seen. The IFN gene cluster was homozygously deleted in 2 of 15 (13%) analyzed cases, whereas the MTAP gene was deleted in 6 of 15 cases (40%). In addition, hemizygous deletions of the CDKN2 region were identified in 6 ALL cases using interphase FISH. No point mutation of the coding region of CDKN2 was detected by SSCP in these cases. We conclude that CDKN2 is the most frequently homozygously deleted marker on 9p. The absence of point mutations in the coding region of CDKN2 in cases with hemizygous 9p deletions and the frequent codeletion of MTAP, CDKN2B, and other yet unidentified neighboring genes suggest that the simultaneous deletion of these genes may be necessary for the selective growth advantage of malignant cells.

Adolescent↗
PubMed1995

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Amino Acid Sequence↗
PubMed1995

Detection of CDKN2 deletions in tumor cell lines and primary glioma by interphase fluorescence in situ hybridization.

Deletions of chromosomal band 9p21 have been detected in various tumor types including melanoma, glioma, lung cancer, mesothelioma, and bladder cancer. Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) has been proposed as a candidate tumor suppressor gene because it is frequently deleted in cell lines derived from multiple tumor types. We performed fluorescence in situ hybridization (FISH) with interphase cells using yeast artificial chromosome clones and a cosmid contig of the CDKN2 region. In 10 cell lines (4 glioma, 2 melanoma, 2 non-small cell lung cancer, 2 bladder cancer) with 9p alterations detected by molecular or cytogenetic analysis, interphase FISH with the CDKN2 cosmid contig detected all 9p deletions previously identified by molecular analysis. Using this probe, FISH analysis of primary glioblastoma tumors revealed homozygous deletions of the CDKN2 region in 6 of 9 tumors (67%) whereas a yeast artificial chromosome probe containing the interferon type I (IFN) gene cluster was deleted in only 4 cases (44%). Thus, it is likely that the CDKN2 region is the target of 9p deletions in gliomas. Interphase FISH will play an important role in defining the clinical significance of 9p deletions in primary tumors because it is especially applicable to clinical samples which may be contaminated by normal cells.

Brain Neoplasms↗
PubMed1995

Detection of 9p deletions in leukemia cell lines by interphase fluorescence in situ hybridization with YAC-derived probes.

Hemizygous and homozygous deletions of the type I interferon gene cluster (IFN) have been detected in about 20% of acute lymphoblastic leukemias. A putative tumor suppressor gene (TSG) is thought to be located centromeric to the IFN cluster on chromosomal bands 9p21-22. We studied the accuracy of fluorescence in situ hybridization (FISH) for detecting deletions in interphase cells using yeast artificial chromosome (YAC) clones containing all or part of the IFN cluster. FISH probes were generated from YACs (320-1300 kb in size) by a sequence-independent amplification technique (SIA). Fifteen cell lines (nine T-ALL, three B-cell precursor ALL, one B-ALL, one AML, one CML-BC) that had been well characterized by conventional cytogenetic analysis and molecular techniques were analyzed. We were able to detect all numerical changes of the IFN cluster including homozygous and hemizygous deletions accurately and to define subclones of the cell lines. Moreover, in six cell lines we were able to identify subclones. In dilution experiments the detection thresholds for subpopulations with homozygous and hemizygous deletions were determined to be 5% and 7.5%, respectively.

Base Sequence↗
PubMed1995

CDKN2 gene deletion is not found in chronic lymphoid leukaemias of B- and T-cell origin but is frequent in acute lymphoblastic leukaemia.

Homozygous deletions of the cyclin-dependent kinase 4 (CDK4) inhibitor gene CDKN2 (p16, MTS1) have been demonstrated to occur frequently in human cancer cell lines of different origin. However, in most primary tumours the frequencies of CDKN2 deletions are not well defined. We studied primary samples of 100 patients with lymphoid leukaemias [B-lineage acute lymphoblastic leukaemia (ALL), n = 23; T-ALL, n = 7; B-cell chronic lymphocytic (B-CLL) or prolymphocytic (B-PLL) leukaemia, n = 50; T-CLL/T-PLL, n = 20] using fluorescence in situ hybridization (FISH) with eight overlapping cosmid clones covering the region on chromosome band 9p21 containing CDKN2. We did not observe any CDKN2 deletions in the 70 patients with chronic lymphoid leukaemias of B- or T-cell origin. Of the 23 patients with B-lineage ALL, one (4%) exhibited a CDKN2 deletion: in this patient, two clones were detected, one exhibiting a hemizygous and the other a homozygous deletion. On chromosome banding analysis, four patients with B-lineage ALL had a 9p aberration, whereas all CDKN2 copies were retained. In contrast, six of the seven (86%) patients with T-ALL exhibited CDKN2 deletions (homozygous, n = 4; hemizygous, n = 2). We conclude that hemizygous or homozygous deletions of the CDKN2 gene occur at high frequency in T-ALL and at low frequency in B-lineage ALL, supporting the role of this gene as a tumour suppressor, especially in T-ALL. However, from our data there is no evidence that CDKN2 is involved in the pathogenesis of chronic lymphoid leukaemias of B- or T-cell origin.

Adolescent↗
PubMed1994

Mapping a putative tumor suppressor gene on chromosome 9 bands p21-p22 with microdissection probes.

Deletions of the short arm of chromosome 9 have been observed in a number of malignant cell lines and primary tumor samples using cytogenetic and molecular techniques. These tumors include acute lymphoblastic leukemias, lymphomas, gliomas, melanomas, mesotheliomas, bladder cancer, and lung cancer. The smallest region of overlap (SRO) of these deletions is thought to contain a tumor suppressor gene. A microdissection library was constructed from bands 9p21-p23 to obtain DNA probes that would be useful in further defining the limits of the deletions. Eight single-copy probes were found to be homozygously deleted in at least 1 of the 10 cell lines examined. The mapping of these 8 clones using a panel of cell lines with deletions revealed that 3 probes mapped telomeric to the SRO and 5 clones mapped centromeric to the SRO.

Base Sequence↗