- [Show abstract] [Hide abstract] ABSTRACT: At least 15 different translocations have been described activating RET in papillary thyroid carcinomas. A break-apart fluorescence in situ hybridization (FISH) assay should detect many translocations involving the RET gene without requiring knowledge of the partner gene. G-banding and spectral karyotyping was performed to further characterize the papillary carcinoma cell line TPC-1. BAC clones flanking the 5' and 3' regions of RET were labeled with SpectrumRed and biotin detected with avidin-AMCA (blue). In addition to the previously described chromosomal t(1;10;21), TPC-1 was found to have del(7)(q22q31) and der(8)t(8;8)(p21;q11.2). With the BAC probes, TPC-1 interphase nuclei showed the expected signal pattern of one paired red-blue signal as well as separated red and blue signals from the rearranged RET gene in 93% of cells. Interphase nuclei from normal human lymphocytes showed two paired red-blue signals in 97% of cells. TPC-1 cells were found to have the previously described chromosomal rearrangement that involves chromosome 10, with few other cytogenetically detectable genomic alterations. RET rearrangement can be detected by a break-apart BAC FISH probe set in the interphase nuclei of TPC-1 cells. This assay can potentially detect clinically relevant translocations involving RET.
- [Show abstract] [Hide abstract] ABSTRACT: The high level of karyotypic complexity found in epithelial neoplasms hinders the characterization of their cytogenetic evolution. Derivation of such pathways in adenocarcinoma of the pancreas has been particularly limited, because only a few pancreatic carcinomas are resected at an early stage of disease and so the number of primary carcinomas for which analysis of abnormal karyotypes has been reported is small. Here we report the clonal karyotypic abnormalities identified by G-banding analysis of 36 primary pancreatic carcinomas obtained from patients undergoing a Whipple resection with curative intent. The majority of the 36 carcinomas were diploid or triploid (33 of 36; 91%). Numerical alterations were found in all carcinomas for which a complete karyotype was determined. All the chromosomes were involved in gain, loss, or both gain and loss of the entire chromosome, in at least 8 and up to 28 of the carcinomas. Most commonly lost were chromosomes 18 (in 78% of the 36 carcinomas), 17 (56%), 6 (44%), 21 (42%), 22 (42%), Y (36%), and 4 (33%). Gain of chromosome 20 was observed in 10 of the 36 carcinomas. Structural abnormalities were common, resulting in partial chromosomal gains and losses, with a median number of 7 partial imbalances per carcinoma (range, 1-15). Sixteen carcinomas contained double-minute chromosomes, homogeneously staining regions, or both, indicating gene amplification. Pooling data for these 36 carcinomas with the primary carcinomas with karyotypes published in the Mitelman database (http://cgap.nci.nih.gov/Chromosomes/Mitelman), we defined pathways of karyotypic evolution. The most frequent chromosomal imbalances were -18 (67.6%), -10 (34.3%), -4 (31.4%), +20 (31.4%), -15p (23.8%), -14p (22.9%), +2 (21.9%), -5 (21.9%), -13p (20%), +16 (20%), -21p (19%), -17p (19%), +1q (19.0%). Recurrent imbalances identified as occurring early were -1p, -15p, -18, -7q, -8p, -17p, and -5; late recurrent imbalances were +11q, +7q, +6p, -19p, and +2. In contrast to reports from similar analyses in other epithelial carcinomas, we did not find evidence for multiple karyotypic evolutionary pathways.
- [Show abstract] [Hide abstract] ABSTRACT: Inflammatory myofibroblastic tumors (IMTs) are rare soft tissue tumors occurring primarily in children and young adults. ALK gene rearrangements have been identified in this neoplasm, with fusion of the ALK gene at 2p23 to a number of different partner genes. Metaphase cytogenetic analyses of these tumors have been relatively few, however, and may help to identify additional variant partners. We report on an IMT from a 2-year-old boy with a karyotype of 45,XY,der(2)inv(2)(p23q12)del(2)(p11.1p11.2),-22. FISH showed ALK-RANBP2 fusion in this tumor. The breakpoint was cloned and the fusion was confirmed, making this the third reported case of IMT with ALK-RANBP2 fusion. In addition, we identified the ALK fusion partner in a previously reported IMT with t(2;17)(p23;q23) as CLTC, a gene reported to be involved in four other IMTs, and showed that the breakpoint involved a novel ALK-CLTC fusion. FISH evaluation of nine other IMTs identified CLTC as the fusion partner in one additional case, but RANBP2 was not involved in the remaining eight IMTs, suggesting that the variant partners involved in ALK rearrangements in IMTs are diverse.
- [Show abstract] [Hide abstract] ABSTRACT: Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.
- [Show abstract] [Hide abstract] ABSTRACT: Cutaneous T-cell lymphoma (CTCL) is a clonally derived lymphoproliferative disorder that preferentially involves the skin. The two major clinical expressions of CTCL, mycosis fungoides (MF) and Sézary syndrome (SS), have poorly understood pathogenesis. Chromosome abnormalities, mostly complex karyotypes, are seen in about 50% of patients with MF/SS, and there have only been a few instances of recurrent rearrangements. We analyzed 19 blood samples from patients with MF/SS with cytogenetics and multicolor FISH (SKY) to better describe the complex karyotypes and search for recurrent abnormalities or breakpoints. Comparison of phytohemagglutinin (PHA)-stimulated cultures versus a combination of interleukin 2 plus interleukin 7 showed similar efficiency in detecting abnormal clones; however, the PHA cultures yielded more analyzable metaphases. Nine of 19 patients (47%) had an abnormal karyotype. The most frequent abnormalities, in 7 of 9 cases, involved chromosome 10; followed by chromosome 6, in 6 of 9 cases; chromosomes 3, 7, 9, 17, and 19, in 5 of 9 cases; chromosomes 1 and 12, in 4 of 9 cases; and chromosomes 8, 11, and 13, in 3 of 9 cases. Most abnormalities were structural. Recurrent rearrangements included deleted chromosomes 6 and 13, in three cases each, and recurrent breakpoints at 1p32-36, 6q22-25, 17p11.2-13, 10q23-26, and 19p13.3, occurring in three or more cases. One patient had a pseudodicentric translocation between the short arms of chromosomes 8 and 17, confirmed by dual-color FISH and interpreted as psu dic(17;8)(p11.2;p11.2). Two patients with SS reported in the literature seem to have a similar translocation. If confirmed, a psu dic(17;8) could be the first recurring translocation detected in at least three patients with MF/SS.
- [Show abstract] [Hide abstract] ABSTRACT: The BCR/ABL gene rearrangement is the causing factor in chronic myeloid leukemia (CML). In most cases, it is cytogenetically visualized as a translocation between chromosomes 9 and 22, known as the Philadelphia (Ph) translocation. About 5-10% of CML patients lack cytogenetic evidence of the Ph translocation but show BCR/ABL fusion by fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction. Deletions around the breakpoints on the derivative 9 including ABL and or BCR sequences occur in 10-15% of Ph+ CML patients and are thought to have prognostic significance. We describe two patients with CML and normal karyotype in whom cryptic rearrangements involving chromosomes 9 and 22 resulted in the causative BCR/ABL gene. FISH with a three-color probe combination revealed BCR/ABL fusion on chromosome 9 without deletion in one patient; the other patient had BCR/ABL on chromosome 22 with an associated derivative 9 deletion. We discuss the proposed mechanisms in the formation of BCR/ABL in the setting of a normal karyotype. Some authors reported that patients with the chimeric gene located on the derivative 9 have a poor clinical course. We suggest that deletion rather than location of the chimeric gene alone is more likely to be associated with prognosis.
- [Show abstract] [Hide abstract] ABSTRACT: Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.
Conference Paper: How one oligodendroglioma lost both 1p and 19q.
- [Show abstract] [Hide abstract] ABSTRACT: Imatinib has impressive activity against chronic myeloid leukaemia (CML), but does not appear to completely eradicate the disease. Although responses to interferon-alpha (IFN) are slower and less dramatic than those to imatinib, they can be durable even after discontinuation of the drug. Unlike imatinib, the specific mechanisms responsible for IFN's clinical activity in CML are unknown. We found that IFN induced a G1 cell cycle arrest, as well as terminal differentiation, of the CML cell line KT-1 and CML CD34+ cells from clinical specimens. Myeloid growth factors augmented the antileukaemic activity of IFN, and neutralising antibodies directed against myeloid growth factors inhibited IFN's antileukaemic activity. We next directly compared the effects of imatinib and IFN against differentiated and primitive CML progenitors from newly-diagnosed patients. Although less active against CML granulocyte-macrophage colony forming units than imatinib, IFN was significantly more toxic to primitive CML progenitors responsible for the maintenance of long-term cultures. Imatinib and IFN appear to have divergent effects on CML progenitors at different stages of maturation, with imatinib more active against differentiated CML progenitors and IFN more active against primitive CML progenitors. The different target cells for these agents may explain the disparities in the kinetics and durability of their clinical responses. At least part of the clinical effect of IFN in CML appears to result from its ability to differentiate primitive CML progenitors.
- [Show abstract] [Hide abstract] ABSTRACT: We report a 4-year-old female with a de novo complex karyotype with multiple chromosomal rearrangements and a distinctive phenotype. Her medical history is significant for having been a twin born at 35 weeks gestation, breech presentation, with feeding problems and poor growth as an infant, gastroesophageal reflux disease, peripheral pulmonic stenosis, omphalocele, high myopia, and severe mental retardation. She is small for her age with microcephaly, posteriorly sloping forehead, shallow orbits, long palpebral fissures, prominent nose, wide mouth, absent uvula, kyphosis, brachydactyly, bridged palmar crease, and hypertonia. Peripheral blood lymphocytes revealed a karyotype of 46,XX,t(1;12)(p22.3;q21.3),inv(6)(p24q23),t(7;18)(q11.2;q21.2) in all cells. Parental karyotypes and that of her twin were normal. Spectral Karyotyping (SKY) and fluorescence in situ hybridization (FISH) with whole chromosome paints for chromosomes 1, 6, 7, 12, and 18 did not reveal additional rearrangements. Prometaphase G-banding analysis suggested that the "inverted" chromosome 6 might contain a cryptic rearrangement. Although no deletion nor duplication was detected using metaphase comparative genomic hybridization (CGH), multicolor high resolution banding (mBAND) demonstrated a double inversion of chromosome 6, resulting in a final karyotype as above but including der(6)(pter --> p23::q21 --> q22.3::q21 --> p23::q22.3 --> qter).
- [Show abstract] [Hide abstract] ABSTRACT: Clear cell sarcoma (CCS) is a rare and aggressive tumor, arising mainly in the soft tissue of the extremities in young adults. A distinctive chromosomal translocation, t(12;22)(q13;q12), has been found in most reported cases. We performed cytogenetic analyses on a primary and subsequent metastatic CCS that contained the t(12;22), along with other complex karyotypic changes. G-banding chromosome analysis was supplemented by spectral karyotyping (SKY), a 24-color chromosome-paint FISH technique, thus allowing identification of three marker chromosomes, unbalanced translocations, and other complex abnormalities. Four of these involved additional copies and structural abnormalities of chromosome 8. Clarifying such secondary karyotypic changes in CCS may prove valuable to the understanding of tumor cell biology and clinical behavior.
- [Show abstract] [Hide abstract] ABSTRACT: Malignant triton tumor (MTT) is a highly malignant neoplasm, classified as a variant of malignant peripheral nerve sheath tumor (MPNST) with rhabdomyoblastic differentiation. Few cytogenetic studies of MTT have been reported using conventional cytogenetic analysis. Here, we report a comprehensive cytogenetic study of a case of MTT using G-banding, Spectral Karyotyping(), and fluorescence in situ hybridization (FISH) for specific regions. A complex hyperdiploid karyotype with multiple unbalanced translocations was observed: 48 approximately 55,XY,der(7)add(7)(p?)dup(7),der(7) t(7;20)(p22;?)ins(20;19),der(7)ins(8;7)(?;p22q36)t(3;8)t(8;20),-8,-8,r(8)dup(8), +der(8)r(8;22),-9,der(11)t(11;20)(p15;?)ins(20;19),der(12)t(8;12)(q21;p13),der(13) t(3;13)(q25;p11),-17,-19,der(19)t(17;19)(q11.2;q13.1),-20,-22,+4 approximately 7r[cp24]/46,XY. The 1995 International System for Human Cytogenetic Nomenclature was followed where possible. Note that breakpoints were frequently omitted where only SKY information was known for a small part of an involved chromosome. Our analysis revealed some breakpoints in common with those previously reported in MTT, MPNST, and rhabdomyosarcoma, namely 7p22, 7q36, 11p15, 12p13, 13p11.2, 17q11.2, and 19q13.1. FISH showed high increase of copy number for MYC and loss of a single copy for TP53.
- [Show abstract] [Hide abstract] ABSTRACT: The myelin P2 protein, a 14,800-Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin-producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin-producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P2 protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human-hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P2 gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P2 gene as a candidate gene for autosomal recessive Charcot-Marie-Tooth disease type 4A.
- [Show abstract] [Hide abstract] ABSTRACT: The aromatic fatty acid sodium phenylbutyrate (PB) promotes cytostasis and differentiation in a wide variety of tumor types; among several molecular activities, inhibition of histone deacetylase (HDAC) may account for many of its pharmacodynamic effects. A Phase I study demonstrated promising preliminary evidence of clinical activity in acute myeloid leukemia and myelodysplastic syndrome; however, plasma concentrations achieved at the maximum tolerated dose were less than those targeted based on in vitro studies. Because prolonged exposure to suboptimal concentrations of PB in vitro led to pharmacodynamic changes similar to a more brief exposure to higher concentrations, a study of the feasibility of prolonged administration of sodium PB was performed. Selected patients with acute myeloid leukemia and myelodysplastic syndrome were treated with sodium PB as a continuous i.v. infusion via ambulatory infusion pump. Sequential cohorts were treated for 7 consecutive days out of 14 or with 21 consecutive days out of 28. Prolonged infusions were well tolerated; dose-limiting central nervous system toxicity developed in 1 of 23 patients treated. End-of-infusion plasma concentrations were maintained within a range sufficient to inhibit HDAC. Two patients on the 21/28 schedule developed hematological improvement. Prolonged infusions of PB are well tolerated making this an attractive platform for the clinical investigation of HDAC inhibition.
- [Show abstract] [Hide abstract] ABSTRACT: The extent of chromosomal rearrangements correlates positively with the level of expression of the nuclear matrix high mobility group (HMG) proteins HMGI(Y) when tested in three human prostate cancer cell lines (PC-3 > DU-145 > LNCaP). HMGI(Y), topoisomerase II, and A-T-rich sequences have been reported to be located at the base of the DNA loop domains in both the nucleus and chromosome and are juxtapositioned for chromosomal rearrangement. Transfecting and expressing full-length HMG-I into the LNCaP cell markedly enhanced the presence and heterogeneity of unbalanced (nonreciprocal) chromosomal rearrangements but not of balanced rearrangements. Unbalanced chromosomal rearrangements are common in solid human tumors.
- [Show abstract] [Hide abstract] ABSTRACT: We have identified a novel c-Myc-responsive gene, named JPO1, by representational difference analysis. JPO1 responds to two inducible c-Myc systems and behaves as a direct c-Myc target gene. JPO1 mRNA expression is readily detectable in the thymus, small intestine, and colon, whereas expression is relatively low in spleen, bone marrow, and peripheral leukocytes. We cloned a full-length JPO1 cDNA that encodes a 47-kDa nuclear protein. To determine the role of JPO1 in Myc-mediated cellular phenotypes, stable Rat1a fibroblasts overexpressing JPO1 were tested and compared with transformed Rat1a-Myc cells. Although JPO1 has a diminished transforming activity as compared with c-Myc, JPO1 complements a transformation-defective Myc Box II mutant in the Rat1a transformation assay. This complementation provides evidence for a genetic link between c-Myc and JPO1. Similar to c-Myc, JPO1 overexpression enhances the clonogenicity of CB33 human lymphoblastoid cells in methylcellulose assays. These observations suggest that JPO1 participates in c-Myc-mediated transformation, supporting an emerging concept that c-Myc target genes constitute nodal points in a network of pathways that lead from c-Myc to various Myc-related phenotypes and ultimately to tumorigenesis.
- [Show abstract] [Hide abstract] ABSTRACT: Arsenic is effective in the treatment of acute promyelocytic leukemia. Paradoxically, it is also carcinogenic. In the process of elucidating a mechanism of arsenic resistance in a leukemia cell line, NB4, we discovered that arsenic exposure causes chromosomal abnormalities, with a preponderance of end-to-end fusions. These chromosomal end fusions suggested that telomerase activity may be inhibited by arsenic. We found that arsenic inhibits transcription of the hTERT gene, which encodes the reverse transcriptase subunit of human telomerase. This effect may in part be explained by decreased c-Myc and Sp1 transcription factor activities. Decreased telomerase activity leads to chromosomal end lesions, which promote either genomic instability and carcinogenesis or cancer cell death. These phenomena may explain the seemingly paradoxical carcinogenic and antitumor effects of arsenic.
- [Show abstract] [Hide abstract] ABSTRACT: Sodium phenylbutyrate (PB) is an aromatic fatty acid with cytostatic and differentiating activity against malignant myeloid cells (ID(50), 1-2 mM). Higher doses induce apoptosis. Patients with myelodysplasia (n = 11) and acute myeloid leukemia (n = 16) were treated with PB as a 7-day continuous infusion repeated every 28 days in a Phase I dose escalation study. The maximum tolerated dose was 375 mg/kg/day; higher doses led to dose-limiting reversible neurocortical toxicity. At the maximum tolerated dose, PB was extremely well tolerated, with no significant toxicities; median steady-state plasma concentration at this dose was 0.29 +/- 0.16 mM. Although no patients achieved complete or partial remission, four patients achieved hematological improvement (neutrophils in three, platelet transfusion-independence in one). Other patients developed transient increases in neutrophils or platelets and decrements in circulating blasts. Monitoring of the percentage of clonal cells using centromere fluorescence in situ hybridization over the course of PB administration showed that hematopoiesis remained clonal. Hematological response was often associated with increases in both colony-forming units-granulocyte-macrophage and leukemic colony-forming units. PB administration was also associated with increases in fetal erythrocytes. These data document the safety of continuous infusion PB and provide preliminary evidence of clinical activity in patients with myeloid malignancies.
University of Maryland, BaltimoreBaltimore, Maryland, United States
Hopkins SchoolNew Haven, Connecticut, United States