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A S T Lim,
T H Lim,
M M Hess,
S K Kee,
Y Y F Lau,
R Gilbert,
T E Hempel,
K J Anderson,
D H Zaleski,
S L Tien,
P Chia,
R Subramaniam,
H K Tan,
A S A Tan, W G Sanger
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ABSTRACT: To assess the clinical utility of fluorescence in-situ hybridisation with chromosomes 13, 18, 21, X and Y as a stand-alone test in detecting chromosomal abnormalities, and the types of chromosomal abnormalities missed.
Retrospective analysis.
A restructured Government hospital in Singapore and an academic hospital in the United States.
Cytogenetic data of prenatal specimens and results of fluorescence in-situ hybridisation of 5883 patients performed between January 2000 and August 2007 were reviewed.
Fluorescence in-situ hybridisation detected 558 (9.5%) patients with chromosomal abnormalities. Abnormal ultrasounds (70%) and maternal serum screens (21%) were the most indicative of chromosomal abnormalities. When comparing fluorescence in-situ hybridisation data with karyotype results for the five chromosomes of interest, the sensitivity and specificity were 99.3% and 99.9%, respectively. When comparing fluorescence in-situ hybridisation data with karyotype results for all chromosomes, the sensitivity decreased to 86.8%, whereas the specificity remained at 99.9%. Of 643 cases with karyotype abnormalities, 85 were fluorescence in-situ hybridisation-negative (false negative rate, 13.2%), which included structural rearrangements, chromosome mosaicism, and other trisomies. Despite abnormal ultrasound indications, fluorescence in-situ hybridisation missed 32 cases which included structural rearrangements, mosaicisms, and other trisomies.
This study does not support fluorescence in-situ hybridisation as a stand-alone test. Institutions supporting fluorescence in-situ hybridisation as a stand-alone test must seriously consider the risks of a missed diagnosis.
Hong Kong medical journal = Xianggang yi xue za zhi / Hong Kong Academy of Medicine 12/2010; 16(6):427-33.
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J Iqbal,
T C Greiner,
K Patel,
B J Dave,
L Smith,
J Ji,
G Wright, W G Sanger,
D L Pickering,
S Jain, [......],
E S Jaffe,
J Delabie,
L Rimsza,
G Ott,
H K Müller-Hermelink,
J M Connors,
J M Vose,
T McKeithan,
L M Staudt,
W C Chan
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ABSTRACT: Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.
Leukemia 12/2007; 21(11):2332-43. · 9.56 Impact Factor
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J Iqbal,
T C Greiner,
K Patel,
B J Dave,
L Smith,
J Ji,
G Wright, W G Sanger,
D L Pickering,
S Jain, [......],
E S Jaffe,
J Delabie,
L Rimsza,
G Ott,
H K M|[uuml]|ller-Hermelink,
J M Connors,
J M Vose,
T McKeithan,
L M Staudt,
W C Chan
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[hide abstract]
ABSTRACT: Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.Keywords: BCL6, mutation, translocation, protein expression, gene expression profiling, diffuse large B-cell lymphoma
Leukemia 07/2007; 21(11):2332-2343. · 9.56 Impact Factor
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Prenatal Diagnosis 12/2004; 24(11):931-2. · 2.11 Impact Factor
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Y Hayami,
S Iida,
N Nakazawa,
I Hanamura,
M Kato,
H Komatsu,
I Miura,
B J Dave, W G Sanger,
B Lim,
M Taniwaki,
R Ueda
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ABSTRACT: Chromosomal band 1p34-36 is a commonly rearranged locus in many types of cancers. We cloned the breakpoint region of a chromosomal translocation, t(1;14)(p34;q32), found in the human multiple myeloma (MM) cell line, ODA. This rearrangement occurred between the nearby switch region of the immunoglobulin heavy chain (IgH) gene (Sgamma3) at 14q32 and the first intron of the human retinoic acid-inducible E3 protein (E3)/lysosome-associated protein, transmembrane-5 (LAPTm5) gene at the 1p34 locus. Consequently, the E3 gene, which is a hematopoietic cell-specific transcript induced by retinoic acid and located at the rearranged allele, was interrupted within its coding region and was not expressed in the ODA cell line in spite of the other allele still being intact. The expression derived from the remaining intact allele in ODA cells was silenced by DNA methylation at sequences within the first intron around a GC-rich EagI site. Interestingly, the silenced expression of E3 mRNA due to DNA methylation of intron 1 sequences was frequently encountered in MM cells [6/10 (60%) of MM cell lines tested], while E3 is expressed in normal plasma cells and in most other hematopoietic cell lines including those of B-cell lineage. Thus, as the E3 protein has been suggested to be involved in cellular differentiation and apoptotic pathways in certain cell types, our results suggest that loss of E3 gene expression might be a crucial event during the progression of human MM.
Leukemia 09/2003; 17(8):1650-7. · 9.56 Impact Factor
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ABSTRACT: The etiology of multiple myeloma (MM) remains obscure, although reports of familial clustering have implicated both a host susceptibility factor and environmental effects. Here we describe the medical histories of members of a family prone to MM.
We developed a pedigree for an MM-prone family by using information obtained from a questionnaire. Protein immunoelectrophoresis of serum and urine from the proband and from 19 family members was performed to detect monoclonal immunoproteins. Peripheral blood obtained from the proband and from five relatives was subjected to standard cytogenetic studies to detect constitutional chromosomal abnormalities. Multifluor-fluorescence in situ hybridization (M-FISH) and standard FISH studies were performed on peripheral blood from the proband and from two other affected living relatives to determine their karyotypes and to detect clonal chromosomal abnormalities frequently seen in patients with MM.
Within this family, a sibship of seven included three individuals (including the proband) with histologically verified MM and two individuals with a monoclonal gammopathy of unknown significance (MGUS), as determined by immunoelectrophoresis of serum and urine. This family also had members with acute lymphocytic leukemia, malignant melanoma, and prostate cancer. In the family members tested, we detected no constitutional chromosomal abnormality. None of the three individuals analyzed by FISH had a deletion of the retinoblastoma (Rb-1) locus, which is frequently deleted in patients with MM, and only one (the proband) had a translocation involving chromosomes 11 and 14, a clonal abnormality commonly seen in MM.
The study of familial MM may provide insights into the pathogenesis and, ultimately, the control and prevention of MM and related disorders.
JNCI Journal of the National Cancer Institute 11/2001; 93(19):1479-83. · 13.76 Impact Factor
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ABSTRACT: A female infant survived 5(1/2) hours after delivery at 33 weeks gestation. Autopsy showed a lobar variant of holoprosencephaly (HPE). Cytogenetic analysis revealed a 2q37.1-->2q37.3 deletion. This case represents the fourth reported case of HPE associated with partial monosomy 2q37 and the first with an apparent isolated 2q37 deletion. Chromosome segment 2q37.1-->2q37.3 may harbor yet another locus important in forebrain development, which, when disrupted, can lead to brain malformations within the HPE spectrum.
American Journal of Medical Genetics 05/2001; 100(3):179-81.
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ABSTRACT: Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.
Blood 03/2001; 97(3):809-11. · 9.90 Impact Factor
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ABSTRACT: Although mantle cell lymphoma (MCL) is considered a distinctive disease entity within non-Hodgkin's lymphoma (NHL), the cytology and growth pattern of MCL can be quite variable and the clinical significance of these features is unclear. Also, the role of anthracyclines in the management of MCL is unclear. Therefore, we examined our experience with MCL in an effort to clarify these important issues. We identified 68 patients with MCL who were evaluated clinically and treated by the Nebraska Lymphoma Study Group. Treatment consisted of combination chemotherapy containing an anthracycline in 76% of the patients. The cases were grouped by blastic or lymphocytic cytology, and the latter were divided by growth pattern into nodular (or mantle-zone) and diffuse types. The clinical and pathological variables were then evaluated for their prognostic value. The median overall survival (OS) and failure-free survival (FFS) for the entire group were 38 months and 12 months, respectively, and there was no survival advantage for those who received an anthracycline. The cases were grouped as follows: blastic type, 26%; nodular lymphocytic type, 44%; and diffuse lymphocytic type, 30%. Both the cytology and pattern of growth were predictive of OS and FFS. The median OS was as follows: blastic type, 55 months; nodular lymphocytic type, 50 months; and diffuse lymphocytic type, 16 months (P = 0.0038). The clinical features that predicted for a shorter survival included bone marrow involvement, advanced stage disease, B symptoms, a poor performance score, and the International Prognostic Index. We conclude that new therapeutic approaches, with the patients stratified by histologic type and clinical prognostic factors, are clearly needed for MCL.
American Journal of Hematology 08/2000; 64(3):190-6. · 4.67 Impact Factor
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D D Weisenburger,
R D Gascoyne,
P J Bierman,
T Shenkier,
D E Horsman,
J C Lynch,
W C Chan,
T C Greiner,
J M Connors,
J M Vose,
J O Armitage, W G Sanger
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ABSTRACT: Follicular large cell lymphoma (FLCL) is an aggressive disease that responds to anthracycline-containing chemotherapy much like diffuse large B-cell lymphoma (DLBCL). Since the t(14;18) and/or bcl2 protein expression are less common in FLCL than in its low-grade counterparts, we sought to determine whether these features were predictive of survival as in DLBCL. We studied 50 patients with FLCL who were treated with curative intent. The t(14;18) was found by cytogenetic analysis in 56% of the patients and bcl2 protein was expressed by the tumor cells in 73%, but neither was predictive of survival. However, abnormalities of chromosome 17p and the presence of trisomy 21 were adverse predictors of survival, as were a number of clinical features. We conclude that neither the absence of the t(14;18) nor the lack of bcl2 expression explain the good response of a subset of patients with FLCL to curative therapy.
Leukemia and Lymphoma 03/2000; 36(5-6):513-23. · 2.58 Impact Factor
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ABSTRACT: Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.
Journal of Interferon & Cytokine Research 11/1999; 19(10):1207-17. · 3.06 Impact Factor
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ABSTRACT: Identification of clonal chromosomal abnormalities involving 14q32 and its association with specific histological subtypes of non-Hodgkin lymphoma (NHL) has provided substantial insight to the genetic events leading to the disease. However, in some cases with inferior morphology of tumor cell chromosomes, the additional segment on chromosome 14 remains unidentified by cytogenetic banding techniques alone. To elucidate the origin of the additional chromosomal segment and to correlate the newly determined alterations with histology, metaphases from 15 NHL patients with add(14)(q32) were examined using fluorescence in situ hybridization (FISH) techniques after cytogenetic analysis had been performed. We found the duplication of 14q involving the q32 region in 6 cases with a dup(14) (q32) in 4 cases and a dup(14)(q24q32) in 2 cases. In 8 cases, FISH unveiled known NHL associated translocations; a t(14;18)(q32;q21) in 4 cases, a t(11;14)(q13;q32) in 2 cases, a t(8;14)(q24;q32) and a t(9;14)(p13;q32) in 1 case each. We also noted a t(14;17)(q32;q21) in 1 case. The use of FISH was a valuable asset in determining the origin of the additional material on chromosome 14q32, and helped resolve a group of B-cell NHLs with involvement of a duplicated 14q32 region.
Cancer Genetics and Cytogenetics 09/1999; 113(1):19-24. · 1.39 Impact Factor
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ABSTRACT: We studied 850 consecutive cases of histologically ascertained pretreatment non-Hodgkin's lymphoma with cytogenetically abnormal clones. The diagnostic karyotypes revealed that 12% of these cases exhibited structural rearrangements involving chromosome band 1p36. Here, we describe the karyotypes of 53 cases containing a 1p36 rearrangement [often involving translocations of unknown material and presented as add(1)(p36)]. We used fluorescence in situ hybridization to determine the origin of the translocation partners. We report three different recurrent translocations involving 1p36. These include der(1)t(1;1)(p36;q21) (three cases), der(1)t(1;1)(p36;q25) (three cases), and der(1)t(1;9)(p36;q13) (four cases). Using cytogenetic and fluorescence in situ hybridization analyses, we have resolved the translocation partners in 31 cases. Rearrangements of band 1p36 were found among different histopathological subtypes. Alterations of 1p36 never occurred as a sole abnormality, and in 42 of 53 cases, alterations of the band 14q32 were observed. The t(14;18)(q32;q21) translocation was present in 35 cases. The significantly high occurrence of 1p36 breakpoint in structural rearrangements and its involvement in recurrent translocations suggest that the region is bearing gene(s) that are important in lymphomagenesis. Our study also showed that cytogenetically evident deletions were frequent in chromosome 1p, almost always involving the p36 region, whereas duplications were rare and never encompassed the p36 region. Chromosome band 1p36 harbors many candidate tumor suppressor genes, and we propose that one or more of these genes might be deleted or functionally disrupted as a molecular consequence of the rearrangements, thus contributing to lymphomagenesis.
Clinical Cancer Research 07/1999; 5(6):1401-9. · 7.74 Impact Factor
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Q Zhang,
R Siebert,
M Yan,
B Hinzmann,
X Cui,
L Xue,
K M Rakestraw,
C W Naeve,
G Beckmann,
D D Weisenburger, W G Sanger,
H Nowotny,
M Vesely,
E Callet-Bauchu,
G Salles,
V M Dixit,
A Rosenthal,
B Schlegelberger,
S W Morris
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ABSTRACT: Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10, a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD), in MALT lymphomas due to the recurrent t(1;14)(p22;q32). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD. Wild-type BCL10 activated NF-kappaB but induced apoptosis of MCF7 and 293 cells. CARD-truncation mutants were unable to induce cell death or activate NF-kappaB, whereas mutants with C-terminal truncations retained NF-kappaB activation but did not induce apoptosis. Mutant BCL10 overexpression might have a twofold lymphomagenic effect: loss of BCL10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-kappaB activation may provide both anti-apoptotic and proliferative signals mediated via its transcriptional targets.
Nature Genetics 06/1999; 22(1):63-8. · 35.53 Impact Factor
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V H Rao,
R K Singh,
D C Delimont,
R H Finnell,
J A Bridge,
J R Neff,
B P Garvin,
D L Pickering, W G Sanger,
B A Buehler,
G B Schaefer
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ABSTRACT: We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.
International Journal of Oncology 03/1999; 14(2):291-300. · 2.40 Impact Factor
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ABSTRACT: Low grade malignant lymphomas arising from mucosa associated lymphoid tissue (MALT) represent a distinct clinicopathological entity. The cytogenetic findings and molecular genetics of MALT lymphomas remain minimally defined. Cytogenetic studies infrequently constitute part of the diagnostic work-up of MALT lymphomas, most commonly due to small biopsy size and their extranodal localization. Only 28 MALT cases with a clonal karyotype have been published to date. A number of chromosomal abnormalities have been observed with the majority of the cases featuring trisomy of chromosome 3 which is present in up to 78% of the cases.
A total of 116 cases of MALT lymphoma were diagnosed at BCCA between 1988 and 1997. Eleven cases of pathologically confirmed MALT lymphomas were subjected to cytogenetic analysis at the time of the initial evaluation. Eight of 11 cases yielded successful cultures and the presence of a clonal karyotype using standard cytogenetic methodology. In addition, a single case of orbital MALT lymphoma with a clonal karyotype has been obtained through our consultative practice from University of Nebraska Medical Center. These nine cases of MALT lymphoma with a clonal karyotype are the subject of this report.
In this study we report nine cytogenetically studied MALT lymphomas, three of which feature a novel t(11;18)(q21;q21) translocation which has also been observed in five other MALT cases described in the literature. This recurrent translocation is the most common translocation associated with MALT lymphomas being present in 33% (three of nine) of our cases and 18% (five of 28) of the previously published cases. The results suggest that a potentially important gene located at one of these breakpoints may be involved in the pathogenesis of MALT lymphomas.
Annals of Oncology 11/1997; 8(10):979-85. · 6.43 Impact Factor
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P Mathew, W G Sanger,
D D Weisenburger,
M Valentine,
V Valentine,
D Pickering,
C Higgins,
M Hess,
X Cui,
D K Srivastava,
S W Morris
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ABSTRACT: The non-Hodgkin's lymphoma (NHL) subset commonly referred to as large cell lymphoma (LCL) has historically been characterized by it's marked cytological, immunological, and clinical heterogeneity. One potential defining feature of these lymphomas, the t(2;5)(p23;q35), occurs in 25% to 30% of anaplastic LCLs and is also found in cases with diffuse large cell or immunoblastic morphology. We recently identified nucleophosmin (NPM) and anaplastic lymphoma kinase (ALK) as the genes on chromosomes 5 and 2, respectively, that are juxtaposed by this translocation. To provide a complementary approach to the use of classical cytogenetics or polymerase chain reaction-based methods for the detection of this abnormality, we have developed a two-color fluorescent in situ hybridization (FISH) assay for the t(2;5) that may be used for the analysis of both interphase nuclei and metaphase chromosomes. Three overlapping chromosome 5 cosmid clones located immediately centromeric to the NPM gene locus and an ALK P1 clone located telomeric to the chromosome 2 breakpoint were labeled with digoxigenin or biotin, respectively, and used to visualize the derivative chromosome 5 produced by the t(2;5), evident as juxtaposed or overlapping red and green fluorescent signals. This NPM-ALK FISH assay was initially validated by analysis of a series of cytogenetically characterized cell lines, with the presence of the der(5) chromosome showed specifically only in those lines known to contain the t(2;5). The assay was then applied in a blinded fashion to a series of eight cytogenetically t(2;5)-positive clinical specimens and seven known t(2;5)-negative cases, including three NHL and four Hodgkin's disease biopsy samples. Whereas the t(2;5)-negative cases were negative by FISH, all eight t(2;5)-positive cases were positive. One additional case, initially thought to be positive for the translocation by cytogenetics, was proven to not be a classic t(2;5) by interphase and metaphase FISH. These data indicate that the FISH assay described is a highly specific and rapid test that should prove to be a useful adjunct to the currently available methods for detection of the t(2;5).
Blood 04/1997; 89(5):1678-85. · 9.90 Impact Factor
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ABSTRACT: We describe five patients with tetrasomy 12p (one previously reported). These patients exhibit a very wide range of phenotypic features from that of classic Pallister-Killian syndrome to only mild learning disabilities with pigmentary skin changes. As such, these cases highlight the fact that tetrasomy 12p [i(12p)] and Pallister-Killian syndrome are not synonymous, although this combination of genotype and phenotype is often seen. This information is especially important in prenatally ascertained i(12p). The full spectrum of phenotypic possibilities associated with this chromosome aneuploidy should be discussed in prenatal counseling.
Clinical Genetics 03/1997; 51(2):102-8. · 3.13 Impact Factor
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ABSTRACT: Primary CD30(Ki-1)-positive anaplastic large-cell lymphoma (ALCL) is considered by some to be a distinct clinicopathologic entity associated with the t(2;5) (p23;q35). However, the specificity of t(2;5) for ALCL has not been carefully studied. Therefore, we performed a detailed analysis of all cases of ALCL with abnormal cytogenetics results in the Nebraska Lymphoma Study Group registry, as well as all other cases of non-Hodgkin's lymphoma with t(2;5) in the registry. We found the t(2;5) in only five of 10 cases of ALCL, four of whom were young patients. However, we also found the t(2;5) in 11 other cases of nonanaplastic lymphoma, including eight children with typical peripheral T-cell lymphomas of various types. The t(2;5) was also found in three older adults with B-cell lymphomas of various types. Thus, the t(2;5) was not specific for CD30+ ALCL. However, t(2;5) may define a clinicopathologic entity in children and young adults characterized by variable morphologies with a T-cell or indeterminate phenotype, CD30-positivity, nodal disease with frequent extranodal involvement, advanced stage, and an excellent response to therapy, including bone marrow transplantation for relapsed disease. The clinical relevance of the t(2;5) in older patients requires further study.
Blood 06/1996; 87(9):3860-8. · 9.90 Impact Factor
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ABSTRACT: We examined phosphorothioate oligodeoxyribonucleotides (ODNs) directed against bcr in exon 3 or exon 2, which are rearranged with exon 2 of abl (B3A2 and B2A2) at t(9;22) of chronic myelogenous leukemia (CML). Since these ODNs are designed to be CML cell specific, we studied their effects on the human CML cell line K562, which is known to have B3A2 rearrangement, and leukemic cells from patients, as well as normal hematopoietic stem cells in vitro. In vitro experiments were performed to determine a potential role of these two ODNs as ex vivo purging agents. Incubation of B3A2 antisense at 40, 80, and 120 micrograms/ml with K562 CML cells for 72 hours at 37 degrees C resulted in 44%, 56%, and 63% reduction of CFU-L as compared to controls. In contrast, B3A2 sense and B2A2 antisense had no significant growth inhibitory effect on K562 cells. Incubation of B3A2 and B2A2 antisense ODNs at concentration of 80 micrograms/ml at 37 degrees C for 36 hours with normal peripheral blood stem/progenitor cells (PBSC) resulted in 124% and 98% CFU-GM formation as compared to untreated controls, respectively. However, incubation of PBSC with B3A2 and B2A2 sense-ODNs resulted in a 22% and 44% reduction in CFU-GM, respectively. In order to determine the ex vivo purging effects of bcr-abl ODNs, the K562 cells were mixed with PBSC from normal donors at a ratio of 1:20 (CML:PBSC). The mixture of cells was then incubated with B3A2 antisense at 80 micrograms/ml for 36 hrs at 37 degrees C. After incubation, no CML cells were detected by fluorescence in situ hybridization (FISH) as compared to untreated controls. These results were confirmed by RT-PCR using bcr-abl primers and mRNA isolated from the mixture of cells. Further, these results support the hypothesis that bcr-abl antisense ODNs are potentially effective agents for ex vivo purging of autologous stem cells before transplantation to eliminate/reduce the burden of leukemic cells. No significant toxicity to normal hematopoietic stem/progenitor cell population by the bcr-abl antisense ODNs was observed. Although unanticipated reductions in normal hematopoietic progenitor cells (CFU-GM) were observed with sense ODNs, no reduction in CFU-GM was observed with unrelated phosphorothioate ODN controls.
Leukemia and Lymphoma 01/1996; 20(1-2):67-76. · 2.58 Impact Factor
