BCL2 and JUNB abnormalities in primary cutaneous lymphomas.

BACKGROUND: BCL2 is upregulated in nodal and extranodal B-cell non-Hodgkin's lymphomas, with a consequent antiapoptotic effect. However, loss of BCL2 has also been noted in some malignancies, suggesting a different molecular pathogenesis. OBJECTIVES: To investigate genomic and protein expression status of BCL2 and to compare the results with that of JUNB in primary cutaneous lymphomas (PCLs). METHODS: We analysed gene copy number of BCL2 and JUNB in 88 DNA samples from 80 patients with PCL consisting of Sézary syndrome/mycosis fungoides (SS/MF), primary cutaneous B-cell lymphoma (PCBCL) and primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) by the use of real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). Real-time PCR and IHC findings were subsequently compared with the results of additional fluorescent in situ hybridization (FISH) analysis of 23 cases of SS and Affymetrix cDNA expression microarray study of two primary cutaneous T-cell lymphoma (CTCL) cell lines. RESULTS: Real-time PCR analysis showed loss of BCL2 gene copy number in 22 of 80 PCL cases (28%), including 17 of 42 SS/MF, three of 13 C-ALCL and two of 33 PCBCL samples, and gain of BCL2 in four PCBCL samples. Gain of JUNB was identified in 18 of 71 PCL cases (25%), including nine of 35 SS/MF, seven of 13 C-ALCL and two of 31 PCBCL samples. IHC analysis revealed absent nuclear expression of BCL2 protein in 47 of 73 PCL cases, comprising 28 of 36 SS/MF, eight of eight C-ALCL and 11 of 29 PCBCL cases. In contrast, BCL2 protein expression was detected in 26 of 73 PCL cases, consisting of 18 of 29 PCBCL and eight of 36 SS/MF cases. JUNB protein expression was present in tumour cells from 30 of 33 of SS/MF and eight of eight C-ALCL, and was absent in tumour cells from 18 of 27 PCBCL cases. A comparison between BCL2 and JUNB revealed loss of BCL2 and gain of JUNB in five of 35 SS/MF samples, and expression of JUNB protein and absent BCL2 expression in 25 SS/MF and eight of eight C-ALCL cases. In contrast, expression of BCL2 and absent JUNB expression were detected in 67% of PCBCL cases. Additional FISH analysis revealed deletion of BCL2 in 19 of 23 SS cases (83%), including eight cases with BCL2 loss shown by real-time PCR. Furthermore, Affymetrix expression microarray demonstrated decreased expression of proapoptotic and antiapoptotic genes involved in BCL2 signalling pathways such as BOK, BIM, HRK, RASA1 and STAT2 in two CTCL cell lines with BCL2 loss and absent BCL2 expression. Increased expression of JUNB was also identified in the MF cell line. CONCLUSIONS: These findings provide a comprehensive assessment of BCL2 and JUNB status in PCL, and suggest that there is a selection pressure in a subset of CTCL cases for tumour cells showing BCL2 loss and upregulation of JUNB primarily through chromosomal deletion and amplification, respectively.

High level amplification of N-MYC is not associated with adverse histology or outcome in primary retinoblastoma tumours.

Twenty-five primary retinoblastoma tumours were analysed by real-time quantitative polymerase chain reaction to determine the genomic copy number of the N-MYC gene (2p24) relative to the copy number for REL, B2M, ALB, AF10 and MLL. Twenty-one of these tumours were shown by Comparative Genomic Hybridization to contain variable copy number increases of chromosomal material mapping to 2p. High level amplification (>30-fold) of N-MYC was found in three tumours, none of which showed adverse histological features and all patients are surviving at between 54 and 108 months post enucleation. Furthermore, the three tumours associated with metastasis and adverse patient outcome showed normal N-MYC copy number. Although high level amplification of N-MYC is an unfavourable prognostic indicator in neuroblastoma, these data show no evidence of a correlation between amplification of N-MYC and adverse outcome in retinoblastoma.

Genetic susceptibility to Hodgkin's disease and secondary neoplasias: FISH analysis reveals patients at high risk of developing secondary neoplasia.

BACKGROUND: Cytotoxic drugs administered before high-dose therapy (HDT) represent a significant factor in the development of leukemic complications in patients with lymphoid malignancies. This retrospective study was used to detect evidence of abnormal therapy-related myelodysplasia/secondary acute myeloid leukaemia (tMDS/sAML) clones before HDT in a subset of patients who subsequently developed secondary neoplasia. PATIENTS AND METHODS: 230 patients with non-Hodgkin's lymphoma (NHL) underwent HDT comprising cyclophosphamide and total body irradiation (TBI) with autologous hematopoietic progenitor-cell support. Thirty-three patients have developed tMDS/sAML and 20 of these were screened for the presence of emerging therapy-related abnormalities before HDT. A further 24 patients without evidence of secondary neoplasia were screened using fluorescence in situ hybridisation (FISH). RESULTS: Significant levels of abnormal cells were identified in 20/20 patients screened who have developed secondary neoplasia compared with only three of 24 patients in the HDT control group who have not. The latter three patients have since died. CONCLUSIONS: The triple FISH assay was developed to detect loss of chromosomal material from 5q31, 7q22 and 13q14. It can potentially identify those patients at risk of alkylating agent-induced leukaemia before they proceed to HDT. Used in a prospective manner, the triple FISH assay could permit more informed clinical management.

A case of adult T-cell leukaemia/lymphoma characterized by multiplex-fluorescence in situ hybridization, comparative genomic hybridization, fluorescence in situ hybridization and cytogenetics.

Adult T-cell leukaemia/lymphoma (ATLL) is a neoplasm of mature helper (CD4) T lymphocytes. Little is known, however, about the chromosome aberrations associated with the pathogenesis of this malignancy. Using molecular cytogenetic techniques we, therefore, investigated a 44-year-old man who had a 7-year history of ATLL with cutaneous involvement mimicking primary cutaneous T-cell lymphoma. Conventional cytogenetics revealed gross chromosomal changes with chromosome numbers ranging from 71 to 82. There were structural abnormalities of chromosomes 7 and 9, partial deletions of chromosomes 1, 3, 5 and 6, and loss of chromosomes 2, 4, 9, 11--14, 21 and 22. Multiplex-fluorescence in situ hybridization (M-FISH) identified two derivative chromosomes, der(6)t(6;7)(q16;q21) and der(7)t(6;7)(q16;q21)ins(6;12)(q2?;?), and a deletion of chromosome 1p. Conventional FISH confirmed the M-FISH findings. Comparative genomic hybridization of the blood revealed gains of DNA copy number at 1q12--25, 6p24--25, 9p23, 16p13--q13, 17q11--21, 19p13 and 20q13 and loss at 11p15 while lymph nodes showed gains at 3p22--24, 3q27--29, 7q36 and 15q26 and losses at 2p24--25, 2q37, 10p14--15, 11p15, 13q33--34 and 16p13.3. No DNA copy number changes were seen in a skin lesion. These results show the extent of genetic abnormalities within this malignancy.

Detection of chromosome abnormalities pre-high-dose treatment in patients developing therapy-related myelodysplasia and secondary acute myelogenous leukemia after treatment for non-Hodgkin's lymphoma.

PURPOSE: To assess whether pre-high-dose therapy (HDT)-related factors play a critical role in the development of therapy-related myelodysplasia (tMDS) or secondary acute myelogenous leukemia (sAML). PATIENTS AND METHODS: Twenty-nine of 230 patients with a primary diagnosis of non-Hodgkin's lymphoma (NHL) developed tMDS/sAML after HDT comprising cyclophosphamide and total-body irradiation (TBI) supported by autologous hematopoietic progenitor cells. G-banding and fluorescence in-situ hybridization (FISH) were used to detect clonal cytogenetic abnormalities. RESULTS: The majority of patients showed complex karyotypes at diagnosis of tMDS/sAML containing, in particular, complete or partial loss of chromosomes 5 and/or 7. Using single locus-specific FISH probes, significant levels of clonally abnormal cells were found before HDT in 20 of 20 tMDS/sAML patients screened, compared with three of 24 patients screened who currently have not developed tMDS/sAML, at a median follow-up of 5.9 years after HDT. CONCLUSION: Prior cytotoxic therapy may play an important etiologic role and may predispose to the development of tMDS/sAML. Using a triple FISH assay designed to detect loss of chromosomal material from 5q31, 7q22, or 13q14, significant levels of abnormal cells can be detected before HDT and may predict which patients are at increased risk of developing secondary disease. Further prospective evaluation of this FISH assay is warranted to determine its predictive power in this setting.

The use of multicolor fluorescence technologies in the characterization of prostate carcinoma cell lines: a comparison of multiplex fluorescence in situ hybridization and spectral karyotyping data.

Recent studies have identified several chromosome regions that are altered in primary prostate cancer and prostatic carcinoma cell lines. These targeted regions may harbor genes involved in tumor suppression. We used multiplex fluorescence in situ hybridization (M-FISH) to screen for genetic rearrangements in four prostate cancer cell lines, LNCaP, LNCaP.FCG, DU145, and PC3, and compared our results with those recently obtained using spectral karyotyping (SKY). A number of differences was noted between abnormalities characterized by SKY and M-FISH, suggesting variation in karyotype evolution and characterization by these two methodologies. M-FISH analysis showed that hormone-resistant cell lines (DU145 and PC3) contained many genetic alterations (> or =15 per cell), suggesting high levels of genetic instability in hormone-refractory prostate cancer. Most chromosome regions previously implicated in prostate cancer were altered in one or more of these cell lines. Several specific chromosome aberrations were also detected, including a del(4)(p14) and a del(6)(q21) in the hormone-insensitive cell lines, a t(1;15)(p?;q?) in LNCaP, LNCaP, and PC3, and a i(5p) in LNCaP.FCG, DU145, and PC3. These clonal chromosome abnormalities may pinpoint gene loci associated with prostate tumourigenesis, cancer progression, and hormone sensitivity.

Molecular analysis of the genomic inversion and insertion of AF10 into MLL suggests a single-step event.

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions.

Acute Myelogenous Leukaemia in Patients 60 Years and Older: A Retrospective Analysis from St Bartholomew's Hospital 1969-1999.

Between 1969 and 1999, 420 patients (age > 60 years) with newly diagnosed AML were managed at St Bartholomew's Hospital (SBH), London, UK. Sixty-nine percent of patients received therapy with curative intent Eighty-eight patients (31%) of the latter achieved complete remission (CR), representing an overall CR rate of 21%. Treatment failure due to early death (ED) and resistant disease (RD) occurred in 50 and 19%, respectively. With median follow up of 11 years, actuarial survivals at 1,3 and 5 years were 20, 7 and 4%, respectively, the median survival of the entire cohort was 2 months. For patients who achieved CR, median survival was significantly better than that of patients in whom treatment failed (14 vs. 6 months). Over the 30 years, CR rate and the relative incidence of RD both increased from 13 to 45%, and 3 to 27%, respectively, whilst ED rate reduced from 84 to 27%. Multivariate analysis showed that treatment era, hepatosplenomegaly and increasing age predicted for reduced CR rate and OS. Although elderly patients with AML are characterised by a poor response to intensive chemotherapy, significant improvements in supportive care and the delivery of intensive treatment have led to improved CR rates and OS. New therapeutic strategies and a greater awareness of prognostic factors may further improve clinical outcome in this important group of patients.

Upregulation of lipocortin 1 inhibits tumour necrosis factor-induced apoptosis in human leukaemic cells: a possible mechanism of resistance to immune surveillance.

The signal transduction pathway through which tumour necrosis factor (TNF) induces apoptosis in leukaemic cells may involve activation of cytosolic phospholipase A(2) (cPLA(2)). The steroids dexamethasone (Dex) and 1,25(OH)(2) D(3) both render U937 leukaemic cells resistant to TNF-induced apoptosis. In this study, we found that Dex inhibited both spontaneous and TNF-induced activation of cPLA(2). Dex had no direct effect on cellular cPLA(2) levels, but facilitated cPLA(2) degradation upon subsequent stimulation of cells with TNF. In addition, Dex increased synthesis of the endogenous cPLA(2) inhibitor lipocortin 1 (LC1). An antisense oligonucleotide to LC1 could completely abrogate Dex-induced resistance to the cytotoxic action of TNF. Constitutive LC1 levels were relatively higher in myeloid leukaemic blasts showing resistance to TNF than TNF-sensitive myeloid leukaemic cell lines. Our data suggest that Dex confers the resistance of U937 cells to TNF-induced apoptosis by upregulating intracellular levels of LC1 and by facilitating a negative-feedback loop, which is activated upon stimulation with TNF. High constitutive levels of LC1 in leukaemic blasts may protect them against immune-mediated killing.

Cytogenetic and molecular evidence of marrow involvement in extramedullary acute myeloid leukaemia.

A diagnosis of granulocytic sarcoma was made in a 2-year-old child based on the detection of myelomonocytic blasts in tissue obtained from a subcutaneous nodule with no evidence of concomitant disease in the bone marrow. The child responded to systemic chemotherapy and is in remission 3 years later. An identical clone with an in frame fusion of the MLL and AF10 genes was identified from both tissue and bone marrow samples. The generation of an in frame MLL-AF10 fusion requires complex intra- and interchromosomal exchanges between chromosomes 10 and 11. In this case, an intrachromosomal rearrangement of chromosome 5 was also observed. This case illustrates the presence of systemic disease in extramedullary leukaemia, its response to systemic rather than topical therapy and suggests that the events leading to chromosomal translocations in leukaemia may be part of a generalized intracellular event.

Therapy-related myelodysplasia and secondary acute myelogenous leukemia after high-dose therapy with autologous hematopoietic progenitor-cell support for lymphoid malignancies.

PURPOSE: To evaluate the incidence of and risk factors for therapy-related myelodysplasia (tMDS) and secondary acute myelogenous leukemia (sAML), after high-dose therapy (HDT) with autologous bone marrow or peripheral-blood progenitor-cell support, in patients with non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Between January 1985 and November 1996, 230 patients underwent HDT comprising cyclophosphamide therapy and total-body irradiation, with autologous hematopoietic progenitor-cell support, as consolidation of remission. With a median follow-up of 6 years, 27 (12%) developed tMDS or sAML. RESULTS: Median time to development of tMDS or sAML was 4.4 years (range, 11 months to 8.8 years) after HDT. Karyotyping (performed in 24 cases) at diagnosis of tMDS or sAML revealed complex karyotypes in 18 patients. Seventeen patients had monosomy 5/5q-, 15 had -7/7q-, seven had -18/18q-, seven had -13/13q-, and four had -20/20q-. Twenty-one patients died from complications of tMDS or sAML or treatment for tMDS or sAML, at a median of 10 months (range, 0 to 26 months). Sixteen died without evidence of recurrent lymphoma. Six patients were alive at a median follow-up of 6 months (range, 2 to 22 months) after diagnosis of tMDS or sAML. On multivariate analysis, prior fludarabine therapy (P =.009) and older age (P =.02) were associated with the development of tMDS or sAML. Increased interval from diagnosis to HDT and bone marrow involvement at diagnosis were of borderline significance (P =.05 and.07, respectively). CONCLUSION: tMDS and sAML are serious complications of HDT for NHL and are associated with very poor prognosis. Alternative strategies for reducing their incidence and for treatment are needed.

Autologous transplantation with Philadelphia-negative progenitor cells for patients with chronic myeloid leukaemia (CML) failing to attain a cytogenetic response to alpha interferon.

Between October 1993 and March 1999, 29 patients with CML who were ineligible for allogeneic BMT underwent PBSC harvest using idarubicin, cytarabine and G-CSF. In 9/29 (31%) patients all collected stem cells were Ph-negative, and 15/29 patients' (52%) collections were substantially (>95%) Ph-negative. The proportion of patients from whom Ph-negative stem cells were obtained was similar between patients who had, or had not, received prior alphaIFN. Fifteen patients in chronic phase (median age 45) proceeded to PBSCT following busulphan 16 mg/m2 and cyclophosphamide 120 mg/m2. Nine of the 13 patients who had failed to respond to prior alphaIFN proceeded to stem cell transplantation as soon as was feasible and six of the newly diagnosed patients were transplanted after failing to achieve a cytogenetic response after a minimum of 12 months on alphaIFN following progenitor cell harvest. The median number of days to neutrophils >0.5 and platelet >50 was 18 (range 13-69) and 28 (range 13-234), respectively. There was no procedure-related mortality. At median follow-up of 2.3 years post autograft 10 of 15 patients remain alive and in chronic phase. Overall survival for all 27 patients at 5 years after initial diagnosis is 70% and median survival from diagnosis 7.3 years. Survival for alphaIFN non-responders who were transplanted is 74% at 5 years from diagnosis and 75% at 3 years from transplant. Cytogenetic analysis performed 3 months post transplant demonstrated one patient with a complete cytogenetic response, seven with a partial response and three with no response. Six patients remain partially Ph-negative, with one major CR. Survival for all patients in the protocol is favourable compared with conventional therapy and is particularly encouraging following PBSCT for alphaIFN non-responsive patients. Patients not responding to alphaIFN can be induced into Ph-negativity with PBSCT but this may not always be sustainable. There seems to be no obvious disadvantage in harvesting stem cells after prior exposure to alphaIFN, providing an adequate alphaIFN-free rest period is used.

The t(6;11)(q27;q23) translocation in acute leukemia: a laboratory and clinical study of 30 cases. EU Concerted Action 11q23 Workshop participants.

Thirty patients representing 5.5% of those collected by the 11q23 workshop had a t(6;11)(q27;q23). They included 27cases of acute myeloid leukemia (AML) (M1, three cases; M2, two cases; M4, nine cases; M4/M5, one case; M5, 12 cases) of age range 3-72 years and three cases of acute lymphoblastic leukemia (ALL) (B-lineage ALL, two cases; T-ALL, one case) of age range 0.5-13 years. In 20 cases the t(6;11) was the sole abnormality. In 10 cases the recurrent additional abnormalities were extra copies of chromosomes 8, 19, 21, or the der(6). Translocation t(6;11) was identified by cytogenetics alone in 13 cases. In three cases it was confirmed by fluorescence in situ hybridization (FISH) using whole chromosome paints (wcps) 6 and 11. In a further 14 cases involvement of MLL was demonstrated by FISH, by reverse transcriptase polymerase chain reaction (RT-PCR), by Southern blotting (SB) or by a combination of these methods. One case had a direct insertion of 11 into 6-dir ins(6;11)(q27;q13q23). Molecular investigations showed that one case had a 3' deletion of MLL. The median overall survival for the patients was 12 months, indicating a poor prognosis for patients with a t(6;11) translocation.

The t(10;11)(p12;q23) translocation in acute leukaemia: a cytogenetic and clinical study of 20 patients. European 11q23 Workshop participants.

The clinical, haematological and cytogenetic data for 20 patients with an acquired abnormality of 11q23 and 10p have been reviewed at this workshop. Patients predominantly presented with de novo AML M5a and the most common cytogenetic finding was an inversion of part of the long arm of chromosome 11 followed by a translocation between 11q and 10p. Band p12 represented the most common breakpoint on chromosome 10. The t(10;11) subgroup defined a subset of younger 11q23 patients, the majority of whom achieve a first complete remission despite the differing treatment regimens.

1,25-Dihydroxyvitamin D3 protects human leukemic cells from tumor necrosis factor-induced apoptosis via inactivation of cytosolic phospholipase A2.

The mechanism by which tumor necrosis factor (TNF) induces death of cancer cells appears to involve the activation of cytosolic phospholipase A2 (cPLA2). U937 human leukemic cells treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; 10(-8) M] become resistant to TNF, an effect that is independent of cell cycle status and expression of TNF receptors or BCL-2. In this study, TNF produced a dose- and time-dependent enhancement of [3H]arachidonic acid release in U937 cells. The amount of [3H]arachidonic acid release was positively associated with TNF-induced apoptosis. Both immunofluorescence microscopy and Western blotting of cell subcompartments demonstrated translocation of cPLA2 from the cytosol to the cell membrane in response to TNF. In addition, TNF up-regulated expression of cPLA2 mRNA. An antisense oligonucleotide to cPLA2 and the cPLA2 inhibitor 4-bromophenacyl bromide significantly inhibited TNF-induced cytotoxicity. Prior incubation of cells with 1,25(OH)2D3 significantly inhibited (a) TNF-induced [3H]arachidonic acid release and apoptosis, (b) TNF-induced translocation of cPLA2 to the membrane, and (c) the up-regulation of cPLA2 mRNA with TNF. Furthermore, the inhibitory effect of 1,25(OH)2D3 was not reversed by inhibitors of transcription or translation. The data suggest that activation of cPLA2 is involved in TNF-induced apoptosis of leukemic cells. 1,25(OH)2D3 directly inhibits cPLA2 translocation and mRNA up-regulation induced by TNF. Disruption of cPLA2 activation may represent a possible mechanism whereby leukemic cells can become resistant to TNF-mediated killing.

The onset of CML in blastic crisis: molecular features.

The lack of distinguishing characteristics between lymphoid blastic crisis (BC) of Philadelphia (Ph)+ chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) remains an exciting dilemma. Indeed, the genetic defect of approximately half of Ph+ ALL patients is identical to that identified in CML. Here we report the case of one patient admitted with immunological and molecular patterns indicative of Ph+ ALL. The patient was brought into complete remission by chemotherapy and was transplanted with an HLA identical sibling donor, but relapsed a few months later with immunological and molecular evidence of BC of CML, displaying myeloid markers and lacking lymphoid antigens and immunoglobulin heavy chain (IgH) rearrangement. This suggest a CML case with an initial BC without a previous chronic phase or supervening on a subclinical CML.

Gene BR140, which is related to AF10 and AF17, maps to chromosome band 3p25.

The genes AF10 and AF17 have been identified as the basis of the t(10;11) and t(11;17) translocations, events that result in their fusion to the MLL/HRX gene in acute myeloid leukaemias. AF10 and AF17 bear significant homology to each other within their putative zinc finger and leucine zipper domains, although they are diverged outside these regions. The BR140 gene encodes a 140 kDa protein of unknown function that contains a putative zinc finger domain, a leucine zipper region, and, in addition, a bromo domain. The zinc finger and leucine zipper domains of BR140 have significant homology to those of AF10 and AF17, suggesting that it belongs to this newly described gene family and, therefore, could be a target for chromosome translocation. To assess the potential involvement of BR140 in chromosome translocations in leukaemia, the chromosomal location of the BR140 gene has been determined by using several independent methods. A combination of Southern analysis, polymerase chain reactions (PCR) on monochromosomal cell hybrids, and fluorescence in situ hybridisation (FISH) has been used to show that the BR140 gene maps to chromosome band 3p25.

Concurrent activation of MYC and BCL2 in B cell non-Hodgkin lymphoma cell lines by translocation of both oncogenes to the same immunoglobulin heavy chain locus.

Concurrent activation of BCL2 and MYC usually occurs in B cell non-Hodgkin lymphoma (B-NHL) by translocation of both oncogenes to both immunoglobulin heavy chain (IGH) alleles: this abrogates immunoglobulin synthesis. We have studied three B-NHL cell lines (DoHH2, VAL and ROS 50) and show that concurrent activation of BCL2 and MYC may follow translocation of both oncogenes to the same IGH allele. Conventional cytogenetics of DoHH2 suggested the presence of a t(14;18)(q32;q21) translocation. However, fluorescent in situ hybridization (FISH) studies using whole chromosome paints, alpha satellite probes and flow-sorted chromosomes as probes revealed an unexpected complexity of rearrangements involving chromosomes 8, 14 and 18, namely t(8;14;18)(q24;q32;q21). DNA blot and previous PCR analysis confirmed the juxtaposition of BCL2 major breakpoint region (mbr) with IGJH6, but also demonstrated a rearrangement within the first exon of MYC. The centromeric (5') MYC rearranged fragment comigrated with the BCL2-JH6 rearranged fragment in BamHI, EcoRI and Bg/II restriction digests. The der(8)t(8;14;18) therefore comprised 5' MYC (exon I)-Sgamma4-JH6-BCL2 mbr. Similar rearrangements were observed in both ROS 50 and VAL cell lines which contained two and three copies of the der(8)t(8;14;18) respectively. Quantitative flow cytometry for BCL2 and MYC expression showed abundant expression of both proteins in all three lines. These data indicate the der(14)t(14;18)(q32;q21) may itself be the target for any second translocation. The presence of the intact BCL2-JH fusion gene on the der(8)t(8;14;18) allowed concurrent activation of both BCL2 and MYC with no loss of immunoglobulin expression.