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.

Chronic myelogenous leukemia: laboratory diagnosis and monitoring.

Rapid developments have occurred both in laboratory medicine and in therapeutic interventions for the management of patients with chronic myelogenous leukemia (CML). With a wide array of laboratory tests available, selecting the appropriate test for a specific diagnostic or therapeutic setting has become increasingly difficult. In this review, we first discuss, from the point of view of laboratory medicine, the advantages and disadvantages of several commonly used laboratory assays, including cytogenetics, fluorescence in situ hybridization (FISH), and qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We then discuss, from the point of view of clinical care, the test(s) of choice for the most common clinical scenarios, including diagnosis and monitoring of the therapeutic response and minimal residual disease in patients treated with different therapies. The purpose of this review is to help clinicians and laboratory physicians select appropriate tests for the diagnosis and monitoring of CML, with the ultimate goal of improving the cost-effective usage of clinical laboratories and improving patient care.

Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation.

We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11) in the leukemia of a patient previously administered etoposide and dactinomycin by molecular and biochemical approaches to gain insights about the translocation mechanism and the relevant drug exposure. The genomic breakpoint junctions were amplified by PCR. Cleavage of DNA substrates containing the normal homologues of the MLL and AF-4 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha and etoposide, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF-4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoints in MLL and AF-4 were DNA topoisomerase II cleavage sites. Etoposide and its metabolites, but not dactinomycin, enhanced cleavage at these sites. Assuming that DNA topoisomerase II was the mediator of the breakage, processing of the staggered nicks induced by DNA topoisomerase II, including exonucleolytic deletion and template-directed polymerization, would have been required before ligation of the ends to generate the observed genomic breakpoint junctions. These data are inconsistent with a translocation mechanism involving interchromosomal recombination by simple exchange of DNA topoisomerase II subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase II cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposide and/or its metabolites, but not dactinomycin, likely were the relevant exposures in this patient.

t(3;11) translocation in treatment-related acute myeloid leukemia fuses MLL with the GMPS (GUANOSINE 5' MONOPHOSPHATE SYNTHETASE) gene.

The partner gene of MLL was identified in a patient with treatment-related acute myeloid leukemia in which the karyotype suggested t(3;11)(q25;q23). Prior therapy included the DNA topoisomerase II inhibitors, teniposide and doxorubicin. Southern blot analysis indicated that the MLL gene was involved in the translocation. cDNA panhandle polymerase chain reaction (PCR) was used, which does not require partner gene-specific primers, to identify the chimeric transcript. Reverse-transcription of first-strand cDNAs with oligonucleotides containing known MLL sequence at the 5' ends and random hexamers at the 3' ends generated templates with an intra-strand loop for PCR. In-frame fusions of either MLL exon 7 or exon 8 with the GMPS (GUANOSINE 5'-MONOPHOSPHATE SYNTHETASE) gene from chromosome band 3q24 were detected. The fusion transcript was alternatively spliced. Guanosine monophosphate synthetase is essential for de novo purine synthesis. GMPS is the first partner gene of MLL on chromosome 3q and the first gene of this type in leukemia-associated translocations. (Blood. 2000;96:4360-4362)

Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Sarcoidal tissue reaction in Sézary syndrome.

Sézary syndrome (SS) is an erythrodermic and leukemic variant of cutaneous T-cell lymphoma (CTCL). Occasionally, the histology of CTCL exhibits evidence of a granulomatous infiltrate in the skin. A case of SS that showed epithelioid granulomas resembling sarcoidosis in the skin and lymph nodes is presented. The clinical course of this patient has been relatively indolent.

Hepatosplenic gamma-delta T-cell lymphoma as a late-onset posttransplant lymphoproliferative disorder in renal transplant recipients.

We report 2 cases of renal transplant recipients in whom hepatosplenic gamma-delta T-cell lymphoma (gamma-delta HSTCL) developed 5 and 10 years after transplantation. Both patients had marked hepatosplenomegaly, B symptoms (weight loss, fever, and night sweats), and abnormal peripheral blood findings, including anemia in both, thrombocytopenia and leukoerythroblastic changes in 1, and leukocytosis in the other. Markedly atypical lymphoid infiltrate of intermediate to large cells was observed in the spleen, liver, and bone marrow. The malignant cells showed typical immunophenotype of gamma-delta T cells (CD2+, CD3+, CD4-, CD8-, CD7+, gamma-delta T-cell receptor-positive, and alpha-beta T-cell receptor-negative) with clonal T-cell receptor gene rearrangement and were of the V-delta-1 subset. In addition, the cells contained a cytolytic granule-associated protein, TIA-1, and Fas ligand, indicating cytotoxic T-cell differentiation. The malignant T cells in both cases were of host tissue origin. Both cases were negative for Epstein-Barr virus genome using Southern blot analysis. The patients did not respond to reduction of immunosuppression. Despite initial response to chemotherapy, both patients died within 6 months of diagnosis. Our findings indicate that gamma-delta HSTCL can occur as a late complication in transplant recipients.

The immunosuppressive macrolide RAD inhibits growth of human Epstein-Barr virus-transformed B lymphocytes in vitro and in vivo: A potential approach to prevention and treatment of posttransplant lymphoproliferative disorders.

Whereas the standard immunosuppressive agents foster development of posttransplant lymphoproliferative disorders (PTLDs), the impact of RAD, a macrolide with potent immunosuppressive properties, and other immunosuppressive macrolides on these disorders remains undetermined. We found that RAD had a profound inhibitory effect on in vitro growth of six different PTLD-like Epstein-Barr virus+ lymphoblastoid B cell lines. Similar to normal T cells, RAD blocked cell-cycle progression in PTLD-like B cells in the early (G(0)/G(1)) phase. Furthermore, RAD increased the apoptotic rate in such cells. The drug also had a profound inhibitory effect on the growth of PTLD-like Epstein-Barr virus+ B cells xenotransplanted s.c. into SCID mice. The degree of the RAD effect varied among the three B cell lines tested and was proportional to its effects on the cell lines in vitro. In this in vivo xenotransplant model, RAD markedly delayed growth or induced regression of the established tumors. In one line, it was able to eradicate the tumor in four of eight mice. When RAD treatment was initiated before tumor cell injection, a marked inhibition of tumor growth was seen in all three lines. In two of them, the drug prevented tumor establishment in approximately 50% of mice (5/11 and 5/8). In summary, RAD is a potent inhibitor of PTLD-like cells in vitro and in vivo. These findings indicate that, in contrast to the standard immunosuppressive agents, macrolides such as RAD may be effective in prevention and treatment of PTLDs.

Detection of leukemia-associated MLL-GAS7 translocation early during chemotherapy with DNA topoisomerase II inhibitors.

Leukemias with MLL gene translocations are a complication of primary cancer treatment with DNA topoisomerase II inhibitors. How early translocations appear during primary cancer treatment has not been investigated. We tracked the leukemic clone with an MLL gene translocation during neuroblastoma therapy in a child who developed acute myeloid leukemia. The karyotype of the leukemic clone showed del(11)(q23). We used panhandle PCR-based methods to isolate the breakpoint junction involving MLL and an unknown partner gene. Marrow DNA from neuroblastoma diagnosis and DNA and RNA from serial preleukemic marrows were examined for the translocation. The karyotypic del(11)(q23) was a cryptic t(11;17). GAS7, a growth arrest-specific gene at chromosome band 17p13, was the partner gene of MLL. Two different MLL-GAS7 fusion transcripts were expressed. The translocation was already detectable by 1.5 months after the start of neuroblastoma treatment. The translocation was not detectable in the marrow at neuroblastoma diagnosis or in peripheral blood lymphocyte DNAs of six normal subjects. GAS7 is a new partner gene of MLL in treatment-related acute myeloid leukemia. MLL gene translocations can be present early during anticancer treatment at low cumulative doses of DNA topoisomerase II inhibitors. Although MLL has many partner genes and most have not been characterized, panhandle PCR strategies afford new means for detecting MLL gene translocations early during therapy when the partner gene is unknown.

Hepatosplenic and subcutaneous panniculitis-like gamma/delta T cell lymphomas are derived from different Vdelta subsets of gamma/delta T lymphocytes.

Gamma/delta T cell lymphomas (gamma/delta TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most gamma/delta TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor delta (TCRS) gene repertoire of hepatosplenic gamma/delta TCL (gamma/delta HSTCL) and subcutaneous panniculitis-like gamma/delta TCL (gamma/delta SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 gamma/delta HSTCL and 4 gamma/delta SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vdelta subtypes (Vdelta1-6). It is noteworthy that 10 of 11 gamma/delta HSTCL expressed the Vdelta1 gene. The remaining case also expressed T cell receptor delta (TCRS) as determined by flow cytometry and TCRdelta rearrangement in Southern blot. However, the Vdelta gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vdelta gene. In striking contrast to the gamma/delta HSTCL, all 4 gamma/delta SPTCL expressed the Vdelta2 gene. Our data demonstrate that gamma/delta HSTCL are preferentially derived from the Vdelta1 subset of gamma/delta T lymphocytes, whereas gamma/delta SPTCL are preferentially derived from the Vdelta2 subset. The pattern of Vdelta gene expression in HSTCL and SPTCL corresponds to the respective, predominant gamma/delta T cell subsets normally found in the spleen and skin. This finding suggests that gamma/delta TCL are derived from normal gamma/delta T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vdelta gene segment usage may provide a molecular tool to distinguish better among various types of gamma/delta TCL lymphoma particularly in the clinically advanced, widely disseminated cases.

Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) represents a subtype of acute myeloid leukemia with characteristic morphologic, molecular, and immunophenotypic features. Previous immunophenotypic analyses have shown that leukemic cells in APL typically express the myeloid markers CD33 and CD13 but lack expression of the early hematopoietic progenitor cell antigens CD34 and HLA-DR. We analyzed selected immunophenotypic features of APL by flow cytometry and showed that 7 (41%) of 17 cases contained significant subsets of CD34+ leukemic cells: CD34+ myeloid cells predominated in 2 APL cases. By using a fluorescence-activated cell sorter-fluorescence in situ hybridization approach, we confirmed that the CD34+ cells harbored the t(15;17) translocation characteristic of APL. By using the same experimental approach, CD34+ populations were stratified into primitive CD34+ CD38- and committed CD34+ CD38+ progenitor cell subpopulations; cells in both subsets contained the t(15;17) translocation. The knowledge that APL may be partly or largely CD34+ is important for proper diagnosis. Furthermore, identification of the t(15;17) translocation in CD34+ CD38- blasts indicates that, in at least some cases, the leukemogenic mutation in APL occurs within primitive hematopoietic progenitor cells.

Calcium signaling induces acquisition of dendritic cell characteristics in chronic myelogenous leukemia myeloid progenitor cells.

Effective host T lymphocyte sensitization to malignant cells depends on successful antigen presentation. In this study, we examined the capacity of malignant myeloid progenitor cells of patients in the chronic phase of chronic myelogenous leukemia (CML) to acquire characteristics of activated dendritic cells (DCs) after intracellular calcium mobilization, thereby bypassing a need for third-party antigen-presenting cells. Treatment of purified CD33(+) CML cells from 15 patients with calcium ionophore (CI) consistently resulted in de novo expression of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2), CD40 and the DC-specific activation marker CD83, as well as marked up-regulation of MHC class I and II molecules and the adhesion molecule CD54. Most of these changes occurred within 24 hr of treatment. Morphologically, CI-treated CML cells developed long dendritic projections similar to those seen in mature DCs. Functionally, CI-treated CML cells provided stimulation of allogeneic T lymphocytes 10- to 20-fold that of untreated CML cells or untreated monocytes. Fluorescent in situ hybridization of CI-activated CML cells confirmed their leukemic origin by displaying the typical bcr/abl fusion signal. No difference in bcr/abl translocation percentages between untreated and CI-treated CML nuclei was observed. These observations indicate that calcium mobilization may constitute a valuable approach for rapidly and reliably generating CML-derived DCs for immunotherapy of CML.

Duplicated regions of AF-4 intron 4 at t(4;11) translocation breakpoints.

BACKGROUND: AF-4 is a common partner gene of MLL. AF-4 breakpoints occur in introns, but most AF-4 introns are uncharacterized. METHODS AND RESULTS: We cloned AF-4 intron 4 and examined the frequency of breakpoints in this intron. The 5.8-kb intron is rich in repeat sequences and was the site of translocation in 3 of 17 leukemias with t(4;11). We cloned the der (11) and der (4) breakpoints and isolated the fusion transcripts in the cell line MV4-11 and in a de novo acute lymphoblastic leukemia (ALL). Both translocations joined MLL intron 6 and AF-4 intron 4. In MV4-11, 249 bases from AF-4 were present in both derivative chromosomes, indicating duplication. In the de novo ALL, duplication of 446 bases from MLL and AF-4 occurred. Reciprocal fusion transcripts were expressed. CONCLUSIONS: Intronic sequence of AF-4 is useful for molecular diagnosis of t(4;11). Duplicated intronic regions suggest staggered chromosomal breakage.

Association of CYP3A4 genotype with treatment-related leukemia.

Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5' promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher's Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0. 016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone.

MLL genomic breakpoint distribution within the breakpoint cluster region in de novo leukemia in children.

PURPOSE: To assess translocation breakpoint distribution within the MLL genomic breakpoint cluster region (bcr), 40 cases of de novo leukemia in children were examined by karyotype and Southern blot analysis. PATIENTS AND METHODS: Criteria for inclusion were karyotypic or molecular rearrangement of chromosome band 11q23. Of the 40 cases, 31 occurred in infants. Twenty cases were acute lymphoblastic leukemia (ALL), 17 were acute myeloid leukemia (AML), and 3 were biphenotypic. RESULTS: Karyotype identified 27 cases with translocation of chromosome band 11q23 and 2 with abnormalities of band 11q13 but not 11q23. Southern blot analysis showed rearrangement within the MLL genomic bcr in 38 of the 40 cases. In these 38, additional probe-restriction digest combinations localized MLL genomic breakpoints to the 5' portion of the bcr in 14 cases and to the 3' portion in 18; material was insufficient for further localization to 5' or 3' within the bcr in 6 cases. In the two remaining cases, both with t(4;11)(q21;q23), one breakpoint mapped 5' of the bcr between intron 3 and exon 5, whereas the other breakpoint was neither within nor 5' of the MLL genomic bcr. CONCLUSIONS: Suggested trends warranting investigation in more patients were breakpoint sites in the 3' bcr in AML and in patients older than 12 months. The distribution of MLL genomic breakpoints within the bcr in de novo leukemia in children is distinct from that in adults, where the breakpoints cluster in the 5' portion of the bcr.

A pilot trial of 13-cis-retinoic acid and alpha-tocopherol with recombinant human erythropoietin in myelodysplastic syndrome patients with progressive or transfusion-dependent anemias. The Central Pennsylvania Oncology Group.

A pilot study was performed to determine the efficacy of 13-cis-retinoic acid (CRA) and alpha-tocopherol (AT) with increasing doses of recombinant human erythropoietin (rHuEPO) in anemic patients with primary myelodysplastic syndrome (MDS), to determine response rate and to determine the dose requirement and long-term effects of rHuEPO therapy on the transition to acute non-lymphocytic anemia and survival of these patients. Twenty-four consecutive MDS patients were entered into the study. Patients were stratified according to their FAB classification at study entry. Therapy consisted of a 6 month trial of CRA (100 mg m(-2) day(-1) and AT (800 mg day(-1)) with rHuEPO (150 units kg(-1) body weight subcutaneously three times a week). The rHuEPO dose was escalated to daily doses at 2 months, and 300 U kg(-1) body weight given three times a week for another 2 months and continuing therapy after 6 months in responsive patients. Response was measured by elimination of transfusions requirement (partial response, PR) and normal hemoglobin level and complete blood counts (complete response, CR). Observed responses for the 23 evaluable patients were 2 CR and 6 PR (34.8%). Odds ratio analysis showed that patients with anemia alone were 14 times more likely to respond than patients with pancytopenia (p = 0.06). In our study, two patients (8%) transformed to acute leukemia in CRA + AT + rHuEPO-treated patients. Median survival of 34 months with a median follow-up of 17 months (range 3-70 months) was observed. The response rates with the addition of rHuEPO to CRA and AT was similar but occurs earlier at 2 months compared to 6-10 months with CRA and AT alone and did not alter survival. There was no increase in the risk for leukemia in the group treated with rHuEPO. Response to either therapy appeared to be limited to patients in the early stages of MDS.

Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: evidence for multiple critical loci.

Complete or partial deletions of the long arm of chromosome 7 (7q- and -7) are nonrandom abnormalities seen in primary and therapy-induced myelodysplasia (MDS) and acute myelogenous leukemia (AML). Monosomy 7, occurring as the sole cytogenetic anomaly in a small but significant number of cases, may denote a dominant mechanism involving critical tumor suppressor gene(s). We have determined the extent of allele loss in cytogenetically prescreened MDS and AML patients for microsatellite markers from chromosome 7q22 and 7q31. Whereas >80% of these cases revealed allele loss for the entire region, a rare case of the 7q- chromosome showed allele loss for only the proximal 7q31.1 loci flanked by the markers D7S486 and D7S2456, and a case of monosomy 7 revealed allele loss for loci at both 7q31 and 7q22 with retention of sequences between these sets of loci. Furthermore, a case of AML with no cytogenetic anomaly of chromosome 7 revealed a submicroscopic allelic imbalance for a third distal locus, D7S677. These findings suggest the presence of three distinct critical loci that may contribute alone or in combination to the evolution of MDS and AML. The data also provide molecular evidence for unbalanced translocation with noncontiguous deletions, as an alternate mechanism underlying monosomy 7.

Novel approach for simultaneous evaluation of cell phenotype, apoptosis, and cell cycle using multiparameter flow cytometry.

Apoptosis is a vital process for organism development and, when disrupted, can lead to abnormalities including cancer and autoimmune diseases. We demonstrate a novel multicolor flow cytometry approach for quantifying apoptosis and cell cycle information of phenotypically distinct populations, using less than 2 x 10(5) cells per sample. We used incorporation of Cy5-dUTP into DNA strand breaks by the terminal dUTP nucleotide end labeling (TUNEL) method to determine apoptosis, while cell cycle information was assessed with an ultraviolet DNA binding dye, DAPI. To simultaneously determine surface phenotype, we used paraformaldehyde fixation and a gentle permeabilization protocol combined with FITC- and PE-labeled surface antibodies. Using these fluorochromes, and three-laser instrumentation, we quantified apoptosis and cell cycle phase in lymphocyte subpopulations from heterogeneous human and murine cell sources, subjected to various culture conditions. Further, we used this method to detect divergent rates of apoptosis in a human, heterogeneous lymphocyte tumor population, demonstrating a potential application for clinical and/or research settings. Thus, we describe a six-parameter, four-color flow cytometry approach for evaluating apoptosis and cell cycle with dual surface labels. This method may also be useful as a generalized scheme to assess simultaneously two intracellular targets in a mixed cell population.

Potential role for wild-type p53 in leukemias with MLL gene translocations.

We used single-strand conformation polymorphism (SSCP) analysis of p53 exons 4-8 to screen for possible mutations in 25 pediatric de novo leukemias with translocations of the MLL gene at chromosome band 11q23. Of the 25 patients, 21 were infants. Fifteen cases were acute myeloid leukemia (AML), eight were acute lymphoblastic leukemia (ALL), and two cases were biphenotypic. Nineteen cases were studied at diagnosis and six at time of relapse. p53 mutations were absent in all 19 cases studied at the time of diagnosis. The only mutation was a TGC-->TTC transversion (cys-->phe) at codon 141 in exon 5 in a case of infant ALL at relapse that occurred by subclone evolution after MLL gene translocation. We previously showed that p53 mutations are also absent in pediatric treatment-related leukemias with MLL gene translocations. The absence of p53 mutations at initial transformation may suggest that the anti-apoptotic effect of mutant p53 is not important in leukemias with MLL gene translocations. Alternatively, exogenous DNA damage may be the common feature in treatment-related and de novo cases. Since MLL gene translocations may occur through DNA repair and wild-type p53 is central to DNA repair, the absence of p53 mutations raises the possibility that wild-type p53, not mutant p53, may be important in the genesis of leukemias with these translocations.