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.

Treatment of insomnia in patients with mood disorders.

Mood disorders and chronic insomnia share complex theoretical and clinical relationships. This article reviews the subjective symptoms and polysomnographic findings of subjects with mood and insomnia syndromes. The polysomnographic findings reviewed include macro-architectural and micro-architectural data. Various treatments of patients with insomnia and mood disorders will be presented, including both behavioral and pharmacological interventions.

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.

Protecting sleep quality in later life: a pilot study of bed restriction and sleep hygiene.

We tested two interventions for improving sleep consolidation and depth in normal elderly participants: a modification of sleep-restriction therapy and sleep-hygiene education. Twenty-one elderly participants without sleep disorders were randomized to sleep hygiene plus bed restriction (i.e., restricting time in bed by 30 minutes nightly for one year) or to sleep hygiene alone. Participants in the bed-restriction group showed a median increase in sleep efficiency of 6.1% versus 1.8% in participants receiving sleep hygiene instruction, and an increase in allnight delta EEG power. Self-reported mood on awakening in the morning showed greater improvement over the first eight weeks in the sleep-hygiene condition. The use of sleep hygiene was associated with initial improvement in daytime well-being, whereas bed restriction led to sustained improvements in sleep continuity and sleep depth.

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.

Disruption of the IFN-gamma cytokine network in chronic lymphocytic leukemia contributes to resistance of leukemic B cells to apoptosis.

Recent evidence indicates that the slowly expanding population of CD5+ B cells that characterizes chronic lymphocytic leukemia (CLL) results primarily from defects in responses to cytokines that regulate apoptosis (e.g. I1-4, TGF-beta, IFN-alpha, IFN-gamma). We have now demonstrated not only that the enhanced anti-apoptotic effect of IFN-gamma on these neoplastic B cells is apparently mediated through increased levels of IFN-gamma receptors but also that there are increased numbers of IFN-gamma-expressing CD4 and CD8 T cells in these patients. This is the strongest evidence to date that multiple alterations in the IFN-gamma cytokine network contribute to the pathogenesis of CLL.

Symptoms of stress and depression as correlates of sleep in primary insomnia.

OBJECTIVE: Previous studies have not evaluated the clinical correlates of the electroencephalographic spectral profile in patients with insomnia. In the preliminary study described here, we evaluated the extent to which symptoms of stress and depression are associated with subjective sleep complaints and quantitative measures of sleep in individuals with chronic insomnia. METHODS: Subjects were 14 healthy adults who met criteria for primary insomnia as specified in the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders. Measures of stress, depression, and subjective sleep quality were collected before subjects participated in a two-night laboratory sleep series. We hypothesized that elevated symptoms of stress and depression would be associated with subjective sleep complaints and electroencephalographic evidence of hyperarousal during sleep. Hyperarousal during sleep was defined as decreases in delta power and elevations in alpha and beta power throughout non-rapid eye movement sleep, and symptoms of stress were defined as the tendency to experience stress-related intrusive thoughts and the interaction between intrusion tendency and the number of recent stressful events (subjective stress burden). RESULTS: A stronger tendency to experience stress-related intrusive thoughts was associated with greater sleep complaints and a trend toward higher beta power, whereas increases in subjective stress burden were associated with decreases in delta power. In addition, elevations in subclinical symptoms of depression were associated with greater sleep complaints and elevations in alpha power. CONCLUSIONS: Observed relationships among symptoms of stress, depression, subjective sleep complaints, and electroencephalographic power may be relevant to the discrepancy between subjective and objective measures of sleep in patients with insomnia and may be more broadly applicable to sleep complaints in association with stressful life events and major depression.

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.

Sleep in neuropsychiatric disorders.

Sleep disorders have been recognized for millennia as a common complication of medical and neurologic disease. Virtually all neuropsychiatric disorders carry with them the potential for disturbances of sleep. When such complications do exist, they are typically associated with decreased quality of life, increased morbidity, and, in some cases, increased mortality rates. The prevalence of major sleep disorders among neurologic patients is high, but the rate of detection and treatment is quite low. The major sleep-related problems in this population can be divided into six areas: insomnia, circadian rhythm (sleep-wake schedule) disorders, hypersomnia, sleep-related breathing disorders, motor disturbances in sleep, and parasomnias. In this brief review, general clinical principles, diagnostic assessment and management guidelines for each of these areas are considered and their specific manifestations in neuropsychiatric disorders identified.

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.

Paroxetine in the treatment of primary insomnia: preliminary clinical and electroencephalogram sleep data.

BACKGROUND: Primary insomnia is a persistent and recurrent disorder as well as a risk factor for depression. The goal of this study was to determine whether paroxetine, a nonsedating antidepressant, would be effective in the treatment of patients with primary insomnia. METHOD: Fifteen patients meeting DSM-IV criteria for primary insomnia received paroxetine at bedtime for 6 weeks in an open, flexible-dose trial (median dose = 20 mg). Patients were assessed with daily sleep diaries, baseline and treatment polysomnography, and weekly standardized clinical evaluations. RESULTS: Of the 14 patients who completed the study (1 dropped out owing to side effects), 11 improved with treatment, and 7 of these 11 no longer met diagnostic criteria for insomnia. Although self-reported sleep quality (measured by the Pittsburgh Sleep Quality Index) and daytime well-being (measured by the Profile of Mood States) improved with treatment, the quantity of sleep, measured by diary and by polysomnography, did not change consistently with these improvements. Power spectral analysis suggested that paroxetine treatment may be associated with decreases in power in frequencies within the delta and alpha frequency ranges. CONCLUSION: These results support the effectiveness of paroxetine in the acute treatment of primary insomnia. Further evaluation with controlled and longitudinal designs is warranted.

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.