Incidence of central nervous system involvement in chronic lymphocytic leukemia and outcome to treatment.

Leptomeningeal involvement in patients with CLL is relatively rare and the prognosis is usually considered to be poor. The authors reviewed all CLL patients treated in a tertiary referral center to assess the incidence and outcome of leptomeningeal involvement (LI) in CLL. They found an incidence of 1-2% of LI. Most of the patients with LI had a longterm survival, despite failure to clear the cerebrospinal fluid from tumor cells.

Low level IGF-1 and common variable immune deficiency: an unusual combination.

A relation between growth hormone (GH) deficiency and immunoglobulin deficiency has been suggested previously in a few cases. We describe a patient with an insulin-like growth factor 1 (IGF-1) deficiency and common variable immune deficiency and briefly review earlier publications on the possible interaction between IGF-1 and the immune system. IGF-1 is the downstream mediator of GH. In this patient, GH and IGF-1 levels were both low. The GH response to a GH-releasing hormone test was normal whereas no subsequent IGF-1 response was seen. In our cohort of 14 patients with hypogammaglobulinaemia, two turned out to have slightly decreased IGF-1 serum levels and one patient with a thymoma had an increased IGF-1 level. Even though IGF-1 may be connected to B lymphocyte differentiation, in this patient we hypothesise there is a common impairment in the IGF-1 and IgG pathways.

Aberrant expression of miR-9/9* in myeloid progenitors inhibits neutrophil differentiation by post-transcriptional regulation of ERG.

Aberrant post-transcriptional regulation by microRNAs (miRNAs) has been shown to be involved in the pathogenesis of acute myeloid leukemia (AML). In a previous study, we performed a large functional screen using a retroviral barcoded miRNA expression library. Here, we report that overexpression of miR-9/9* in myeloid 32D cell line (32D-miR-9/9*) had profound impact on granulocyte colony-stimulating factor-induced differentiation. Further in vitro studies showed that enforced expression of miR-9/9* blocked normal neutrophil development in 32D and in primary murine lineage-negative bone marrow cells. We examined the expression of miR-9/9* in a cohort of 647 primary human AMLs. In most cases, miR-9 and miR-9* were significantly upregulated and their expression levels varied according to AML subtype, with the highest expression in MLL-related leukemias harboring 11q23 abnormalities and the lowest expression in AML cases with t(8;21) and biallelic mutations in CEBPA. Gene expression profiling of AMLs with high expression of miR-9/9* and 32D-miR-9/9* identified ETS-related gene (Erg) as the only common potential target. Upregulation of ERG in 32D cells rescued miR-9/9*-induced block in neutrophil differentiation. Taken together, this study demonstrates that miR-9/9* are aberrantly expressed in most of AML cases and interfere with normal neutrophil differentiation by downregulation of ERG.

Cytogenetic clonality analysis in myelodysplastic syndrome: monosomy 7 can be demonstrated in the myeloid and in the lymphoid lineage.

Bone marrow and blood from three patients with myelodysplastic syndrome (MDS) and monosomy 7 were studied for cell lineage involvement of the chromosomal abnormality. Cytogenetic involvement of the myeloid and erythroid cell lineages in MDS with monosomy 7 has been shown before. Lymphoid subpopulations have also been investigated but generally with negative results. A combined technique of May-Grünwald-Giemsa (MGG) for cell cytology and interphase fluorescence in situ hybridization (FISH) using a chromosome 7 specific DNA probe was applied. Further, immunophenotype and genotype of the cells were simultaneously examined with alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining and FISH. The monosomy 7 was found in the blasts and in all or in subpopulations of myeloid and erythroid cells. T cells (CD3+, CD5+) did not appear to be involved. B cells (CD19+, CD22+) showed a normal distribution of FISH spots in two patients. In one patient however the loss of a chromosome 7 was found in approximately 70% of the cells positive for B cell markers including CD79a. The results of this study show that in some cases MDS is a disease arising in a progenitor cell with repopulative abilities restricted to myelopoiesis and erythropoiesis. In other cases, the pluripotent progenitor cells in MDS may show the capacities to differentiate into B lineage lymphoid cells, as well as suggesting that in those instances MDS represents a condition of more primitive transformed hematopoietic ancestor cells.

Clonality analysis of hematopoietic cell lineages in acute myeloid leukemia and translocation (8;21): only myeloid cells are part of the malignant clone.

Bone marrow from six patients with acute myeloid leukemia (AML) and t(8;21) (q22;q22) or a variant t(8;13;21) was studied by simultaneous analysis of cell morphology and karyotype. Combination of May-Grünwald-Giemsa (MGG) and fluorescence in situ hybridization (FISH) using probes specific for the breakpoint regions of chromosome 8 and 21 allowed us to establish the extent of cell-lineage involvement of the translocation. The translocation was found in all myeloid blasts and in high percentages of the more mature neutrophilic cells. In one patient we could demonstrate the translocation in the eosinophils as well. Erythroblasts and lymphocytes did not show the t(8;21) abnormality. These results indicate that the t(8;21) in AML is restricted to the myeloid (granulocytic) lineage.

Erythroid defects and increased retrovirally-induced tumor formation in Evi1 transgenic mice.

Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.

Induction of clinical remission in T-large granular lymphocyte leukemia with cyclosporin A, monitored by use of immunophenotyping with Vbeta antibodies.

A 54-year-old woman presented with a severe autoimmune anemia, thrombocytopenia, neutropenia (Evans' syndrome), and CD8+ lymphocytosis, without signs of lymphadenopathy or splenomegaly. A diagnosis of T cell large granular lymphocyte (T-LGL) leukemia was made, based on cytomorphology, the typical CD3+/CD4-/CD8+/CD16+/CD56-/CD57-/HLA-DR(+/-) immunophenotype of the lymphocytosis (9 x 10(9)/l), and biallelic clonally rearranged T cell receptor beta (TCR beta) genes. Clonality of the TCR alphabeta+ T-LGL was also demonstrated with a panel of antibodies against variable domains of TCR beta chains, which showed single Vbeta7.1 expression on the CD3+ T-lymphocytes. After treatment failure with corticosteroids, splenectomy, and cyclophosphamide, respectively, a complete clinical remission was induced and sustained with cyclosporin A. Vbeta7.1/CD8/CD3 triple immunofluorescence stainings appeared to be valuable for titrating the cyclosporin A dosage by monitoring the T-LGL cells during treatment.

High expression of the multidrug resistance P-glycoprotein in high-risk myelodysplasia is associated with immature phenotype.

The expression of the multidrug resistance (MDR-1) gene product, P-170 glycoprotein (P-170) was investigated in 26 patients with low-risk (n = 9) or high-risk (n = 17) myelodysplastic syndrome (MDS), using a panel of monoclonal antibodies to P-170 (C219, JSB1, C494, MRK16) and quantitative analysis of MDR-1 mRNA. P-170 membrane staining was demonstrated in bone marrow blast cells of 14/17 HR-MDS and in 2/9 LR-MDS patients (p < 0.01). P-170 expression was associated with the presence of blast cells characterized by an immature or early myeloid phenotype as defined by CD34 expression (p = 0.034), CD13 or CD33 expression (p = 0.0006), or CD13/33 plus terminal deoxynucleotidyl transferase (TdT) double expression (p = 0.04). With double fluorescence analysis, P-170 expression was observed in a subset of CD34+ cells, but not in CD34- cells. P-170 expression was present in 13/15 (86%) patient samples with an abnormal karyotype as compared with 3/10 samples (30%) with a normal karyotype (p < 0.05). Nine of these 15 patients had a loss or a deletion of chromosome 7. Thirteen out of 16 (81%) MDR-1 positive patients developed acute leukemia versus two of ten (20%) MDR-1 negative patients (p = 0.025). It is concluded that MDR-1 expression in MDS is present in cells with an immature phenotype and is frequently observed in patients who have an abnormal karyotype and a high risk of leukemic transformation.

Nitrous oxide selectively reduces the proliferation of the malignant cells in experimental rat leukemia.

A considerable reduction of hepatosplenomegaly and leucocytosis in leukemic rats of the Brown Norway Myeloid Leukemia (BNML) can be achieved by exposure to 50% nitrous oxide/50% oxygen. In this study the differential antiproliferative effect of nitrous oxide, inactivating vitamin B12, on normal and leukemic hemopoiesis was investigated in this rat model. Rats injected with leukemic cells and exposed to nitrous oxide for 10 days showed 30% reduction of hepatosplenomegaly and 50% reduction of leukocytosis. Similarly treated healthy rats showed no signs of impaired hemopoiesis as measured by peripheral blood parameters. Clonogenic assays of erythroid and myeloid progenitors from both healthy and leukemic rats revealed that exposure to nitrous oxide did not suppress normal bone marrow functioning. On the contrary, the reduction of leukemic proliferation by nitrous oxide retarded the leukemic infiltration of the bone marrow compartment.

Effect of cobalamin inactivation on folate metabolism of leukemic cells.

Exposure to nitrous oxide inactivates the cobalamin coenzyme of methionine synthetase, an essential enzyme in folate metabolism. Hemopoietic cells are especially dependent on the function of cobalamin for the folate-dependent synthesis of thymidylate (dTMP). Inhibition of methionine synthetase may therefore be of potential value in the treatment of hematological malignancies. In the present study we investigated the effect of nitrous oxide induced cobalamin inactivation on folate metabolism of fresh leukemic cells and the human myelomonocytic cell line U937. Cells were exposed to nitrous oxide for 20 h. Subsequently they were subjected to the deoxyuridine suppression test (dU test), which measures the disturbance of folate-dependent dTMP synthesis. In all bone marrow samples, cobalamin inactivation resulted in a 200% increase of the dU test value, implicating a decreased de-novo synthesis of dTMP. Incubation of leukemic cells with methotrexate, 5-fluorouracil or cycloleucine induced similar increases of the dU test values which could be further raised to 400% with the addition of N2O exposure. Prolonged experiments with U937 cells revealed that the disturbance of folate metabolism aggravated up to 48 h of nitrous oxide exposure. It can be concluded that cobalamin inactivation in human leukemic cells results in disturbed folate-dependent dTMP synthesis. Moreover, effects of several drugs interfering with folate metabolism can be enhanced.

Liver imaging at 1.5 tesla: pulse sequence optimization based on improved measurement of tissue relaxation times.

In order to predict the most sensitive MR imaging sequence for detecting liver metastases at 1.5 T, in vivo measurements of T1 and T2 relaxation times and proton density were obtained using multipoint techniques. Based on these measurements, two-dimensional contrast contour plots were constructed demonstrating signal intensity contrast between hepatic lesions and surrounding liver parenchyma for different pulse sequences and pulse timing parameters. The data predict that inversion recovery spin echo (IRSE) imaging should yield the greatest contrast between liver metastases and liver parenchyma at 1.5 T, followed by short tau inversion recovery (STIR) and spin-echo (SE) pulse sequences. T2-weighted SE images provided greater liver/lesion contrast than T1-weighted SE pulse sequences. Calculated T1, T2, and proton density values of the spleen were similar to those of hepatic metastatic lesions, indicating that the signal intensity of the spleen may be used as an internal standard to predict the signal intensity of hepatic metastases on T1- and T2-weighted images at 1.5 T.

[Diagnosis and treatment of malignant histiocytosis]. / Diagnostiek en behandeling van maligne histiocytose.

Of twelve patients with malignant histiocytosis admitted between 1974 and 1987, clinical symptoms, diagnostic procedures and the course of the disease were retrospectively evaluated. Predominant findings at physical examination were fever (11/12), splenomegaly (12/12), hepatomegaly (8/12), and lymphadenopathy (8/12). Laboratory findings included anaemia, leukocytopenia, thrombocytopenia, high lactate dehydrogenase, and jaundice. Positive diagnostic procedures included biopsies or aspirates of bone marrow (11/12), spleen (6/10), liver (7/9), lymph node (4/4), skin (1/2), lung (1/1) and blood (2/12). In seven patients treated with combination chemotherapy an average survival of 540 days was observed, while two long-term disease-free survivals were accomplished.

Quality control of bone marrow cytology; organization and over 7 years experience in the south-west Netherlands.

To asses the quality of bone marrow cytology of hospital laboratories in the south-west Netherlands a proficiency testing program was implemented. Two sets of bone marrow and blood smears from two patients were sent to 20 hospital laboratories using a tight time schedule biannually. Required results consisted of differential counts of 500 bone marrow cells and 100 peripheral blood cells, together with the description of morphological abnormalities and final conclusions. Twice a year the collected review data were discussed in a plenary session which was also used for continuous education. Over the past 7 years 30 bone marrow samples were evaluated. The coefficient of variations of specific cells counts was large. The amount of correct conclusions ranged from 12% to 100% (median: 61%). Participant attendance of the meetings was 90-100%. The total cost of this scheme of proficiency testing approximately amounted euro7000 per year. The presented formulae for both proficiency testing and haematopathological/cytological education is feasible and fulfilled the need of the participants.

miRNA analysis in B-cell chronic lymphocytic leukaemia: proliferation centres characterized by low miR-150 and high BIC/miR-155 expression.

Several miRNAs have been reported to be associated with immunoglobulin heavy chain (IgH) mutation and ZAP-70 expression status in blood samples of B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (B-CLL/SLL). In the bone marrow and lymphoid tissues, proliferation centres (PCs) represent an important site of activation and proliferation of the neoplastic cells, suggesting that these tissues better reflect the biology of CLL than circulating blood cells. We collected 33 lymph nodes and 37 blood CLL samples and analysed IgH mutation status and ZAP-70 expression status. Expression of 15 miRNAs was analysed by qRT-PCR and RNA-ISH. Sixty-three per cent of the lymph node cases contained mutated IgH genes and 49% of the lymph node cases were ZAP-70-positive, and a significant correlation was observed between ZAP-70 expression and IgH mutation status. Of the blood CLL samples, 49% contained mutated IgH sequences. The miRNA expression pattern in CLL lymph node and blood samples was very similar. Three of 15 miRNAs (miR-16, miR-21, and miR-150) showed a high expression level in both blood and lymph node samples. No difference was observed between ZAP-70-positive or -negative and between IgH-mutated or unmutated cases. No correlation was found between miR-15a and miR-16 expression levels and 13q14 deletion in the blood CLL samples. RNA in situ hybridization (ISH) revealed strong homogeneous staining of miR-150 in the tumour cells outside the PCs. In reverse BIC/pri-miR-155 expression was observed mainly in individual cells including prolymphocytes of the PCs. This reciprocal pattern likely reflects the different functions and targets of miR-150 and miR-155.

Eosinophilia and granulocytic dysplasia terminating in acute myeloid leukaemia after 24 years.

Eosinophilia of variable duration, and subsequent progression to granulocytic sarcoma and acute myeloid leukaemia, has been infrequently reported in the literature. We report a patient with eosinophilia and normal cytogenetics who, after 24 years, showed transformation to a granulocytic sarcoma of the brain. Haematological counts were normal but the marrow revealed the cytogenetic abnormality trisomy 8 in 25% of mitoses. Subsequently an AML-M2 developed, showing a complex karyotype including the trisomy 8 in all metaphases. FISH analysis combined with cytological examination identified the trisomy 8 in blasts, eosinophils and dysplastic granulocytes only. Thus progressive leukaemic transformation selectively involved the myeloid compartment.

Acute myeloid leukaemia with +i(12p) shortly after treatment of mediastinal germ cell tumour.

We report a patient who developed acute myeloid leukaemia (M2) shortly after successful treatment of a mediastinal germ cell tumour. The leukaemia was preceded by a documented myelodysplastic phase. Complex cytogenetic abnormalities were found in bone marrow and peripheral blood cells including +i(12p), typical of germ cell malignancy. Fluorescence in situ hybridization revealed the presence of +i(12p) in myeloblasts, erythroblasts and neutrophils but not in lymphocytes. This case provides further evidence for a common clonal origin of haematological malignancies and mediastinal germ cell tumours.

Clinicopathological diagnosis and treatment of malignant histiocytosis.

The diagnostic findings of malignant histiocytosis (MH) were analysed in 12 consecutive patients in a single institution. Most patients presented with systemic symptoms and lymphadenopathy (92%), splenomegaly (100%) and hepatomegaly (67%). Neurologic symptoms were present in three patients, while involvement of other organs was present in five patients. The incidence of severe thrombocytopenia was 92% of anaemia 92% and of leucocytopenia 67%. Serum angiotensin converting enzyme, alpha 1-antitrypsin and lysozyme were independently increased in 6/9, 3/10 and 1/9 patients respectively. High serum levels of tumour necrosis factor (TNF) were present in 3/10 patients, while serum levels of interleukin-1 were normal in 10/10 patients. Histologic evidence of MH was obtained in all patients by repeated biopsies of involved tissues. Four patients died prior to treatment. Seven patients were treated with combination chemotherapy, consisting of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or MOPP (chloromethine, vincristine, procarbazine, prednisone), in some cases followed by non-cross-resistant second line chemotherapy, if no complete response was attained. The response rate of treated patients was 57%, and progression was observed in two patients. The median duration of response was 38 months. Three patients are alive without evidence of disease and off therapy (30+, 83+, 85+ months). Although MH is a potentially lethal disease, combination chemotherapy may offer a chance for cure in some patients.

Intraosseous and intradiscal gas in association with spinal infection: report of three cases.

The detection of a vacuum phenomenon within the intervertebral disk usually confirms the diagnosis of degenerative disease rather than an infective process. A similar phenomenon in the vertebral body generally indicates ischemic necrosis. However, spinal infection may rarely be accompanied by intradiscal or intraosseous gas so that the latter finding does not entirely exclude the possibility of infection. Three cases are reported to illustrate gas formation in association with vertebral infection.

In situ hybridization on May-Grünwald Giemsa-stained bone marrow and blood smears of patients with hematologic disorders allows detection of cell-lineage-specific cytogenetic abnormalities.

Bone marrow and blood from patients with acute myeloid leukemia and myelodysplastic syndrome were studied by simultaneous analysis of cell morphology and karyotype. A combined technique of May-Grünwald Giemsa (MGG) for cell morphology and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of cytogenetic aberrations allowed us to investigate cell-lineage-specific chromosomal abnormalities. We introduced video recordings to examine large numbers of cells. Briefly, evaluation was first performed on MGG slides, during which cell position and morphology were recorded on an S-VHS recorder. Subsequently, the same slides were used for FISH. This resulted in the identification of MGG-stained cells on the video screen and, at the same time, the interpretation of FISH signals in the fluorescence microscope. Specimens of bone marrow or blood samples from four patients with different hematologic malignancies were studied. One of these patients was studied before and after cytotoxic treatment. The gain or loss of chromosomes could be detected easily and morphologically assigned to the blasts in all patients and to a variable proportion of the myelomonocytic lineage in two patients, but not to the lymphocytes. Thus, this method provides new possibilities for investigating the clonality of hematologic malignancies.