Chronic myeloproliferative diseases, such as chronic myeloid leukemia and polycythemia vera, are associated with neutrophil dysfunction. Very little data is available on essential thrombocythemia (ET). In the current study we evaluated 21 patients with ET. All patients were studied at least 16 weeks after any cytostatic therapy and 10 days after any other therapy. Neutrophil functions were investigated as follows: flow cytometric evaluation of whole blood phagocytosis of opsonized FITC-conjugated E. coli; whole blood chemiluminescence after stimulation with opsonized zymosan and evaluation by an automated, computer-assisted luminometer (LB 950, Berthold); and chemiluminescence and superoxide anion generation by purified neutrophils after f-MLP and PMA stimulation. Chemiluminescence and superoxide anion generation after f-MLP stimulation were found to be significantly lower than in normal subjects, whereas values within the normal ranges were registered after PMA stimulation. Phagocytosis-associated chemiluminescence was found to be impaired both by using zymosan opsonized with autologous plasma and zymosan opsonized with normal plasma, despite a normal phagocytic activity. These data show the presence in ET of a complex neutrophil dysfunction that may be related to an impaired signal transduction during both the phagocytic process and f-MLP stimulation.
The cellular contact between B cells and follicular dendritic cells (FDCs) in the germinal center is thought to play a key role in B-cell maturation and proliferation. The adhesion pathway through the very late antigen 4 (VLA-4) on the B cells and the vascular cell adhesion molecule 1 (VCAM-1) on the FDCs support this binding process. The neoplastic follicular centers in follicular non-Hodgkin's lymphomas (FNHLs) have similar structures and cellular components to those of normal germinal centers, but their interaction between B cells and FDCs may be functionally disturbed. In view of this we analyzed the interaction between VLA-4 and VCAM-1 molecules in the germinal center microenvironment, both in neoplastic and normal follicles. The structural characterization of FNHLs and reactive lymph nodes was studied with indirect immunohistochemical stainings using monoclonal antibodies against VLA-4, VCAM-1, and fibronectin, with special reference to the reaction pattern in the normal and neoplastic follicles. In the reactive follicular centers most B cells did not show a positive reaction for VLA-4, except for moderate reaction products in the B cells of the light zone. In FNHLs, on the other hand, most follicular center B cells were positive for VLA-4. The reaction patterns of VCAM-1 and fibronectin in both normal and neoplastic follicular centers were not basically different. To investigate the interaction of VLA-4 with VCAM-1 in both neoplastic and normal follicular centers, we performed a frozen-section binding assay, which found decreased binding between VLA-4 and VCAM-1 in FNHLs. The results of this study indicated that the microenvironment in neoplastic follicular centers is different from that in their normal counterparts, in terms of the characteristic distribution pattern of the VLA-4-positive B cells, and the functional deterioration of the VCAM-1 on FDCs.
Antisense technology offers the potential to create compounds specific enough to support highly isotypically selective pharmacology (i.e., pharmacologic agents specific enough to affect only one isotype in a multigene family of related proteins). However, enthusiasm about the potential of the technology has been appropriately tempered by questions about its ability to deliver on its promise. The fundamental issue is: Can oligonucleotides that have acceptable drug properties be created? This review discusses recent progress that helps to define the pharmacokinetic, pharmacologic, and toxicologic properties of phosphorothioate oligonucleotides. Progress in the medicinal chemistry of oligonucleotides that has resulted in a wide range of new chemical classes is also described.
This study assesses the value of immunologic and ultrastructural methods in disclosing the lineage commitment of cells from acute leukemias (ALs). Two hundred and fifty-one ALs were characterized morphologically, cytochemically, and immunologically. Myeloperoxidase (MPO) positivity in > 3% of blasts was regarded as evidence of the myeloid origin of leukemic cells, cytoplasmic CD22 (cCD22) expression was taken as an indication for B-lineage acute lymphoblastic leukemia (ALL), and CD3+ (membrane or cytoplasmic) cases were classified as T-ALL. Diagnosis of minimally differentiated acute myeloid leukemia (AML-M0) was made when blast cells had undifferentiated features by light microscopy, reacted with at least one of the antibodies to myeloid-specific antigens (CD13, CD33, MPO), and lacked CD19, cCD22, and c/mCD3. Megakaryoblastic differentiation was demonstrated by the expression of CD41 and/or CD61. Following these criteria, 209 cases were classified as acute myeloid leukemia (AML) and 39 as ALL. Expression of lymphoid antigens was detected in 45% of AML cases and 30% of ALLs showed myeloid antigens. One case was regarded as a true biphenotypic leukemia because of the combined expression of MPO and CD33 for the myeloid lineage, and cCD3, CD2, and CD5 for the T-cell lineage. Two cases lacked signs of myeloid or lymphoid differentiation and were studied by electron microscopy methods. One displayed platelet peroxidase (PPO) activity and was classified as a megakaryoblastic variant, one other reacted with anti-CD33 and was considered AML-M0. We conclude that light microscopy and standard immunologic methods can accurately demonstrate the lineage orientation in greater than 99% of ALs. Integration with ultrastructural analysis can define the cell nature of virtually all cases of AL.
Paraffin wax sections of lymph node biopsies from a total of thirteen patients with the morphologic and clinical features of Castleman's disease were analyzed for the presence of the Epstein-Barr virus (EBV) by in situ hybridization for the noncoding EBV early RNAs (EBERs) and by immunohistochemistry for the EBV-encoded latent membrane protein-1 (LMP-1). Of twelve cases of localized Castleman's disease EBER-positive cells were identified in five, and in these cases were only rarely found and were always confined to the interfollicular regions. LMP-1 was not detected in any of these cases, either alone or after dual staining for EBERs and LMP-1. (A similar pattern of EBER expression is seen in nonneoplastic lymphoid tissue from EBV-positive individuals.) No EBER-positive or LMP-1 positive cells were identified in a single case of multicentric Castleman's disease. In two additional patients initially diagnosed with Castleman's disease of localized plasma cell type, repeat biopsy showed Hodgkin's disease. In both cases Reed-Sternberg cells and their variants were identified in the original biopsy on which the diagnosis of Castleman's disease was made. In one of these cases these cells showed expression of EBERs and LMP-1, indicating latent infection with EBV. The results suggest that EBV is not generally associated with Castleman's disease. Further analysis of a series of cases of multicentric Castleman's disease is indicated.
The rearrangement of the immunoglobulin heavy chain gene can be used as a marker of B-cell lineage and clonality. By using polymerase chain reaction (PCR) with variable-region and joining-region specific primers it is possible to detect the rearrangement of a small amount of clonal B cells, as described by several groups. The specific PCR product can be detected after amplification with gel electrophoresis and ethidium bromide staining. The DNA fragments obtained from different clones are, however, approximately of the same size, making it difficult to distinguish between the clones by simple electrophoresis and ethidium bromide staining, as described in many reports. Single-strand conformational polymorphism (SSCP) was evaluated as a method to detect specific clonal amplicons in a mixture of PCR-amplified products. Unique patterns were obtained for different B-cell clones, detectable in mixtures of 0.25% clonal cells in normal cells. It is concluded that SSCP is a valuable method for the specificity control of PCR in B-lymphocyte clonality analyses. The advantages of the described method over previously published techniques are increased specificity, simplicity without radioactivity, and rapidity.
p21 is induced by and mediates the effects of p53 in response to DNA damage arresting the cell in G1 or G2, by inhibiting multiple cyclin-cyclin-dependent kinases (CDK) or binding to proliferating-cell nuclear antigen (PCNA), respectively. To determine whether p21 mutants occur in tumors we examined DNA from 188 primary non-Hodgkin's B-cell lymphoma (NHL) tumors and 84 chronic myelogenous leukemia samples for mutational changes in the coding region of p21 by single-strand conformation polymorphism (SSCP) analysis and direct sequencing of polymerase chain reaction (PCR)-amplified DNA. We did not find mutations in the coding region in these two tumor types. We identified a polymorphic nucleotide change in codon 31 in which a transversion from C to A substituted amino acid arginine for serine. Three of 188 NHL tumors were homozygous for this change, but they were not identified in 84 CMLs or in 97 normal controls. On the other hand, in one CML case a transition from G to A in codon 64 substituted amino acid threonine for alanine. These data do not indicate that derangements in the coding region of p21 contribute to the initiation and/or progression of these tumors.
High-dose methylprednisolone (HDMP) has been shown to induce differentiation of myeloid leukemic cells with a remarkable antileukemic effect in children with various subtypes of acute myeloblastic leukemia (AML), therefore we used HDMP in the treatment of four children with myelodysplastic syndrome (MDS). Two patients had refractory anemia with an excess of blasts in transformation (RAEB-t) with extramedullary infiltration (EMI), one had chronic myelomonocytic leukemia with pleural effusion, and one had RAEB. HDMP was administered orally at a single dose of 20-30 mg/kg/day combined with low-dose cytosine arabinoside (LD Ara-C) (10 mg/m2, 12-hourly s.c.) for 2 weeks. The treatment continued with mitoxantrone (10 mg/m2, i.v.) and Ara-C (5 mg/kg, i.v.) once a week for four doses followed by maintenance chemotherapy. All patients achieved hematologic remission 2-4 weeks after initiation of treatment. Extramedullary infiltration disappeared in all cases within 2 weeks to 3 months after initiation of therapy. With the exception of two patients who relapsed 6 and 24 months after remission, treatment could be stopped in others who remained in remission for 36 months without evidence of EMI; 6 months later one of them developed myelodysplastic relapse (RAEB). No side effects related to HDMP treatment were noted, but hyperleukocytosis developed in two patients who initially had high WBC counts. We suggest that the addition of HDMP with or without LD Ara-C to cytotoxic chemotherapy offers a promising alternative in cases not considered suitable for bone marrow transplantation.
Fourteen patients with hypocellular acute leukemia (HAL) were reviewed. The median age was 72 years, with an equal male-to-female ratio. Severe granulocytopenia with marrow hypocellularity and increased marrow blasts and absence of physical findings were common features. The median peripheral blood blast count was 2%. All except 3 cases of erythroleukemia had marrow blast count that exceeded 30% of all nucleated marrow cells. All cases were classifiable with the FAB criteria. FAB classification revealed a preponderance of the M1 category followed by M2 and M6 types. The majority of blasts were type I and the median myeloperoxidase positivity was 14%. Immunophenotyping of bone marrow cells by flow cytometry in 9 cases showed expression of myeloid antigens (CD13, CD33); 6 cases also expressed CD34 antigen. Significant dysplasia involving erythroid and megakaryocytic lineages was seen in most of the cases. Trilineage dysplasia was observed in 5 cases. Median survival of the entire group was 10.5 months. Eleven patients underwent induction therapy consisting of daunorubicin and cytosine arabinoside +/- 6 thioguanine; 8 patients achieved complete remission (72.6%). Remission duration was 14.5 months. Three patients (27.4%) died secondary to infections during induction therapy. Higher frequencies of trilineage dysplasia and FAB M6 type together with low percentage of peripheral blasts and presence of antecedent hematologic disorders suggest that some of these cases might represent the hypocellular form of acute myeloid leukemia with trilineage dysplasia.
In paraffin-embedded sections of bone marrow fixed in B5-formalin, proerythroblasts from patients with marked erythroid hyperplasia displayed a characteristic nuclear chromatin pattern. This pattern consisted of an "arm" of chromatin extending from the center of the nucleus to the inner aspect of the nuclear envelope. In some instances the arm showed an "elbow bend." Depending on the plane of section, approximately 30% of proerythroblasts demonstrated these chromatin patterns. Ultrastructurally a similar "arm" of chromatin was seen in proerythroblasts. Embedded within the chromatin was a nucleolus. Although the significance of this pattern is as yet unknown, it provides a morphologic clue to identification of proerythroblasts in tissue sections.
The next technologic revolution may well be the miniaturization of solution phase experimentation through the marriage of microelectronics and molecular biology. Already researchers leading this rapidly emerging technology are developing prototype biochemical "microlaboratories" that offer a millionfold reduction of scale; that is, biochemical analyses are being conducted with picoliter sample volumes rather than conventional microliter formats. Such miniaturization is achieved by exploiting the well-honed tools of microelectronics that accommodate highly parallel automated assays, ultrasensitive detection, high throughput, integrated data acquisition, computation, and distributed data storage/retrieval. The subject of this paper is to survey this evolving bioelectronic miniaturization technology with applications to gene-based diagnostics. Several pioneering microchips are described and compared to traditional biochemical methods. Specifically, miniaturized examples of various diagnostic processes such as sample preparation (PCR), assays (electrophoresis and probe arrays), and detection (integrated CCDs) are presented. Although these microdevices are rather embryonic they represent the first steps toward fabricating a fully integrated diagnostic system on a microchip.
It is well known that some of the widely used antibodies directed against hemopoietic antigens exhibit cross-reactivity with normal and neoplastic nonhemopoietic cells. By contrast, relatively little is known about the immunoreactivity of hemopoietic cells with antibodies that detect nonhemopoietic antigens. In this study 43 routinely processed bone marrow biopsy specimens containing infiltrates of acute leukemia of different subtypes were stained with a panel of 20 antibodies that detect nonhemopoietic antigens in formalin-fixed and paraffin-embedded tissue. Thirteen of the antibodies applied (KL1; BMA 120; and antibodies against epithelial membrane antigen, alpha-fetoprotein, prostate-specific acid phosphatase, prostate-specific epithelial antigen, placental alkaline phosphatase, alpha-amylase, serotonin, bombesin, beta-human chorionic gonadotrophin, desmin, and S-100 protein) did not stain blast cells in any of the cases. However, anti-vimentin, HMB45, and anti-myoglobin stained blast cells in the majority of the cases; the antibodies against thyroglobulin, actin, and carcinoembryonic antigen stained blast cells in 10% to 25% of the cases; and anti-neuron-specific enolase stained blast cells in less than 10% of the cases. No correlation was found between the leukemia subtype and the pattern of immunoreactivity. The staining specificity, (i.e., the specificity of binding of the primary antibody--immunologic vs. nonimmunologic binding), was tested by increasing the dilution of the primary antibody and comparing the staining intensity in the bone marrow specimens and control tissue. Staining specificity was confirmed only for staining with the antibodies against neuron-specific enolase and vimentin. The findings show that immunoreactivity of tumor cells in bone marrow biopsy specimens for nonhemopoietic antigens does not exclude a diagnosis of acute leukemia.
Polymorphonuclear leukocytes (PMNL) from chronic myeloid leukemia (CML) patients are defective for chemotaxis in response to the synthetic chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) as compared to normal PMNL. The present study investigated whether the defective chemotactic response was mediated through altered actin polymerization induced with fMLP. Granulocytes isolated from seven normal subjects and seven CML patients were stimulated with fMLP and lysed with Triton containing buffer at time points of 0, 30 seconds, and 1, 2, and 10 minutes. The Triton insoluble cytoskeleton containing polymerized actin was analyzed by SDS-PAGE and densitometry. The CML PMNL polymerized significantly lesser actin than normal PMNL on stimulation with 10 nM (p > 0.05) and 1 nM (p > 0.01) fMLP. This lower actin polymerization observed in fMLP-stimulated CML PMNL may be responsible for the defective chemotaxis seen in these cells.
Sequential bone marrow aspirates and sections from patients with acute myeloid leukemia (AML) were examined to determine if a correlation exists between bone marrow morphology during induction phase of therapy and outcome. Of 95 patients of AML diagnosed between July 1987 and December 1991, 53 uniformly treated patients (induction therapy with cytosine arabinoside and daunorubicin) had sequential bone marrow examinations performed in the 2- to 5-week period following initiation of induction therapy. Four morphologic patterns were recognized in these 53 patients: Group I (22 patients)--hypocellularity or normal regeneration (> or = 15% cellularity and < 5% blasts) on the initial 2-week marrow followed by marrows showing normal regeneration; Group II (10 patients)--hypocellularity followed by "reactive myeloblastosis" (> or = 15% cellularity, 5% to 34% blasts, with promyelocytes = or > blasts); Group III (12 patients)--residual blasts (> or = 5% blasts with blasts >> promyelocytes) in the initial posttherapy marrow; Group IV (9 patients)--atypical patterns not fitting any of the other categories. Complete remission was achieved in all 32 patients in Groups I and II without additional induction therapy, but was achieved eventually in only 10 of 21 patients in Groups III and IV combined (p < 0.005), 15 of whom received additional induction therapy. Remission duration and actuarial survival for each group were as follows: Group I: 344/596 days; Group II: 443 days/> 660 days; Group III and IV combined: 351/311 days (p = 0.017 for actuarial survival). Seven of 21 patients in Groups III and IV had unfavorable initial morphology (MO, hypoplastic AML and AML preceded by myelodysplasia) compared to only 3 of 32 patients in Groups I and II (p = 0.039). It was thus observed that "reactive myeloblastosis" with up to 34% blasts on the third or fourth week bone marrow following an initial hypocellular marrow does not require additional induction therapy to achieve durable remissions or favorable survival. Also, residual blasts that outnumber promyelocytes, and atypical patterns of regeneration correlate with lower remission induction rates, shortened survival, and unfavorable morphology on the initial diagnostic bone marrow.
The concept of ex vivo expansion of the human stem/progenitor cell population was tested in this study. Cord blood mononuclear cells were isolated and cultured in the presence of SCF, IL-3, and either cord blood serum (CBS) or fetal calf serum (FCS). After 8 days of incubation with CBS or FCS, the number of CFU-GM was increased ninefold and four-and-a-half-fold, respectively. However, the enhancing effect lasted for only a short period. This study demonstrates that CBS could contain growth factors expanding progenitor cells. Whether the ability of CBS to enhance the CFU-GM expanding capacity is attributable to a novel factor or factors, or represents effects of other known cytokines, remains to be determined.
T cells play a central role in the immune system as effectors and regulators. They become activated upon antigen recognition by their T-cell receptors (TCR). The TCR repertoire is established by developmentally regulated TCR gene rearrangements and shaped by predominantly intrathymic selection processes. Failure of this system can lead to autoimmune disease. TCR gene probes and primers are widely used to distinguish polyclonal from abnormal clonal T-cell proliferations in Southern blot or polymerase chain reaction (PCR) procedures. T cells are normally activated by signal transduction through the TCR/CD3 complex and accessory molecules such as CD4 and CD8. Protein tyrosine kinases (PTKs) and nuclear transcription factors (TFs) are important intracellular signaling molecules. Chromosomal abnormalities in T-cell leukemia often affect the gene loci of TFs, PTKs, and sometimes other growth regulatory proteins. Aberrant activation of these molecules may lead to alteration of the signaling cascade and interference with ordered T-cell development and differentiation. The increasing knowledge about different functional aspects of TCR physiology thus contributes to the diagnosis and understanding of reactive and malignant T-cell disorders. This will eventually lead to new diagnostic concepts and novel therapeutic strategies.
Chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL) are differentiated B-cell leukemias with well-described clinical, morphologic, and immunologic characteristics. We encountered two patients with indolent chronic B-cell leukemia showing overlapping features of these malignancies. The patients had progressive splenomegaly, minimal lymphadenopathy, and abnormal lymphoid cells with abundant cytoplasm and villi, which were strongly positive for surface antigens CD22 and CD11c, features associated with HCL. However, blood counts showed lymphocytosis without neutropenia and monocytopenia, and the bone marrow biopsies demonstrated tightly aggregated nodules of lymphocytes. In addition, the lymphoid cells were dual positive for CD19 and CD5, displaying weak-to-moderately positive monoclonal surface immunoglobulin, findings strongly suggestive of CLL. One patient failed to respond to therapy with chlorambucil and prednisone. The second patient showed a partial response to treatment with 2-chlorodeoxyadenosine. We compare our patients with similar variants of differentiated B-cell leukemias reported in the literature, including disorders described as hairy cell variant (HCL-V) or splenic lymphoma with villous lymphocytes (SLVL).
The association of plasmacytosis and lymphocytosis with acute myeloid leukemia (AML) has been documented in isolated case reports. We examined 149 cases (134 adults, 15 children) of newly diagnosed AML and found 9 adults (6%) with greater than or equal to 5% plasma cells and 1 child and 1 adult with greater than or equal to 20% lymphocytes. Lymphocytes constituted 25% and 42% of marrow cellularity in the adult and child respectively and persisted throughout remission in the child's marrow. The percentage of morphologically normal plasma cells ranged from 5% to 13% (mean 7%). Monoclonal immunoglobulins were not detected with immunostaining or flow cytometry. Hypergammaglobulinemia was present in 3 cases, and a monoclonal increase in IgG-kappa in 1. Plasmacytosis was not seen in remission marrows from these patients (n=4). Lymphocytosis or plasmacytosis occurs in approximately 7% of patients with AML, appears reactive in nature, and may represent an immunological response to tumor. Monoclonal paraproteins may occur without other evidence of B-cell neoplasia.
