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Clonality of myeloid and lymphoid cell fractions obtained from peripheral blood (PB) or bone marrow (BM) of five patients with a myelodysplastic syndrome (MDS), was studied by combined immunophenotypic analysis and DNA in situ hybridization. This novel technique enables quantitative and direct analysis of cytogenetic alterations in nondividing cells of distinct cell lineages. Four patients with a trisomy 8 and one patient with a translocation (1;7) were studied. For cell lineage determination, antibodies specific for progenitor cells (CD34), myeloid cells (CD15), monocytes (63D3), T cells (CD3), and B cells (CD19,20,22) were used. In one patient with a trisomy 8, BM cells were available and the erythroid lineage could be studied. For detection of cytogenetic aberrations, we used chromosome- specific repetitive DNA probes. In three patients, all nonlymphoid cells carried the cytogenetic abnormality; in two patients, mosaicism within these lineages was suggested by the relative low numbers (35% to 55%) of aberrant cells. None of the T or B cells of the five patients carried the chromosomal aberrations. We conclude that combined immunophenotyping and in situ hybridization is a feasible technique to study lineage involvement. Our data suggest that the chromosomal aberrations studied in MDS are restricted to the myeloid lineages.

Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T-cell receptor gamma-polymerase chain reaction (TCR gamma-PCR). Subsequently, in 6 patients, the CDR3 length and conformation from 22 different TCRBV subfamilies were analyzed after PCR amplification of cDNA using TCRBV subfamily-specific primers. T cells from 15 of 25 HCL patients showed clonal excess by the TCR gamma-PCR. In fluorescence-activated cell sorted T-cell subsets, more clonal bands were observed than in the unseparated T cells, with most of these in CD8+ subsets, but also in CD4+, CD3+ gamma/delta+, and a double-negative CD3+ alpha/beta+ subset. In other B-cell malignancies, 6 of 16 samples showed oligoclonal T cells, whereas only 2 of 18 normal spleen and blood samples showed abnormal bands. Analysis of the TCRBV subfamilies disclosed in all 6 HCL patients a markedly abnormal pattern, with many clonal bands in 5 to 15 subfamilies, and absent or abnormal weak patterns in another 1 to 8 subfamilies. In comparison, 6 normal samples (2 spleens and 4 blood samples) showed in only 1 blood donor 1 clonal band. Two patients with active HCL but without infections or treatment were tested several times during the course of the disease and showed a complete identical skewed T-cell repertoire with the same oligoclonal T-cell populations. In conclusion, T cells in the blood and spleen of HCL patients show impressive abnormalities with many oligoclonal T-cell populations and a very restricted and skewed TCRBV repertoire.

An International Prognostic Index (IPI) for patients with aggressive non-Hodgkin's lymphoma (NHL) has recently been published. The IPI is based on pretreatment clinical characteristics and developed on clinical trial patients, classified as intermediate grade according to the Working Formulation (WF). We applied this IPI in a population-based registry of NHL patients. This registry does not have the restrictions that usually hold for patients in clinical trials, eg, with respect to age and performance status. Moreover, it covers all the three WF classes (low, intermediate, and high). The IPI turned out to be of prognostic value for response rate and survival in our unselected cohort of 744 patients, as well. In each of the three WF classes separately, the four IPI classes showed going from low to high substantially decreasing response rates and survival percentages. For our cohort of WF intermediate grade patients 5-year survival levels were lower in all four IPI classes (59%, 34%, 14%, and 10%, respectively), probably reflecting the selection of clinical trial patients in the original study (73%, 51%, 43%, and 26%).

Large cell lymphomas (LCLs) are heterogeneous in morphology, clinical presentation, and behavior. We studied 52 de novo LCLs of B-cell type for rearrangements of the bcl-2 and c-myc oncogenes by Southern blot analysis and related these data to the primary site of presentation, stage, and cytomorphology. Thirteen tumors had comigrating rearrangements of JH and bcl-2, indicative of a t(14;18). Far more primary nodal lymphomas than extranodal lymphomas carried a t(14;18) (40% v less than 5%). Additionally, almost all lymphomas with a t(14;18) versus 41% of the tumors without a bcl-2 rearrangement presented with lymphadenopathy. c-myc rearrangements were seen in 35% of the extranodal lymphomas and 5% of the nodal lymphomas. No differences were observed in bone marrow involvement and staging according to Ann Arbor. bcl-2 rearrangements were found in 50% of the LCLs with cleaved nuclei, whereas c-myc rearrangements were relatively frequent (25%) in the noncleaved subtype. Our data support the hypothesis that primary nodal and extranodal lymphomas have a different genetic origin.

Forty-four B-chronic lymphocytic leukemias (CLL) were studied by Southern blot analysis using probes for the Ig genes and bcl-1, bcl-2 (major, minor and 5' breakpoint region), bcl-3, c-myc, and retinoblastoma (Rb) loci. Eight cases had three or more rearranged JH bands, indicating oligoclonality, clonal evolution, or chromosomal translocation. One case had a rearrangement of the bcl-1 locus and three of the bcl-2 locus. In the first case, comigration of the rearranged bcl-1 and JH sequences indicated a t(11;14)(q13;q32) translocation, which, in contrast to previously described cases, seems to be completely reciprocal. One case with a bcl-2 rearrangement showed comigration of the bcl-2 major breakpoint region and a rearranged JH band. This indicates a t(14;18) (q32;q21). The two other cases showed rearrangements of the bcl-2 5' breakpoint region without apparent comigration. No rearrangements were detected of c-myc and bcl-3, located at chromosome 19, nor was a deletion of Rb found. All but three cases had CD5 expression. The exceptions included the t(11;14) and the t(14;18) cases. Our results confirm recent data on rearrangements at the 5' site of bcl-2 in CLL. Additionally, they corroborate the presumption that CD5-negative chronic B-cell leukemias should be considered apart from classical CLL.

B-cell colony growth of malignant and normal B-cells has been studied in a double layer (agar-fluid) colony assay. Stimulatory factors consisted of irradiated blood leukocytes, phytohaemagglutinin (PHA), interleukin 2 (IL2) and 12-0-tetradecanoylphorbol-13-acetate (TPA) in various combinations. B-cell colonies have been obtained in all cases tested, i.e., 7/7 cases with chronic lymphocytic leukaemia, 7/7 cases with non-Hodgkin's lymphoma, 5/5 cases with hairy cell leukaemia and 7/7 normal B-cell suspensions, obtained from blood (X 3), bone marrow (X 2) and spleen (X 2). The plating efficacy ranged from 0.02-0.35, with a median of 0.07. Colony formation was found to be linear (r = 0.96) in the plating range of 0.5-8 X 10(5) cells. Secondary colonies could be obtained in 2 cases tested. DNA synthesizing cells in colonies were determined in 4 cases using monoclonal antibodies against DNA-incorporated bromodeoxyuridine (BrdUrd). In most cases the combination of PHA (with or without IL2) and irradiated leukocytes yielded the highest number of colonies, but in some experiments stimulation with TPA + IL2 was found to be optimal.

Serum neural cell adhesion molecule (NCAM) has been described as a prognostic marker in multiple myeloma (MM). Both C-reactive protein (CRP) and beta 2-microglobulin (beta 2M) are established prognostic markers in MM. We tested the diagnostic value of these markers in 212 serum samples of patients with paraproteinemia registered prospectively in a population-based registry. Sixty patients had MM and 152 had other monoclonal gammopathies (hematologic diseases [48], paraneoplastic disease [35], autoimmune disease [15], and monoclonal gammopathy of undetermined significance [56]). CRP and beta 2M had wide and overlapping ranges in all diagnostic categories. However, serum neural cell adhesion molecule (NCAM) was low (< 20 U/mL) in all but 4 of 152 nonmyeloma cases and high (> or = 20 U/mL) in 31 (52%) of the 60 MM cases. Two patients with non-Hodgkin's lymphoma, 1 with chronic lymphatic leukemia, and 1 with autoimmune disease had serum NCAM values between 20 and 30 U/mL. In a discriminant analysis in which serum NCAM, CRP, beta 2M, paraprotein type and concentration, hemoglobin, leukocyte and thrombocyte counts, creatinine, corrected calcium, lactate dehydrogenase, and alkaline phosphatase were included, paraprotein type and concentration and serum NCAM turned out to be the best combination of parameters predicting whether a patient had MM, with 89% of cases being correctly classified. Even without bone marrow and x-ray examinations, serum NCAM, in combination with paraprotein type and concentration, can differentiate between MM and nonmyeloma patients.

In the present study we demonstrate that Aza-dC in combination with Amsacrine has major antileukaemic properties in patients who have not already received extensive Ara-C therapy. Eight out of 11 patients in their first relapse of acute leukaemia achieved complete remission. Cross resistance between Ara-C and Aza-dC was revealed by the lack of antileukaemic activity in five patients with with Ara-C resistant leukaemia. Combination therapy with Aza-dC/Ams-acrine induced a considerable period of a granulocytopenia (28-35 days), while the toxic effect on erythro- and megakaryopoiesis was comparable to that reported for high dose Ara-C/Amsacrine chemotherapy. Remarkable is the long disappearance time for leukaemic blast cells in bone marrow, i.e. 3-5 weeks in some cases. Analysis of cell membrane markers showed a loss of the early differentiation antigens CD34 and CD33 from leukaemic bone marrow cells after 7 days of Aza-dC treatment, which is suggestive of leukaemic cell differentiation. In the small group of patients tested for DNA hypomethylation no association existed between the degree of hypomethylation and clinical response. Non-haematologic side effects were considerable in patients receiving the highest dosages of Aza-dC and consisted of severe, although usually reversible, gastrointestinal and neurological complications. In comparison with Ara-C, Aza-dC causes less nausea and vomiting and is therefore better tolerated.