No benefit from adding GM-CSF to induction chemotherapy in transforming myelodysplastic syndromes: better outcome in patients with less proliferative disease.

In this prospective randomized multicenter trial 93 patients, median age 72 years, with RAEB-t (n=25) and myelodysplastic syndrome (MDS)-AML (n=68) were allocated to a standard induction chemotherapy regimen (TAD 2+7) with or without addition of granulocyte-macrophage-CSF (GM-CSF). The overall complete remission (CR) rate was 43% with no difference between the arms. Median survival times for all patients, CR patients, and non-CR patients were 280, 550, and 100 days, respectively, with no difference between the arms. Response rates were significantly better in patients with serum lactate dehydrogenase (S-LDH) levels

Long-term follow-up of 18 patients with myelodysplastic syndromes responding to recombinant erythropoietin treatment.

The outcome of continued EPO therapy was studied in 18 responding MDS patients. The EPO dose was reduced in a stepwise fashion to find the lowest possible maintenance dose. Relapses of anemia were associated with either progressive disease or reduction of the administered EPO dose. In the latter group second responses to renewed EPO therapy were readily achieved. Long-term responses were seen in about a third of the patients. Thus, it seems safe to reduce the EPO dose among responding patients. This approach may have advantages both from a medical and a socio-economic perspective.

Gain of chromosome 7, which marks the progression from indolent to aggressive follicle centre lymphomas, is restricted to the B-lymphoid cell lineage: a study by FISH in combination with morphology and immunocytochemistry.

In a previous fluorescent in situ hybridization (FISH) study of patients with high-grade follicle centre lymphomas (FLCs), we often found additional copies of chromosome 7 in bone marrow (BM) cell nuclei even though obvious malignant tumour cells could not always be morphologically identified in the corresponding cell smears. This raised the question whether the gains of chromosome 7 are really confined to B-lymphoid tumour cells or whether other cell lineages are also of clonal origin. In the present investigation we employed FISH in combination with immunomarkers and morphological studies on BM smears and lymph node imprints from seven patients with high-grade FCLs and diffuse large B-cell lymphomas (DLBCLs). Three out of seven BM samples were found to contain clonal CD20-positive B-lymphoid cells (range 0.4-96% of the cells) and no extra copies of chromosome 7 were detected in the myelomonocytoid or erythroid cells or in the CD3-positive T lymphocytes. All seven patients showed additional copies of chromosome 7 in the lymph nodes and, again, this cytogenetic abnormality was also restricted to the CD20-positive cells (range 0.7-80% of the cells). Thus the present findings confirm that high-grade B-cell lymphomas with or without BM engagement involve the CD20-positive B-lymphoid cells exclusively and not the T lymphocytes, erythroid or myelomonocytoid cell lineages. These findings may indicate that anti-CD20 immunotherapy could be of value in high-grade B-cell lymphomas.

Addition of etoposide to CHOP chemotherapy in untreated patients with high-grade non-Hodgkin's lymphoma.

BACKGROUND: Second- and third-generation chemotherapy protocols for the treatment of aggressive non-Hodgkin's lymphomas (NHL) have considerable, and age-related, toxic effects. In addition, they do not seem to prolong overall survival in comparison to standard CHOP chemotherapy. In this phase II study we investigated the feasibility and efficacy of the addition of etoposide to the conventional CHOP regimen. PATIENTS AND METHODS: Toxicity and clinical efficacy were determined in 132 patients with previously untreated high-grade NHL. There were 51 patients in clinical stage I and II and 81 patients in stage III and IV, with a median age of 54 years (range 17-85). Patients received standard-dose CHOP plus etoposide 100 mg/m2 i.v. on day 1 and 200 mg/m2 p.o. on days 2-3. RESULTS: The overall response rate was 84%, with 70% complete and 14% partial responses. The predicted three- and five-year survivals for the group as a whole were 60% and 53%, respectively, and the corresponding disease-free survivals for patients achieving complete remissions were 65% and 56%, respectively. Outcome was not different from that of CHOP-treated patients in a recently completed Nordic study performed during the same time period. Myelosuppression (WHO grade 3-4), observed in 87% of patients and infectious complications (WHO grade 3-4) in 33%, dominated the toxicity profile of this regimen. Fifty-seven of 92 complete responders (62%) received 6-8 CHOP-E cycles with no reductions in planned dose intensity. LDH level higher than normal, extranodal sites = 2, stage III-IV at diagnosis were all indicators of a poor survival. CONCLUSIONS: We conclude that CHOP-E treatment is effective in high-grade NHL. However, mainly due to severe myelosuppression frequent schedule modifications were required and the results are not obviously superior to those of conventional CHOP.

[Genetic origin of malignant hematopoiesis well defined]. / Den maligna hematopoesens genetiska ursprung väl kartlagt.

It is vital to determine which cell lines are affected in haematological malignancies, since such information is important to an understanding of the biology of neoplastic stem cells and their capacity to differentiate and mature. Parallel studies of cellular morphology and of chromosomal anomalies is an approach permitting determination of lineage specificity for different haematological neoplasms. Findings in current studies suggest that acute myeloid leukaemia and myelodysplastic disorders generally involve cells of myeloid lineage only, whereas myeloproliferative disorders may also involve lymphoid cell lines. Lymphoid malignancies such as non-Hodgkin's disease or acute lymphoid leukaemia usually involve lymphoid cell lines.

Gain of chromosome 7 marks the progression from indolent to aggressive follicle centre lymphoma and is a common finding in patients with diffuse large B-cell lymphoma: a study by FISH.

Gain of chromosome 7 represents one of the most frequent cytogenetic findings in B-cell lymphomas with a follicular growth pattern. We used fluorescence in situ hybridization (FISH) and a probe specifying chromosome 7 on lymph node imprints and/or bone marrow (BM) and peripheral blood (PB) smears from six consecutive patients with follicle centre lymphomas (FCLs) grade I or II (low-grade lymphomas), four patients with FCLs grade III and 11 patients with diffuse large B-cell lymphomas (DLBCLs) (high-grade lymphomas). We found gains of chromosome 7 in 14/18 successfully analysed cases (i.e. 2/6 FCLs grade I-II, 3/3 FLCs grade III and in 9/9 DLBCLs) using lymph node imprints. Moreover, the FISH technique demonstrated gains of chromosome 7 in 1/4 BM and 0/4 PB samples from FCLs grade I-II, in 2/4 BM and 2/4 PB specimens from FCLs grade III and in 4/9 BM and 2/9 PB samples from the DLBCLs. In contrast, morphologically recognizable lymphoma cells were seen in only 1/4 BM and 0/4 PB samples from the FCLs grade III and in 1/11 BM and 1/11 PB samples from the DLBCLs. We conclude that: (i) gain of chromosome 7 marks the progression from indolent to aggressive FCL and would appear to be a common finding in patients with FCLs grade III and in DLBCLs, (ii) clonal lymphoid cells occur frequently in BM and PB in high-grade lymphomas, making traditional staging by cytomorphology uncertain, and (iii) using gains of chromosome 7 as a marker of lymphoma cells, FISH is a useful method to detect minimal residual disease in FCLs grade III and DLBCLs.

Promising activity of gemcitabine in refractory high-grade non-Hodgkin's lymphoma.

Three patients (aged 68-75 years) with histologically confirmed relapsed or refractory high-grade non-Hodgkin's lymphoma were entered in this pilot study in which gemcitabine 800 mg/m2 was given as a 30 min i.v. infusion once a week for 3 weeks. One patient responded with complete remission and the other two with partial remission and stable disease for 2 and 3 months, respectively. Haematological toxicity was modest with grade 4 leucopenia (one cycle) and grade 4 thrombocytopenia (two cycles). The activity and mild toxicity seen with gemcitabine suggest that this agent should be further evaluated in the treatment of high-grade non-Hodgkin's lymphoma.

Differences in cell lineage involvement between MDS-AML and de novo AML studied by fluorescence in situ hybridization in combination with morphology.

We have employed fluorescence in situ hybridization (FISH) in combination with standard morphology (MGG/FISH) to identify the clonal involvement of different bone marrow cell lineages in 20 AML patients (14 MDS-AML, 6 de novo AML). Even though the number of cells belonging to the abnormal clone varied between individual cases, the percentage of clonal blasts was similar in MDS-AML and de novo AML patients. The erythropoietic cells appeared to be part of the abnormal clone in 13 of 14 patients with MDS-AML, but only in 1 of 6 with de novo AML. Similarly, clonal granulocytes were detected in 13 of 14 patients with MDS-AML, compared to 2 of 6 with de novo AML. Lymphocytes consistently displayed normal, diploid karyotype. The results suggest that it is possible to distinguish between MDS-AML and de novo AML by the use of MGG/FISH; in de novo AML the abnormal chromosomal clone is generally confined to the immature myeloid cells, while in MDS-AML mature granulocytes and erythroid cells are of clonal origin. It is, however, not possible to conclude that MDS-AML is a "multipotent" type of leukaemia, since it cannot be ruled out that the chromosomally aberrant erythroid cells and granulocytes represent surviving cells from the original MDS clone.

Fluorescence in situ hybridization in combination with morphology detects minimal residual disease in remission and heralds relapse in acute leukaemia.

Fluorescence in situ hybridization in combination with morphology (MGG/FISH) was used to detect minimal residual disease (MRD) in complete remission (CR) in 12 cases of acute leukaemia (six MDS-AML, five de novo AML, one pre-B ALL) with numerical chromosomal aberrations at diagnosis. Residual leukaemic cells could be detected in the remission bone marrows by MGG/FISH in five patients, whereas the other seven showed no abnormalities. All five patients with signs of MRD at CR relapsed in the bone marrow with 2-9 months, in contrast to two of seven with a normal finding by MGG/FISH at CR. In both these patients a second MGG/FISH analysis showed that a subpopulation of leukaemic blasts had reappeared, 4 and 5 months prior to the leukaemia becoming clinically overt. One patient suffered a CNS relapse, but without any evidence of bone marrow involvement. The remaining four patients with no evidence of MRD at CR were still in haematological remission at follow-up after 4, 11, 12 and 13 months, respectively. We conclude that MGG/FISH seems to be a clinically useful method to detect MRD in acute leukaemia and to predict relapses, particularly when repeat studies are performed during CR.

Classical morphology, esterase cytochemistry, and interphase cytogenetics of peripheral blood and bone marrow smears.

We used peripheral blood (PB) and bone marrow (BM) smears in the development of two methods based on cytomorphology and esterase cytochemistry in combination with fluorescence in situ hybridization (FISH). The first method involves photodocumentation of May-Grünewald-Giemsa (MGG)-stained cells, followed by destaining in methanol-acetic acid, fixation in paraformaldehyde, and digestion with protease and RNAse before FISH using alpha-satellite probes that specify chromosomes X, 7, 8, and 17. On average, two hybridization signals were seen in 94.5% of disomic BM cells. The hybridization sensitivity was found to vary, however, both among morphologically defined hematopoietic cell lineages and among differentation levels within a lineage. In the second method, an esterase staining technique was followed by the same treatment as for MGG-stained cells. The esterases and FISH signals could be simultaneously visualized and the method was found suitable for rapid screening of in situ signals in cytochemically defined granulocytes and lymphocytes but not in monocytes. The combined methods proved very useful in elucidating the clinical significance of chromosomal abnormalities seen in two cases of leukemia.

A sequential erythropoietin and GM-CSF schedule offers clinical benefits in the treatment of anaemia in myelodysplastic syndromes.

In order to reduce anaemia in patients with myelodysplastic syndromes (MDS) a stepwise treatment protocol including erythropoietin (EP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was designed. Thirty-seven MDS patients (stages I-III) with symptomatic anaemia were first given EPO 10,000 U s.c. 3 times weekly for 6 weeks. Those not responding, i.e. increased their haemoglobin levels > 15 g/l, proceeded into the second phase of the study where GM-CSF (200 micrograms/d. s.c. on weeks 1-6) was combined with EPO (10,000 U s.c. 3 times weekly on weeks 5-14). Following the initial EPO treatment phase, 14 of the 37 patients (38%) responded with increased haemoglobin levels. Responders were significantly different from non-responders in that their pre-treatment values of s-EPO, s-LDH and bone marrow blast cell counts were lower, their baseline haemoglobin levels higher and their transfusion dependency less pronounced. Eighteen of the 23 non-responders proceeded into the second phase, 13 of those were evaluable having completed the entire schedule. Three of the 13 initially EPO resistant patients (23%) responded to the GM-CSF/EPO combination with increased haemoglobin levels, suggesting a positive synergy between the two cytokines. Thus, the overall response rate to the present protocol was 46% (17 of 37 cases), but only a limited subset of the patients did clearly benefit from the combined GM-CSF/EPO administration. Therefore, we believe this step-wise approach to multiple growth factor treatment in MDS, starting with EPO alone and reserving the combination for refractory cases, has considerable advantages, taking into account both medical and socio-economical aspects.

Clonal cell lineage involvement in myelodysplastic syndromes studied by fluorescence in situ hybridization and morphology.

We have used DNA fluorescence in situ hybridization (FISH) in combination with morphology to study cell lineage involvement in peripheral blood and bone marrow smears from 15 patients with myelodysplastic syndromes (MDS) and known numerical chromosomal aberrations. Eleven cases were investigated at diagnosis of MDS, and four at transformation to acute myeloid leukemia (MDS-AML). Using conventional cytogenetics, monosomy 7 was detected in nine cases, monosomy 17 in two, and trisomy 8 in five, either as a single aberration or as part of a complex clone. One case had both monosomy 7 and 17. Three biotinylated DNA probes directed against the pericentromeric region of chromosomes 7, 8 and 17, respectively, were used. Bone marrow smears were first stained with May-Grünewald-Giemsa (MGG) for morphologic evaluation, and regions of interest documented by photography. Later the same regions were targeted after hybridization, which allowed FISH analysis of selected bone marrow cells. In each patient between 267 and 921 cells (mean 453) were studied. In most cases a majority of the non-lymphoid bone marrow cells appeared to be of clonal origin, ie showed either monosomy or trisomy by FISH, while in contrast both lymphocytes and plasma cells generally were disomic, ie displayed two fluorescent spots for each probe. The percentages of clonal bone marrow cells differed greatly between patients, but in a single case there was a good agreement between the extent of granulocytic, monocytic and erythrocytic cell lineage involvement. In relation to the clinical stage of MDS, patients with more advanced disease had a significantly higher mean percentage of bone marrow blasts showing monosomy or trisomy, while disomic, possibly non-clonal cells were more frequent among the earlier forms of MDS. In the four cases of MDS-AML nearly all non-lymphoid bone marrow cells belonged to the abnormal clone. The FISH technique offers information regarding the distribution of clonal and non-clonal bone marrow cells in MDS, which might have prognostic and possibly, therapeutic implications.

Aplastic anaemia with a trilineage response to erythropoietin therapy.

An 89-year-old man with a severe aplastic anaemia is described. The patient was proven to be cortico-steroid and cyclosporin resistant, but had a trilineage response during subsequent treatment with recombinant human erythropoietin.

Recombinant human granulocyte-macrophage colony-stimulating factor in combination with standard induction chemotherapy in acute myeloid leukemia evolving from myelodysplastic syndromes: a pilot study.

Acute myeloid leukemia preceded by a myelodysplastic syndrome (MDS-AML) is generally regarded as a high-risk type of AML, where remissions are rare and of short duration. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is suggested to increase the sensitivity of leukemic cells to cycle-specific drugs. In this study 14 MDS-AML patients were given rhGM-CSF together with standard induction chemotherapy (TAD). rhGM-CSF was started 48 h prior to chemotherapy and given for up to 3 weeks. The results showed eight (58%) complete and two (14%) partial remissions, while another two (14%) patients had minor responses. One patient relapsed after 1 year, and then responded a second time. rhGM-CSF had to be stopped owing to local allergic reactions in two patients, both non-responders, but was otherwise well tolerated. Compared with our historical group of controls we found significantly higher remission rates, fewer early deaths, fewer fever days, and fewer days with both neutropenia and thrombocytopenia among the patients treated with rhGM-CSF and TAD. The estimated median over-all survival was 332 days. The severity of initial myelodysplastic changes did not correlate to the outcome of therapy but the degree of peripheral blood dysplasia decreased among responding patients. MDS-AML patients in this pilot study did respond better, and with minimal toxicity, when standard induction chemotherapy was given in combination with rhGM-CSF.

Increase of serum interleukin-2 and regression of myeloma after rhGM-CSF treatment of drug induced bone marrow aplasia.

Recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) was administered to a patient with multiple myeloma (IgA, stage IIA) who had a chemotherapy-induced bone marrow aplasia with granulocytopenia complicated by severe pneumonia and septicemia. The rhGM-CSF was given as i.v. infusions, 300-400 micrograms daily, for three weeks. The patient responded both hematologically and clinically with improved granulocyte counts and clearance of massive pulmonary infiltrates. We also observed a partial remission of the myeloma with decreasing s-IgA levels and reduced plasma cell infiltration of the bone marrow during a period of up to four months after the rhGM-CSF treatment. Immunological studies performed during and after cytokine administration showed an increase in serum interleukin-2 (IL-2) levels and HLA-DR positive T-lymphocytes indicating an activation of the immune system. It is suggested that rhGM-CSF induced immunological changes which may have contributed to the partial regression of the myeloma.

Tumor necrosis factor-alpha and interferon-gamma in serum of multiple myeloma patients.

The levels of TNF-alpha and IFN-gamma were examined in serum from 32 patients with multiple myeloma and 33 healthy controls using sensitive enzyme-linked immunosorbent assays (ELISA). The detection limits for TNF-alpha and IFN-gamma were 80 pg/ml and 200 pg/ml, respectively. All samples were obtained at the time of diagnosis, before treatment. In sera from 8 of the myeloma patients the TNF-alpha concentrations were above the detection limit with a maximum value of 1.0 ng/ml. Overall, the TNF-alpha levels of the myeloma patients did not differ from the levels of the control group. Detectable amounts of IFN-gamma were found in 17 of the patient sera with 10.7 ng/ml as the top value. In contrast, the control group showed significantly lower s-IFN-gamma levels without detectable amounts in any of the samples (p less than 0.01). High IFN-alpha levels in 4 patients coincided with intercurrent infections but were not accompanied by a parallel increase of the TNF-alpha levels. The TNF-alpha and IFN-gamma values were compared with the serum levels of beta 2-microglobulin, calcium and creatinine, the M-component, the erythrocyte sedimentation rate, the degree of plasma cell infiltration of the bone marrow, the degree of skeletal destructions and with patients survival. No significant correlations could be observed between TNF-alpha or IFN-gamma and these variables of myeloma activity. We conclude that detection of serum TNF-alpha and IFN-gamma levels in multiple myeloma appears to be without any clinical value.

Cimetidine as an immune response modifier.

Cimetidine, a selective histamine-2 receptor antagonist, has attracted interest because of its potential as an immune response-modifying drug. Most data suggest that cimetidine has a stimulatory action on the immune system, possibly by blocking of receptors on subsets of T-lymphocytes and inhibiting histamine-induced immune suppression. Several studies have shown that cimetidine can affect the relative number of CD8 + ve lymphocytes and increase the NK cell activity as well as the antibody-dependent cellular cytotoxicity. Cimetidine has also been used successfully to restore immune functions in patients with malignant disorders, hypogammaglobulinemia and AIDS-related complexes.