Biochemical markers of bone metabolism reflect osteoclastic and osteoblastic activity in multiple myeloma.

In order to evaluate the use of recently developed assays of bone metabolism in multiple myeloma we performed a histomorphometric study of bone biopsies in 16 myeloma patients. Furthermore, we measured the levels of interleukin-6 (IL-6), soluble IL-6 receptor (IL-6sR), IL-1beta, tumour necrosis factor (TNF) alpha, TNFbeta, and transforming growth factor (TGF) beta in marrow plasma aspirated from the biopsy area. MARKERS OF BONE RESORPTION: The N-terminal telopeptide of collagen I (Ntx) in urine showed a strong positive correlation with the dynamic histomorphometric indices of bone resorption (r=0.68-0.72). Slightly weaker correlations were observed between the dynamic indices of bone resorption and the C-terminal telopeptide of collagen I (ICTP) in serum (r= 0.57-0.62) and deoxypyridinoline (Dpyr) in urine (r= 0.54), whereas urinary pyridinoline (Pyr) did not correlate with the histomorphometric findings. MARKERS OF BONE FORMATION: Serum C-terminal propeptide of procollagen I (PICP) and serum bone-specific alkaline phosphatase (bAP) showed significant correlations with the dynamic parameters of bone formation (r=0.57-0.58), whereas serum osteocalcin and serum total AP did not. CYTOKINES: Highly significant correlations were observed between marrow IL-6 and rates of bone resorption and activation frequency (r=0.76-0.82) and with serum ICTP (r=0.63). Minor, but also significant correlations were observed between the resorptive indices and IL-6sR and IL-1beta. The data indicate that measurements of the biochemical markers of bone metabolism may be useful in monitoring myeloma bone disease, and might thus be of use for dose titration of bisphosphonate therapy.

The effect of CVVHD and endotoxin on the oxidative burst, adhesion molecules and distribution in tissues of granulocytes.

OBJECTIVE: Extracorporeal circulation, such as cardiopulmonary bypass and haemodialysis, has been associated with an activation of the immune system, especially the granulocytes. Continuous veno-venous haemodiafiltration (CVVHD) is used in critically ill septic patients. During CVVHD cytokines are excreted in the ultrafiltrate. But when the membranes used in CVVHD are cultured with granulocytes, the granulocytes are slightly activated. This effect is potentiated by endotoxin. We therefore, in vivo, compared the effect on granulocyte activation of CVVHD with an endotoxin group and a control group. METHODS: Thirty-one pigs were anaesthetized and mechanically ventilated. In ten pigs CVVHD was performed. Eleven pigs received an infusion of Escherichia coli endotoxin 30 mu/kg(-1) and ten pigs served as a control group. The adhesion molecules CD18 and CD62L were measured using monoclonal antibodies. The oxidative burst activity was assayed as superoxide dismutase-inhibitory reduction of cytochrome c. The number of granulocytes in peripheral blood and in the lungs and liver were counted. RESULTS: The infusion of endotoxin was followed by granulocytopenia, reduced oxidative burst activity, increased expression of CD18 and decreased expression of CD62L on granulocytes. Accumulation of granulocytes in liver and lung tissue was also noted in this group. CVVHD was only associated with a non-significant decrease in CD62L expression on granulocytes. It did not affect any of the other measured immunological parameters. CONCLUSION: In contrast to endotoxin-induced sepsis, the granulocytes were not activated during CVVHD.

Non-Hodgkin's lymphoma subtypes over time in an unselected population of 646 patients: a study of clinico-pathological data and incidence based on a review using the REAL-classification.

Biopsies from 646 consecutive unselected cases of non-Hodgkin's lymphoma from a Danish population-based registry were reclassified according to the REAL classification 1) to study the distribution of subtypes over time, and 2) to correlate a number of clinical parameters with the various subtypes. Two cohorts from 1986 and 1992, of 292 and 354 cases, respectively, were studied. From 1986 to 1992 diffuse large B-cell lymphoma showed a change in incidence of + 43.1%, as opposed + 2.5% for all other subtypes combined (p = 0.05), suggesting that the increasing general incidence of non-Hodgkin's lymphoma is due primarily to an increasing incidence of diffuse large B-cell lymphoma. A higher rate of cell proliferation was associated with an increasing chance of having extranodal disease. For the various subgroups there was good agreement between survival and the International prognostic index.

Bone marrow fibrosis and disease activity in multiple myeloma monitored by the aminoterminal propeptide of procollagen III in serum.

Simple bone marrow fibrosis is seen in 10-30% of multiple myeloma (MM) patients. We investigated the incidence and characteristics of the bone marrow stromal alterations, in order to characterize the collagens involved by immunohistochemistry, and to evaluate the use of serum aminoterminal propeptide of type III procollagen (PIIINP) as a marker of marrow fibrogenesis and disease activity in MM. 34 consecutive patients with newly diagnosed MM were included prospectively, and followed for 12-30 months. Compared with the findings in 15 normal individuals we found increased interstitial deposits of collagen III in 48% of MM patients, whereas deposits of collagen I were not increased. Interstitial fibrosis appeared to be restricted to areas of severe plasma cell infiltration, but it could also have a more dispersed presentation in the severely infiltrated marrow. There was a high co-distribution of collagen III fibrils and reticulin fibres. Serum PIIINP levels were elevated in most patients, and in the follow-up study serum PIIINP showed a good correlation with the response to treatment. Patients with resistant or progressive disease had continually elevated levels of PIIINP. In most patients with responsive disease serum PIIINP normalized, and we observed no relapses in patients who had normal serum PIIINP levels. Other patients who responded to treatment by reduced M-component level, but had persistently elevated serum levels of PIIINP, had either early relapses or developed progression of osteolytic lesions in spite of unchanged M-component levels. Therefore an elevated serum PIIINP during treatment might indicate an active malignant clone. Serum PIIINP does not simply follow the M-component, but gives further information of potential therapeutic value.

The monosomy 7 clone in interphase and metaphase cell population: a combined chromosome and primed in situ labeling study.

Loss of a chromosome 7 is associated with a poor prognosis in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Some studies have shown higher frequencies of monosomy 7 (-7) in dividing than nondividing myeloid cells, which might indicate that -7 confers a proliferative advantage on the host cell. As other groups have not been able to confirm this, we compared the -7 frequencies in bone marrow metaphases as studied with conventional cytogenetics and in interphase cells using primed in situ (PRINS) labeling. We found significantly higher -7 frequencies in metaphase than in interphase cells irrespective of diagnosis and presence or absence of additional chromosome aberrations. Further, we found a significant correlation between the -7 percentages in resting and dividing cells. Finally, as our material showed a clear male preponderance, Mitelman's Catalog of Chromosome Aberrations in Cancer was searched for -7. Of 815 cases with AML or MDS, 491 (60.3%) were found to be men. To our knowledge, this is the first observation of a clear deviation from the 1:1 sex ratio in -7 patients.

Studies of the initial anticoagulant response in tissue-thromboplastin induced intravascular coagulation.

The very early anticoagulant response was analysed in non-pregnant female New Zealand rabbits infused with rabbit brain tissue thromboplastin for a period of 10 min (n = 6), 20 min (n = 6), and 30 min (n = 6). The rabbits infused with thromboplastin responded with a significant drop in mean arterial pressure (P < 0.05), an increase in blood PaO2 (P < 0.05) and a decrease in PaCO2 (P < 0.05), while control animals remained stable with respect to these variables. The thromboplastin-treated animals had an immediate drop in platelet count (P < 0.05), plasma fibrinogen (P < 0.05) and a prolongation in prothrombin time (P < 0.05) and activated partial thromboplastin time (P < 0.05). The concentrations in a number of proteins involved in the anticoagulant response (antithrombin, plasminogen, antiplasmin) as well as global fibrinolytic activity did not change significantly following 10, 20 and 30 min infusion of thromboplastin, while the concentration of protein C decreased continuously during the infusion periods (P < 0.05) to reach the lowest level (approximately 60%) in animals infused with thromboplastin for 30 min. The animals infused with tissue thromboplastin had microthrombi in 1-6% of the renal glomeruli, but the number of microthrombi did not differ significantly between animals infused for 10, 20 and 30 min. It is concluded that the protein C system may play a key role during the initial phase of intravascular coagulation and immediate activation of protein C may protect against excessive deposition of fibrin.

The effect of iron chelation on haemopoiesis in MDS patients with transfusional iron overload.

Long-term follow-up data are presented on changes in peripheral blood counts and Hb requirements of 11 patients with myelodysplastic syndromes (MDS) during iron chelation treatment with desferrioxamine for up to 60 months. The erythroid marrow activity was indirectly evaluated by repeated determinations of the serum transferrin receptor concentration. The efficacy of iron chelation was evaluated by repeated quantitative determination of the liver iron concentration by magnetic resonance imaging. Reduction in the Hb requirement ( > or = 50%) was seen in 7/11 (64%) patients. Five patients (46%) became blood transfusion independent. Platelet counts increased in 7/11 (64%) patients and the neutrophil counts in 7/9 (78%) evaluable patients. All patients in whom iron chelation was highly effective showed improvement of erythropoietic output accompanied by an increase in the serum transferrin receptor concentration. It is concluded that reduction in cytopenia in MDS patients may be accomplished by treatment with desferrioxamine, if the iron chelation is efficient and the patients are treated for a sufficiently long period of time. Exactly how treatment with desferrioxamine works remains a challenge for further investigation.

Myelodysplastic syndrome in a child with constitutional trisomy 8 mosaicism and normal phenotype.

Trisomy 8 is a frequently acquired cytogenetic abnormality in myeloid malignancies, but may also represent a constitutional chromosome abnormality with a wide phenotypic variation. We report a case of myelodysplastic syndrome (MDS) that developed in a child with trisomy 8 mosaicism and normal phenotype. Bone marrow (BM) cells all showed trisomy 8 with additional clonal abnormalities in most cells. Based on the present case and a review of previously published cases of myeloid malignancies in patients with trisomy 8 mosaicism, it appears likely that the malignant cells developed from the trisomic cell population, suggesting that constitutional trisomy 8 may be a predisposing condition to myeloid malignancies. Trisomy 8 in malignant cells is usually considered an acquired abnormality, but this implies a risk of ignoring a constitutional trisomy 8 mosaicism. Examination for constitutional trisomy 8, despite a normal phenotype, may therefore be warranted in hematologic malignancies with trisomy 8 of BM cells to evaluate further the possible association and to preclude erroneous use of trisomy 8 as a tumor marker.

Myeloperoxidase deficient polymorphonuclear leucocytes in leukaemia and allied disorders.

This thesis is a survey of nine previously published articles on MPO deficient PMN. The incidences in leukaemia and allied disorders of the presence of this abnormal subpopulation of mature neutrophils and the relationship to clinical course in AML, susceptibility to infections in AML, FAB classification in AML and MDS, cytogenetically defined aberrations in MDS and morphometrical characteristics were investigated. The aims of the studies were to examine the diagnostic as well as the prognostic value of the parameter, to examine the usefulness of the parameter as an predictive indicator of CR and relapse in AML and to examine the concept that MPO deficient PMN may originate from leukaemic precursors. MPO deficient PMN were found to occur in a minor number (less than 4% of the total number of PMN) in normal humans and the incidences of an abnormal number (greater than 4%) were found to be about 40% in AML (I, II, III, IV, VIII), 60% in CML (I, VII), 30% in MPD other than CML (VII) and 30% in MDS (V). The highest incidences in AML were found in the FAB subtypes possessing the most myeloid differentiation potential i.e. FAB M2 and FAB M4 (IV). In ALL, CLL, HCL, Hodgkin's disease, anaemia not related to leukaemia and leukaemoid reactions the incidences all were 0% (I, unpublished data). The abnormal MPO deficient PMN subpopulation, if present, disappeared when CR was achieved and reappeared when relapse eventually was developed (II, VIII). In both situations serial determinations showed that the change occurred before the usual routine blood examinations predicted CR and relapse; several days and several months prior, respectively (VIII). The probability of obtaining CR was lower in the AML patients with the abnormal subpopulation and the risk of developing relapse higher than in AML patients without the anomaly (II, VIII). These differences were not statistically significant, however. AML patients, showing an increased number of MPO deficient PMN, revealed a statistically significant increased susceptibility to infections (P less than 0.01) during the preremission phase accounting for 18% to 67% of the total number of infections in this period (III). This increase was positively correlated to the extent of the anomaly (P less than 0.002). The spontaneous occurrence of a subpopulation of MPO deficient PMN in MDS went together with a simultaneous progression in cytogenetically determined clonal chromosomal aberrations and were related to progression in FAB subtype as well (VI). Morphometrically MPO deficient PMN were characterized by a decreased total cell size and an increased nucleus size of the projected images (IX).(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of leucocyte differentiation antigens in blood and bone marrow in patients with refractory anaemia (RA) and RA with sideroblasts. Prognostic implications of sequential and follow-up data.

34 patients with primary myelodysplastic syndrome (MDS), initially diagnosed as subtypes refractory anaemia (RA) and RA with ringed sideroblasts (RA-S), were followed to investigate the distribution of lymphoid and myeloid differentiation antigens in the blood and bone marrow in search of potential prognostic significance with regard to progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia, and for relations to clinical, morphological and cytogenetic findings. Patients who later progressed to RAEB had significantly decreased percentages of anti-T8 positive T-suppressor cells in the blood at diagnosis compared to those who did not (p = 0.05). Sequential analysis showed a decrease with time also in the percentages of anti-T8-positive cells (p = 0.05). In the bone marrow, progressing patients initially showed significantly increased percentages of anti-My9-positive immature myeloid cells (p less than 0.001), and the percentages of anti-My9-positive cells in the bone marrow increased with time (p less than 0.005). Analysis of the pooled data revealed a statistically significant relation between increasing percentages of anti-My9-positive cells and the frequencies of clonally abnormal (p less than 0.001) and abnormal (p = 0.004) metaphases.

Specific minor chromosome deletions in myelodysplastic syndromes: clinical and morphologic correlations.

Thirty-four patients with myelodysplastic syndrome (MDS) subtypes refractory anemia (RA) and RA with ringed sideroblasts (RA-S) (26 and eight patients, respectively) were investigated for clinical and morphologic significance of the most frequently occurring minor chromosome deletions [del(17)(p12), del(8)(p22), del(2)(p24), and del(9)(p22)]. The occurrence of 17p- was statistically significantly related to a low initial bone marrow cellularity (p = 0.01) and severe granulocytopenia (p = 0.05). A diagnosis of RA was seen more frequently among patients with 17p-, also. 17p- alone was not related to progression to RA with an excess of blasts (RAEB). The occurrence of 8p- was statistically significantly related to a higher initial frequency of bone marrow myeloblasts (p = 0.05), but not to bone marrow cellularity or initial granulocytopenia. 8p- alone was not related to initial diagnosis, but a statistically significant relation to progression to RAEB was found (p = 0.05). 2p- was related to progression to RAEB, also (p = 0.02), but not to any of the other investigated parameters. No significant relations between the occurrence of 9p- and other parameters were demonstrated. The simultaneous occurrence of 17p- and 8p- was also statistically significantly related to progression to RAEB (p = 0.02). These relationships suggest that 17p- is involved in the development of bone marrow and peripheral blood granulocytopenia and that 8p-, and possibly 2p-, interferes with differentiation of primitive granulocyte precursors and, thus, play a part in the process leading to RAEB and acute myeloid leukemia.

The prognostic significance of cytological, histological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts. A follow up study.

Two independent observers performed a double review of cytological and histological bone marrow material obtained at diagnosis and during follow up in 34 patients with the myelodysplastic syndrome (MDS), subtypes refractory anaemia (RA) and RA with ringed sideroblasts (RA-S) (26 and 8 patients, respectively). Average values were used for the analyses. Data obtained at diagnosis confirmed earlier observations that a worse prognosis was indicated by high blast cell counts (P less than 0.01), presence of blast foci and clonal cytogenetic abnormalities (P = 0.08). Data obtained during follow up, in addition, showed that an increased probability of progression to FAB-subtype RA with an excess of blasts was related to both the occurrence of blast foci (P less than 0.05) and the occurrence of new or additional clonal abnormalities (karyotype shift) (P less than 0.01). The relationship between parameters investigated at diagnosis, during follow up, and in the pooled material, points to RA-S being a separate entity having a better prognosis than RA, and further substantiates an earlier observed relationship between blast cell accumulation and the frequency of cytogenetically abnormal metaphases.

13-cis retinoic acid treatment of myelodysplastic syndromes.

Of eight patients with primary myelodysplastic syndrome (MDS) treated with Roacutane (13-cis retinoic acid, Roche, Basel, (13-RA)) 20 mg/m2 for 6 weeks and an additional 100 mg/m2 for 4 weeks (3 patients), 4 responded either with a slight increase in peripheral blood neutrophil count or a decrease in myeloperoxidase deficient neutrophils. In agar cultures 2 patients showed a concurrent increase in growth of day 11 colonies and clusters. In 2 of the patients a decrease in the number of immature bone marrow cells positive for the myeloid antibody anti-My7 was observed. Only minor alterations were seen in natural killer cell activity. In 4 patients showing clonal chromosomal abnormalities before treatment a disappearance of minor clonal abnormalities during treatment was observed, and in 3 chromosomal abnormalities reappeared after cessation of therapy. Even though the overall impact of 13-RA on the hematopoietic system was minor, the increase in myeloperoxidase normal granulocytes in the blood and the decrease in My7 positive cells and in clonal chromosomal abnormalities warrants further interest in this agent as a treatment modality in MDS. The side effects, especially experienced by the patients receiving 100 mg/m2, were, in spite of symptomatic treatment, of such a degree that only low dose treatment (10-20 mg/m2) administered for prolonged periods of time (3-6 months) would seem recommendable.

Myeloperoxidase-deficient polymorphonuclear leucocytes. Longitudinal study during the preremission--and the remission phase in acute myeloid leukaemia. Comparison to neutrophil alkaline phosphatase (NAP) activity.

Serial determinations of MPO and NAP activities in granulocytes were performed during the preremission phase and the remission phase in patients with AML. Of 18 patients examined during the preremission period, 9 showed an increased number of MPO deficient PMN. Complete remission was attained in 4 of these, in 3 the number of abnormal granulocytes changed to normal 7, 7 and 14 days before and in 1 simultaneously with the attainment of complete remission. In the other patients no changes in granulocyte MPO activity occurred during the preremission period. All 20 patients examined during complete remission showed a normal MPO activity in granulocytes. Of eight patients, who at diagnosis had shown abnormal granulocyte MPO activity, three developed relapse. In two of these, an increased number of MPO deficient PMN reappeared two and eight months prior to and in one simultaneous with clinical and laboratory suspicion of relapse. A statistically significant relation between low NAP scores and an increased number of MPO deficient PMN was found (P = 0.011). Serial determinations of MPO activities in PMN, although restricted to cases of AML with initially abnormal values, may prove helpful in predicting achievement of complete remission and may furthermore prove to be useful as an indicator of early relapse.

Myeloperoxidase deficient polymorphonuclear leucocytes: computerized planimetric estimations of cellular and nuclear size.

Quantitative estimations of the mean areas of cell, nucleus and cytoplasm in polymorphonuclear leucocytes (PMN) were performed by automated image analysis of blood smears from six patients with acute myeloid leukaemia. The PMN were qualitatively separated by a cytochemical staining method into two well-defined subpopulations i.e. myeloperoxidase (MPO)-normal and MPO-deficient PMN. MPO-deficient PMN were characterized by a decreased size of the total cell (P less than 0.01), an increased size of the nucleus (P less than 0.01) and a decreased size of the cytoplasm (P less than 0.01). The resulting highly increased nucleus-to-cytoplasm ratio in this specific PMN subpopulation bears a striking resemblance to cells in malignant tumours. The planimetric results in this study further support the concept that MPO-deficient PMN may be the progeny of leukaemic precursors.

Myeloperoxidase-deficient polymorphonuclear leucocytes (VI): Relation to cytogenetic abnormalities in primary myelodysplastic syndromes.

Relations between cytogenetic status, FAB-classification and an abnormal subpopulation of myeloperoxidase (MPO)-deficient polymorphonuclears (PMN) in 45 patients with myelodysplastic syndrome (MDS) are reported. Clonal abnormalities were demonstrated in 85% of the patients, with a lower incidence in the RA+ group (refractory anaemia with ring sideroblasts) compared to the others (p = 0.004). In 12 patients a spontaneous progression in cytogenetic aberrations occurred and in 7 of these (60%) a simultaneous progression in FAB-subtype was seen. The appearance of MPO-deficient PMNs was observed in 6 of these patients (55%). A progression in FAB-subtype was noted in further 4 patients and 2 additional patients developed MPO-deficient PMNs. Only one sufficient cytogenetic investigation was available in these patients. Thus 100% of the fully studied patients showed progression in cytogenetic abnormalities when a progression in FAB-subtype or a development of MPO-deficient PMNs was seen. 3 (49%) of the 8 patients developing MPO-deficient PMNs too showed a progression in FAB-subtype. Although no significant correlation to specific categories of structural aberrations or abnormalities in specific chromosome pairs could be demonstrated, clonal cytogenetic aberrations seem to be involved when the disease progresses and when MPO-deficient PMN develop.

Myeloperoxidase-deficient polymorphonuclear leucocytes (VII): Incidence in untreated myeloproliferative disorders.

In 98 patients with chronic myeloproliferative disorders (45 chr. myeloid leukaemia (CML), 19 myelofibrosis primaria (MP), 28 polycythaemia vera (PV) and 6 idiopathic thrombocythaemia (IT)) the incidences of increased numbers of MPO-deficient polymorphonuclear (PMN) were 60% in CML, 32% in MP, 7% in PV and 0% in IT patients. The CML figure differed significantly from the others (p less than 0.001). This study confirms the finding of low NAP scores in CML compared to normal or high NAP scores in the other groups of the myeloproliferative syndrome. The incidences of increased numbers of MPO-deficient PMN in this study are comparable to those found in the primary myelodysplastic syndromes and in acute myeloid leukaemia. The finding supports the view that some of the CML cases and may be other cases of the chronic myeloproliferative disorders may be fundamentally the same disease as in primary myelodysplastic syndromes and in acute myeloid leukaemias.

Myeloperoxidase-deficient polymorphonuclear leucocytes. (IV): Relation to FAB-classification in acute myeloid leukaemia.

In 148 consecutive cases of untreated acute myeloid leukaemia (AML) the relation between an increased number of myeloperoxidase (MPO)-deficient polymorphonuclear leucocytes (PMN) and a classification modified after the proposals outlined by the French-American-British Co-operative Group, the FAB classification, was investigated. In 22 cases with minimal granulocytic component, 8 M5, 13 M6, 1 M7, no one (0%) showed an increased number of MPO-deficient PMN. In 3 (13%) of 23 cases with slight granulocytic component, 3 M0, 20 M1, and in 49 (57%) of 86 cases with granulocytic differentiation (53 M2, 1 M3, 32 M4), an increased number was demonstrated. This difference was statistically significant at a high level (P less than 0.0001). The number of MPO-deficient PMN could not be estimated in 15 cases of AML and 2 additional cases were mixed leukaemias. It is concluded that an increased number of MPO-deficient PMN in acute leukaemia speaks in favour not only of AML, but suggests the diagnosis of subtypes with some granulocytic component, most likely M1, M2, M3 or M4. Furthermore, the results support the concept that in AML at least some of the mature myeloid cells may be involved in the leukaemic process.