Abstract: 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‐1β, tumour necrosis factor (TNF) α, TNFβ, and transforming growth factor (TGF) β 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‐1β. 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.

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 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.

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 (≥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.

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.

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 < 0.001), and the percentages of anti‐My9‐positive cells in the bone marrow increased with time (p < 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 < 0.001) and abnormal (p = 0.004) metaphases.

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.

British Journal of HaematologyVolume 65, Issue 2 p. 127-129 ENZYME CYTOCHEMISTRY OF NEUTROPHIL GRANULOCYTES Knud Bendix-Hansen, Corresponding Author Knud Bendix-Hansen University Institute of Pathology, and University Department of Medicine and Haematology, Aarhus Amtssygehus.Dr Knud Bendix-Hansen, Universitg Institute of Pathology, and University Department of Medicine and Haematology, Aarhus Amtssygehus, DK-8000 Aarhus C. DenmarkSearch for more papers by this author Knud Bendix-Hansen, Corresponding Author Knud Bendix-Hansen University Institute of Pathology, and University Department of Medicine and Haematology, Aarhus Amtssygehus.Dr Knud Bendix-Hansen, Universitg Institute of Pathology, and University Department of Medicine and Haematology, Aarhus Amtssygehus, DK-8000 Aarhus C. DenmarkSearch for more papers by this author First published: February 1987 https://doi.org/10.1111/j.1365-2141.1987.tb02253.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES Avstrøm. S., Ralfkær, E., Winkel. P. & Nissen. N.I. (1985) The relative merit of various cytochemical quantities and manual differential count in predicting remission following chemotherapy of patients with de novo acute myeloblastic leukaemia. American Journal of Clinical Pathology. 83, 73–76. Baggiolini. M., Horisberger. 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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.

6 patients with primary MDS were treated with 20 mg/m2 13‐cis retinoic acid (13‐RA) (RoacutaneR, Roche, Basel) daily for 6 weeks and with an additional 100 mg/m2 daily for 4 weeks (3 patients). 4 patients responded either with an increase in peripheral blood neutrophil count (2 patients) or with a decrease in MPO‐deficient neutrophils (2 patients). These effects were not seen until 3 weeks after the onset of the treatment and they were accompanied by an increase in the growth of day 11 granulocytemonocyte colonies, and, in the patients showing clonal cytogenetic abnormalities, by a disappearance of minor clonal abnormalities during the treatment. These findings may be the result of a stimulation of the growth of normal or only slightly defective bone marrow progenitor cells in combination with a differentiational effect.The clinical side effects were limited to hyperkeratosis and dryness of mucous membranes in 5 patients. I patient developed a dermatitis. None showed signs of hepatotoxicity. Because of the pronounced subjective discomfort experienced by the patients receiving 100 mg/m2, it seems recommendable to treat patients with MDS with low dosages of 13‐RA (10 to 20 mg/m2) for longer periods (3–6 months) in order to evaluate the effect of 13‐RA on the long term basis.

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.

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.

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.

In 45 cases of primary myelodysplastic syndrome; 16 refractory anaemia (RA), 11 RA with ring sideroblasts (RA+), 13 RA with excess of blasts (RAEB), 5 chronic myelomonocytic leukaemia (CMML), the relations between myeloperoxidase (MPO) activity in polymorphonuclear leucocytes (PMN), neutrophil alkaline phosphatase (NAP) activity, absolute number of PMN and thrombocytopenia were investigated. 11 patients (26%) showed abnormal numbers (greater than 4%) of MPO-deficient PMN and 27 (75%) showed abnormal NAP activity (NAP score; greater than 134.0, less than 15.0), mostly decreased. No significant correlations between MPO activity and NAP activity were demonstrated, nor were any significant correlations found with the other parameters investigated. The FAB-subtypes, RAEB and CMML, showed a significant correlation to thrombocytopenia (p = 0.028) and to pancytopenia (p = 0.024). The findings may support the view that at least some of the myelodysplastic syndromes may be fundamentally the same disease as acute myeloid leukaemia.

Neutrophil alkaline phosphatase (NAP) activity was estimated in 50 healthy humans and 89 patients with leukaemia; 41 cases of acute myeloid leukaemia (AML), 22 cases of chronic myeloid leukaemia (CML), and 26 cases of lymphoid leukaemia (LL). The groups proved to be separate entities (p less than 0.000 25) and the differences between the groups were statistically significant (p less than 0.001) except for the difference between AML and LL. The 95% confidence limits for normal NAP scores were 15.0-132.6. Decreased scores were demonstrated in 73% of CML, 7% of AML but never in LL patients. Increased scores were found in 37% of AML, 31% of LL but never in CML patients. Evaluation of the distribution of the single cell NAP activity (negative, weak positive, strong positive) showed decreased activity in 77% of CML, 15% of AML but never in LL patients. Increased activity was demonstrated in 63% of AML, 54% of LL and 9% of CML patients. The evaluation of single cell activity is a time-saving method, which furthermore proved superior to the scoring method in discriminating between the types of leukaemia investigated.

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 < 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.