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Serum laminin P1 in idiopathic myelofibrosis and related diseases
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Pergamon
Leukemia Research Vol. 18, No. 8, pp. 623-628, 1994
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145-2126/94 $7.00 + 0.00
0145-2126(94)E0044-A
S E R U M L A M I N I N P1 I N I D I O P A T H I C M Y E L O F I B R O S I S RELATED DISEASES
AND
HANS HASSELBALCH* and PETER JUNKERt *Department of Medicine and Haematology L, Herlev University Hospital, Denmark; and tDepartment of Medicine C, Odense University Hospital, Denmark (Received 8 September 1993. Revision accepted 5 March 1994) Abstract--The serum concentration of laminin P1 antigen was determined in 32 patients with various myeloproliferative disorders, including 19 patients with idiopathic myelofibrosis. The serum concentration of the aminoterminal propeptide of type III procollagen (PIIINP) was measured concomitantly in 27 patients. Serial laminin measurements were carried out in 25 patients. The median serum laminin concentration in patients with acute disease, i.e. acute myelofibrosis and patients in a transforming disease phase was significantly higher (1.58 U/ml; range 1.15-2.07) as compared with patients with chronic disease (1.02 U/ml; range 0.75-1.76; p = 0.012) and healthy control subjects (1.13 U/ml; range 0.75-1.67; p = 0.00015). In individual patients serum laminin covariated closely with serum PIIINP, the leucocyte count and LDH. The median serum laminin concentration in patients with a huge spleen was significantly lower than in the patient group with a normal spleen size/previous splenectomy. Pronounced splenomegaly was particularly prevalent in patients with chronic disease implicating splenic enlargement as a significant extrahepatic site of laminin uptake/degradation in myeloproliferative disorders.
Basement membranes are subject to metabolic turnover leading to release of antigens related to, for example, type IV collagen and laminin into the circulation. Increased serum levels of laminin P1 fragment have been found in various conditions with active tissue remodelling, such as late in normal pregnancy [10], alcoholic liver disease with cirrhosis [11=13], diabetes with microangiopathy [14, 15], progressive systemic sclerosis [16] and various malignancies [17, 18]. We and others have recently shown that the myeloproliferative process is reflected in the systemic circulation by increased levels of type III and type IV collagen-related antigens [19-24]. The aims of the present study were: (1) to study the serum concentration of laminin in the myelofibrosis/osteomyelosclerosis syndrome and other myeloproliferative disorders; (2) to investigate: the relationship between serum laminin and conventional markers of disease activity in IMF; (3) to investigate t h e relationship between interstitial collagen and basement membrane metabolism in IMF, as assessed by concomitant measurements
Introduction IDIOPATHIC MYELOFIBROSIS (IMF) is a myeloproliferative disorder which in its classical form is featured by leucoerythroblastic anaemia, splenomegaly and myelofibrosis with large numbers of megakaryocytes [1, 2]. Immunohistochemical studies have shown that the fibrous deposits consist of excessive amounts of interstitial collagens as well as basement membrane material (type IV collagen and laminin) [3,4]. Bone marrow fibrogenesis is accompanied by marked endothelial proliferation [3-5]. Laminin is a high molecular weight cross-shaped glycoprotein which occurs in lamina rara of all basement membranes [6-8]. It serves structural as well as important physiological and pathophysiological purposes, for example, as an anchoring site for cell attachment, in cell growth and development and metastatic spread of malignant tumours [9]. Correspondence to: Hans Hasselbalch, Department of Medicine B, Frederiksberg Hospital, 2000 Frederiksberg, Denmark. 623
624
H. HASSELBALCH and P. JUNKER TABLE 1. CLINICAL AND HAEMATOLOGICAL DATA AND CORRESPONDING SERUM LAMININ AND SERUM PIIINP VALUES IN PATIENTS WITH IDIOPATHIC MYELOFIBROSIS AND RELATED DISEASES
Patient
Sex
Age
Diagnosis
Laminin U/ml
PIIINP ng/ml
LEU MIA/1
LDH U/1
Spleen size*
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
M M F F M F F F F M M F M M F F F F F F M F F M F F F F F F F M
65 62 65 47 74 73 85 65 77 63 63 72 60 77 65 42 28 57 48 55 54 41 53 68 60 64 63 68 46 65 58 41
OMS OMS OMS OMS OMS OMS OMS IMF IMF IMF IMF IMF IMF IMF IMF IMF AMF AMF AMF TMD TMD TMD TMD PV PV CML CML CML CML CML CML CML
0.75 1.02 1.09 1.20 1.14 1.40 1.18 1.60 0.92 0.82 2.07 1.29 1.36 0.89 1.76 0.90 1.15 1.58 1.88 1.17 0.90 2.03 1.13 1.03 1.05 1.50 1.56 1.15 1.53 1.74 1.01 1.49
9.6 6.5 ND 9.6 10.4 11.1 7.9 14.0 6.9 ND 9.9 4.0 11.6 ND 2.7 7.5 7.9 8.3 11.2 6.4 ND 4.1 4.6 4.4 4.0 10.4 7.8 4.4 ND 8.0 9.0 8.1
11.4 12.3 7.9 25.1 22.8 23.0 41.0 1.2 36.5 35.8 99.0 18.1 3.1 4.1 3.7 5.0 14.3 1.9 2.7 55.0 15.5 11.4 73.0 13.1 34.5 104.0 24.9 7.9 3.0 63.6 326.0 126.0
1070 ND 2177 3320 1479 1500 525 1349 1393 ND ND 596 494 686 330 837 995 ND 1268 ND 712 552 569 ND ND ND 594 ND 238 961 2295 926
3+ 3+ 3+ 3+ 3+ 3+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ N N N N N S 3+ S S N 2+ 2+ S N N 2+ 3+ 1+
Abbreviations: PIIINP, aminoterminal propeptide of type III procollagen; L E U , leucocyte count; L D H lactic dehydrogenase; spleen size*, the spleen size was graded according to the following grading scale: normal = not enlarged on 99 mTc-scintigraphy or ultrasound investigation; 1 + = not enlarged on clinical examination but enlarged on scintigraphy or ultrasound; 2 + / 3 + = lower pole above/below the umbilical level on clinical examination; S = splenectomy. of serum concentrations of the N-terminal propeptide o f t y p e I I I p r o c o l l a g e n ( P I I I N P ) a n d l a m i n i n P1, respectively; and (4) to i n v e s t i g a t e t h e i n f l u e n c e o f c y t o t o x i c t r e a t ment on serum laminin.
Materials and Methods Patients Thirty-two patients (11 males and 21 females; age range 28-85 years) with myeloproliferative disorders were investigated, including 19 patients with idiopathic myelofibrosis, four with a transitional myeloproliferative disorder, two patients with polycythaemia vera (PV) and seven patients with chronic myelogenous leukaemia (CML). Patients with the myelofibrosis/osteomyelosclerosis syndrome (n = 19) were subdivided according to the following criteria for disease activity, which have also been used in
previous studies [24, 28]. Acute disease included patients with a syndrome of acute myelofibrosis (AMF) or those in a transforming/blast phase of the disease. Patients without A M F or signs of transformation were considered to have chronic disease. Chronic active disease was defned by the presence of severe constitutional symptoms and/or a demand for blood transfusions at least every second month and/or a rapidly enlarging spleen or rising leucocyte/ platelet counts. Chronic stable disease was defined by the absence of any of the above symptoms and signs of disease activity. When first investigated, seven patients had acute disease. Three of these patients had a syndrome of acute myelofibrosis and the rest were initially studied during the acute transformation from a chronic disease phase. Twelve patients had chronic disease and seven of these latter patients fulfilled the above criteria for chronic active disease. The remaining five patients had chronic stable disease at first investigation. Serial measurements of serum laminin were performed in 25 patients, including 16 patients with the myelofibrosis/osteomyelosclerosis syndrome. Of the latter, one had chronic stable disease. Two
Serum laminin P1 in idiopathic myelofibrosis patients (one OMS, one IMF) had chronic stable disease which transformed into a chronic active disease phase. Three patients (two OMS, one IMF) transformed into an accelerating disease phase followed by terminal blastic crisis. Five had chronic active disease, and one of these terminally entered an accelerating disease phase. Five patients had acute disease from the onset. Twenty-three patients had received cytotoxic agents within 3 months from the day of blood sampling for laminin analysis. Four patients were treated with prednisolone. Measurement of serum laminin was performed in seven patients before and during/after cytotoxic treatment. Four patients received hydroxyurea, one patient busulphan and two patients intensive chemotherapy, both being followed during two treatment courses. Serum laminin and serum PIIINP were measured concomitantly in 27 patients. Control sera for analysis of laminin and PIIINP were obtained from healthy Danish blood donors (n = 39 and 35, respectively).
Methods Laminin P1 was measured using a double antibody RIA based on human laminin antigen and Fab fragments of anti-human laminin P1 antibodies (Hoechst, Behringwerke) [16]. Briefly, patient serum and anti-laminin-P1 antibody were mixed followed by addition of 1-125 labelled laminin P1. After incubation, bound radioactivity was precipitated by a second antibody (goat anti-rabbit IgG) and measured in a gamma counter. The serum concentration of laminin was expressed in arbitrary units/ml, where one unit (U) = the concentration of laminin antigen/ml in a pool of normal sera. The intra-assay coefficient of variation was 3%. The serum concentration of the aminoterminal propeptide of type III procollagen (PIIINP) was analysed by a RIA based on human propeptide as previously described [25]. This assay detects preferentially the authentic propeptide or larger antigen forms. Statistical analysis Statistical comparisons were made by the Mann-Whitney test. Wilcoxon's test for pair differences was used to evaluate differences between serum laminin before and after cytotoxic treatment in individual patients.
Results Serum laminin in healthy subjects ranged from 0.75 to 1.67 U / m l (median 1.13 U/ml). The median serum laminin concentration in the total number of patients under study was 1.18 U / m l (range 0.752.07). Serum laminin exceeded the normal range in only five patients (three IMF; one T M D ; one CML). However, the median serum laminin concentration in myelofibrosis (MF) patients with acute disease (n = 7) was significantly higher than in the healthy control group (1.58 U/ml; (range 1.15-2.07)vs 1.13 U / m l (range 0.75-1.67;) p = 0.00015). The serum laminin concentration in MF-patients with acute disease (n = 7) was also significantly higher than in those with chronic disease (n = 11) (median 1.02
625
U / m l ; range 0.75-1.76; p = 0.012). No significant difference in serum laminin was found between MF patients with chronic stable disease (n = 5) as compared with chronic active disease (n = 7) [28]. Although serum laminin was within the normal range in the majority of patients, the serum values were found to covariate closely with the serum concentration of PIIINP, the leucocyte count and plasma lactic dehydrogenase in individual patients who were followed by serial measurements (n = 25). Figures 1 and 2 illustrate the covariation between the above parameters in two representative patients. When assessed for the whole series of patients no significant correlation was recorded between serum laminin and serum PIIINP (p = 0.24), between serum laminin and plasma lactic dehydrogenase ( p = 0 . 1 9 ) or between serum laminin and the leucocyte count (p = 0.56). The median serum laminin value in patients with a huge spleen (3+) was significantly lower than in the patient group with a normal spleen size/ previous splenectomy (1.03 U / m l (range 0.75-1.40) vs 1.35 (range 0.90-2.03); p = 0.027). Cytotoxic treatment was associated with declining serum laminin concentrations, which was particularly evident in those receiving intensive c h e m o t h e r a p y (n = 2) (Figs 1, 2). In individual patients (n = 7) the serum laminin concentration before cytotoxic treatment (median = 1.35; range 0.9-1.7) was significantly higher than after c h e m o t h e r a p y (median = 0.99; range 0.7-1.3; p = 0.03). Discussion The present study has shown that the serum concentration of laminin is within the normal range in most patients with IMF and related disorders. However, a comparison of patients with acute vs chronic disease showed that serum laminin was higher in the former subset. M o r e o v e r , a close covariation was found between the serum laminin concentration and serum PIIINP and between serum laminin and conventional markers of disease activity, including the leucocyte count and plasma L D H . This association was most evident in patients with blastic transformation, in whom intensive c h e m o t h e r a p y was followed by declining serum antigen levels, whereas rising serum laminin values were found in those with relapsing disease. Several issues should be addressed in the interpretation of these results. First, circulating laminin is heterogeneous consisting of intact as well as various proteinase-resistant fragments. The relationship of these various antigen forms and laminin metabolism still remains to be determined. It has been demonstrated recently, however, that two different elastase
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Serum lamininP1 in idiopathicmyelofibrosis resistant fragments are taken up/degraded by liver endothelial and liver parenchymal cells, respectively. Judged by conventional biochemical markers severe liver dysfunction was not prevalent in the patients under study. However, specific indicators of, for example, Kuppfer and endothelial cell function are not available. Small amounts of laminin are also taken up by the spleen [26]. It follows that the serum concentration of laminin-related antigen depends not only on the amount of laminin protein released to the circulation but also on the extent of uptake and degradation, particularly in the liver. The present finding of an inverse relationship between S-laminin and spleen size suggests an altered function of the enlarged spleen with respect to laminin uptake and degradation. A similar inverse relationship between plasma fibronectin and spleen size has been reported earlier [27]. It is not clear whether the putative splenic uptake of laminin and fibronectin is due to non-specific trapping in th~ enlarged spleen or to a receptor mechanism by endothelial cells [27]. The previously reported low hyaluronan concentrations in splenomegalic patients may reflect a similar mechanism [28]. Although the serum laminin values were found to covariate closely with the serum concentration of PIIINP, the leucocyte count and plasma lactic dehydrogenase in individual patients, following serial measurements no significant correlation existed between serum laminin and the above parameters when assessed for the whole series of patients. This discrepancy may be explained by the lower serum laminin levels in patients with large spleens. With the reservations given above the higher serum laminin levels in patients with active disease most reasonably reflects ongoing basement membrane neoformation. This notion is further supported by our previous findings of elevated serum concentration of type IV collagen antigen in a high proportion of MF-patients and a highly significant correlation between the serum concentrations of type IV collagen antigen (7-S collagen) and PIIINP [22]. Furthermore, our observation of a close covariation between serum laminin and serum PIIINP is in accordance with the current hypothesis for bone marrow fibrogenesis and angiogenesis in idiopathic myelofibrosis and related diseases, implying a common stimulus for both processes by the intramedullary release of growth factors from megakaryocytes (for example, platelet-derived growth factor and transforming growth factor-beta) [29-32]. In conclusion, our observations support the concept that the hyperactive myeloproliferation in IMF and related diseases is accompanied by synchronous changes in the metabolism of laminin and type III collagen.
References 1. Ward H. P. & Block M. H. (1971) The natural history of agnogenic myeloid metaplasia (AMM) and a critical evaluation of its relationship with the myeloproliferative syndrome. Medicine 50, 357. 2. Burkhardt R., Bartl Beil E., Demmler K., Hoffman E., Kronseder A., Irrgang U., Ulrich M., Wiemann H., Langecker H. & Saar U. (1974) Myelofibrosisosteomyelosclerosis syndrome. Review of literature and histomorphology. In Advances in the Biosciences-Dahlem Workshop on Myelofibrosis-Osteomyelosclerosis Syndrome (Burkhardt R., Conley C. L.,
Lennert K., Adler S. S., Pincus T. & Till J. E., Eds), Vol. 16, pp. 9-56, Berlin, 13-15 November. Pergamon Press, Vieweg. 3. Apaja-SarkkinenM., Autio-Harmainen H., Alavaikko M., Risteli J. & Risteli L. (1986) Immunohistochemical study of basement membrane proteins and type III procollagen in myelofibrosis. Br. J. Haemat. 63, 571. 4. Lisse I., Hasselbalch H. & Junker P. (1991) Immunohistochemical study of bone marrow collagen in idiopathic myelofibrosis and related diseases. A P M I S 99, 171. 5. Reilly J. T., Nash J. R. G., Mackie M. J. & McVerry B. A. (1985) Endothelial proliferation in myelofibrosis. Br. J. Haemat. 60, 625. 6. Stanley J. R., Woodley D. T., Katz S. I. & Martin G. R. (1982) Structure and function of basement membrane. J. invest. Dermat. 79, 69. 7. Martin G. R. & Timpl R. (1987) Laminin and other basement membrane components. A. Rev. Cell Biol. 3, 57. 8. Paulsson M. (1987) Noncollagenous proteins of basement membranes. Collagen Relat. Res. 7, 443. 9. Beck K., Hunter I. & Engel J. (1990) Structure and function of laminin: anatomy of a multidomain glycoprotein. F A S E B J. 4, 148. 10. Wurz H. & Crombach G. (1988) Radioimmunoassay of laminin P1 in body fluids of pregnant women, patients with gynaecological cancers and controls. Tumor Biol. 9, 37. 11. Niemela O., Risteli L., Sotaniemi E. A. & Risteli J. (1985) Type IV collagen and laminin-related antigens in human serum in alcoholic liver disease. Eur. J. clin. lnvest. 15, 132. 12. Nouchi T., Worner T. M., Sato S. & Leiber C. S. (1987) Serum procollagen type III N-terminal peptides and laminin P1 peptide in alcoholic liver diseases Alcohol Clin. Exp. Res. 11, 287. 13. Kropf J., Gressner A. M. & Negwer A. (1988) Efficacy of serum laminin measurement for diagnosis of fibrotic liver diseases. Clin. Chem. 34, 2026. 14. Bogemann B., Balleisen L., Rauterberg J., Voss B. & Gerlach U. (1986) Basement membrane components (7S collagen, laminin P1) are increased in sera of diabetics and activate platelets in vitro. Haemostasis 16, 428. 15. Pietschmann P., Schernthaner G., Schnack C. H. & Gaube S. (1988) Serum concentrations of laminin P1 in diabetics with advanced nephropathy. J. clin. Path. 41, 929. 16. Gerstmeier H., Gabrielli A., Meurer M., Brocks D., Braun-Falco O. & Krieg T. (1988) Levels of type IV collagen and laminin fragments in serum from patients with progressive systemic sclerosis. J. Rheumat. 15, 969.
628
H. HASSELBALCHand P. JUNKER
17. Brocks D. G., Strecker H., Neubauer H. P. et al. (1986) Radioimmunoassay of laminin in serum and its application to cancer patients. Clin. Chem. 32, 787. 18. Rochlitz C., Hasslacher C., Brocks D. G. & Herrmann R. (1987) Serum concentration of laminin, and course of the disease in patients with various malignancies. J. clin. Oncol. 5, 1424. 19. Hochweiss S., Fruchtman S., Hahn E. G., Gilbert H, Donovan P. B., Johnson J., Goldberg J. D. & Berk P. D. (1983) Increased serum procollagen III aminoterminal peptide in myelofibrosis. Am. J. Hemat. 15, 343. 20. Arrago J. P., Poirier O., Chomienne C., D'agay M. F. & Najean Y. (1986) Type III aminoterminal propeptide of procollagen in some haematological malignancies. Scand. J. Haemat. 36, 288. 21. Hasselbalch H., Junker P., Lisse I. & Bentsen K. D. (1985) Serum procollagen III peptide in chronic myeloproliferative disorders. Scand. J. Haemat. 35, 550. 22. Hasselbalch H., Junker P., Lisse I., Bentsen K. D., Risteli L. & Risteli J. (1986) Serum markers for type IV collagen and type III procollagen in the myelofibrosisosteomyelosclerosis syndrome and other chronic myeloproliferative disorders. Am. J. Hemat. 23, 101. 23. Barosi G., Costa A., Liberato N., Polino G., Spriano P. & Magrini U. (1989) Serum procollagen-III-peptide level correlates with disease activity in myelofibrosis with myeloid metaplasia. Br. J. Haemat. 72, 16. 24. Hasselbalch H., Junker P., HCrslev-Petersen K., Lisse I. & Bentsen K. D. (1990) Procollagen type III aminoterminal peptide in serum in idiopathic myelofibrosis
and allied conditions: relation to disease activity and effect of chemotherapy. Am. J. Hemat. 33, 18. 25. Risteli J., Niemi S., Trivedi P., Maentausta O., Mowat A. P. & Risteli L. (1988) Rapid equilibrium radioimmunoassay for the aminoterminal propeptide of human type III procollagen. Clin. Chem. 34, 715. 26. SmedsrOd B., Paulsson M. & Johansson S. (1989) Uptake and degradation in vivo and in vitro of laminin and nidogen by rat liver cells. Biochem. J. 261, 37. 27. Hasselbalch H. & Clemmensen I. (1987) Plasma fibronectin in idiopathic myelofibrosis and related chronic myeloproliferative disorders. Scand. J. clin. Lab. Invest. 47, 429. 28. Hasselbalch H., Junker P., Lisse I., Lindquist U. & Engstr0m-Laurent A. (1991) Circulating hyaluronan in the myelofibrosis-osteomyelosclerosis syndrome and other myeloproliferative disorders. Am. J. Hemat. 36, 1. 29. Groopman J. E. (1980) The pathogenesis of myelofibrosis in myeloproliferative disorders. Ann. Intern. Med. 92, 857. 30. Hasselbalch H. (1990) On the pathogenesis of angiogenesis in idiopathic myelofibrosis. A m . J. Hemat. 33, 151. 31. Dolan G., Forrest P., Eastham J., Smith A. & Reilly J. T. (1991) Serum laminin, procollagen terminal peptide III and thrombocyte platelet derived growth factor concentrations in idiopathic myelofibrosis. (Abstr.) Br. J. Haemat. 77, Suppl. 1, 73. 32. Reilly J. T., Barnett D., Dolan G., Forrest P., Eastham J. & Smith A. (1993) Characterization of an acute micromegakaryocytic leukaemia: evidence for the pathogenesis of myelofibrosis. Br. J. Haemat. 83, 58.
Pergamon
Leukemia Research Vol. 18, No. 8, pp. 623-628, 1994
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145-2126/94 $7.00 + 0.00
0145-2126(94)E0044-A
S E R U M L A M I N I N P1 I N I D I O P A T H I C M Y E L O F I B R O S I S RELATED DISEASES
AND
HANS HASSELBALCH* and PETER JUNKERt *Department of Medicine and Haematology L, Herlev University Hospital, Denmark; and tDepartment of Medicine C, Odense University Hospital, Denmark (Received 8 September 1993. Revision accepted 5 March 1994) Abstract--The serum concentration of laminin P1 antigen was determined in 32 patients with various myeloproliferative disorders, including 19 patients with idiopathic myelofibrosis. The serum concentration of the aminoterminal propeptide of type III procollagen (PIIINP) was measured concomitantly in 27 patients. Serial laminin measurements were carried out in 25 patients. The median serum laminin concentration in patients with acute disease, i.e. acute myelofibrosis and patients in a transforming disease phase was significantly higher (1.58 U/ml; range 1.15-2.07) as compared with patients with chronic disease (1.02 U/ml; range 0.75-1.76; p = 0.012) and healthy control subjects (1.13 U/ml; range 0.75-1.67; p = 0.00015). In individual patients serum laminin covariated closely with serum PIIINP, the leucocyte count and LDH. The median serum laminin concentration in patients with a huge spleen was significantly lower than in the patient group with a normal spleen size/previous splenectomy. Pronounced splenomegaly was particularly prevalent in patients with chronic disease implicating splenic enlargement as a significant extrahepatic site of laminin uptake/degradation in myeloproliferative disorders.
Basement membranes are subject to metabolic turnover leading to release of antigens related to, for example, type IV collagen and laminin into the circulation. Increased serum levels of laminin P1 fragment have been found in various conditions with active tissue remodelling, such as late in normal pregnancy [10], alcoholic liver disease with cirrhosis [11=13], diabetes with microangiopathy [14, 15], progressive systemic sclerosis [16] and various malignancies [17, 18]. We and others have recently shown that the myeloproliferative process is reflected in the systemic circulation by increased levels of type III and type IV collagen-related antigens [19-24]. The aims of the present study were: (1) to study the serum concentration of laminin in the myelofibrosis/osteomyelosclerosis syndrome and other myeloproliferative disorders; (2) to investigate: the relationship between serum laminin and conventional markers of disease activity in IMF; (3) to investigate t h e relationship between interstitial collagen and basement membrane metabolism in IMF, as assessed by concomitant measurements
Introduction IDIOPATHIC MYELOFIBROSIS (IMF) is a myeloproliferative disorder which in its classical form is featured by leucoerythroblastic anaemia, splenomegaly and myelofibrosis with large numbers of megakaryocytes [1, 2]. Immunohistochemical studies have shown that the fibrous deposits consist of excessive amounts of interstitial collagens as well as basement membrane material (type IV collagen and laminin) [3,4]. Bone marrow fibrogenesis is accompanied by marked endothelial proliferation [3-5]. Laminin is a high molecular weight cross-shaped glycoprotein which occurs in lamina rara of all basement membranes [6-8]. It serves structural as well as important physiological and pathophysiological purposes, for example, as an anchoring site for cell attachment, in cell growth and development and metastatic spread of malignant tumours [9]. Correspondence to: Hans Hasselbalch, Department of Medicine B, Frederiksberg Hospital, 2000 Frederiksberg, Denmark. 623
624
H. HASSELBALCH and P. JUNKER TABLE 1. CLINICAL AND HAEMATOLOGICAL DATA AND CORRESPONDING SERUM LAMININ AND SERUM PIIINP VALUES IN PATIENTS WITH IDIOPATHIC MYELOFIBROSIS AND RELATED DISEASES
Patient
Sex
Age
Diagnosis
Laminin U/ml
PIIINP ng/ml
LEU MIA/1
LDH U/1
Spleen size*
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
M M F F M F F F F M M F M M F F F F F F M F F M F F F F F F F M
65 62 65 47 74 73 85 65 77 63 63 72 60 77 65 42 28 57 48 55 54 41 53 68 60 64 63 68 46 65 58 41
OMS OMS OMS OMS OMS OMS OMS IMF IMF IMF IMF IMF IMF IMF IMF IMF AMF AMF AMF TMD TMD TMD TMD PV PV CML CML CML CML CML CML CML
0.75 1.02 1.09 1.20 1.14 1.40 1.18 1.60 0.92 0.82 2.07 1.29 1.36 0.89 1.76 0.90 1.15 1.58 1.88 1.17 0.90 2.03 1.13 1.03 1.05 1.50 1.56 1.15 1.53 1.74 1.01 1.49
9.6 6.5 ND 9.6 10.4 11.1 7.9 14.0 6.9 ND 9.9 4.0 11.6 ND 2.7 7.5 7.9 8.3 11.2 6.4 ND 4.1 4.6 4.4 4.0 10.4 7.8 4.4 ND 8.0 9.0 8.1
11.4 12.3 7.9 25.1 22.8 23.0 41.0 1.2 36.5 35.8 99.0 18.1 3.1 4.1 3.7 5.0 14.3 1.9 2.7 55.0 15.5 11.4 73.0 13.1 34.5 104.0 24.9 7.9 3.0 63.6 326.0 126.0
1070 ND 2177 3320 1479 1500 525 1349 1393 ND ND 596 494 686 330 837 995 ND 1268 ND 712 552 569 ND ND ND 594 ND 238 961 2295 926
3+ 3+ 3+ 3+ 3+ 3+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ N N N N N S 3+ S S N 2+ 2+ S N N 2+ 3+ 1+
Abbreviations: PIIINP, aminoterminal propeptide of type III procollagen; L E U , leucocyte count; L D H lactic dehydrogenase; spleen size*, the spleen size was graded according to the following grading scale: normal = not enlarged on 99 mTc-scintigraphy or ultrasound investigation; 1 + = not enlarged on clinical examination but enlarged on scintigraphy or ultrasound; 2 + / 3 + = lower pole above/below the umbilical level on clinical examination; S = splenectomy. of serum concentrations of the N-terminal propeptide o f t y p e I I I p r o c o l l a g e n ( P I I I N P ) a n d l a m i n i n P1, respectively; and (4) to i n v e s t i g a t e t h e i n f l u e n c e o f c y t o t o x i c t r e a t ment on serum laminin.
Materials and Methods Patients Thirty-two patients (11 males and 21 females; age range 28-85 years) with myeloproliferative disorders were investigated, including 19 patients with idiopathic myelofibrosis, four with a transitional myeloproliferative disorder, two patients with polycythaemia vera (PV) and seven patients with chronic myelogenous leukaemia (CML). Patients with the myelofibrosis/osteomyelosclerosis syndrome (n = 19) were subdivided according to the following criteria for disease activity, which have also been used in
previous studies [24, 28]. Acute disease included patients with a syndrome of acute myelofibrosis (AMF) or those in a transforming/blast phase of the disease. Patients without A M F or signs of transformation were considered to have chronic disease. Chronic active disease was defned by the presence of severe constitutional symptoms and/or a demand for blood transfusions at least every second month and/or a rapidly enlarging spleen or rising leucocyte/ platelet counts. Chronic stable disease was defined by the absence of any of the above symptoms and signs of disease activity. When first investigated, seven patients had acute disease. Three of these patients had a syndrome of acute myelofibrosis and the rest were initially studied during the acute transformation from a chronic disease phase. Twelve patients had chronic disease and seven of these latter patients fulfilled the above criteria for chronic active disease. The remaining five patients had chronic stable disease at first investigation. Serial measurements of serum laminin were performed in 25 patients, including 16 patients with the myelofibrosis/osteomyelosclerosis syndrome. Of the latter, one had chronic stable disease. Two
Serum laminin P1 in idiopathic myelofibrosis patients (one OMS, one IMF) had chronic stable disease which transformed into a chronic active disease phase. Three patients (two OMS, one IMF) transformed into an accelerating disease phase followed by terminal blastic crisis. Five had chronic active disease, and one of these terminally entered an accelerating disease phase. Five patients had acute disease from the onset. Twenty-three patients had received cytotoxic agents within 3 months from the day of blood sampling for laminin analysis. Four patients were treated with prednisolone. Measurement of serum laminin was performed in seven patients before and during/after cytotoxic treatment. Four patients received hydroxyurea, one patient busulphan and two patients intensive chemotherapy, both being followed during two treatment courses. Serum laminin and serum PIIINP were measured concomitantly in 27 patients. Control sera for analysis of laminin and PIIINP were obtained from healthy Danish blood donors (n = 39 and 35, respectively).
Methods Laminin P1 was measured using a double antibody RIA based on human laminin antigen and Fab fragments of anti-human laminin P1 antibodies (Hoechst, Behringwerke) [16]. Briefly, patient serum and anti-laminin-P1 antibody were mixed followed by addition of 1-125 labelled laminin P1. After incubation, bound radioactivity was precipitated by a second antibody (goat anti-rabbit IgG) and measured in a gamma counter. The serum concentration of laminin was expressed in arbitrary units/ml, where one unit (U) = the concentration of laminin antigen/ml in a pool of normal sera. The intra-assay coefficient of variation was 3%. The serum concentration of the aminoterminal propeptide of type III procollagen (PIIINP) was analysed by a RIA based on human propeptide as previously described [25]. This assay detects preferentially the authentic propeptide or larger antigen forms. Statistical analysis Statistical comparisons were made by the Mann-Whitney test. Wilcoxon's test for pair differences was used to evaluate differences between serum laminin before and after cytotoxic treatment in individual patients.
Results Serum laminin in healthy subjects ranged from 0.75 to 1.67 U / m l (median 1.13 U/ml). The median serum laminin concentration in the total number of patients under study was 1.18 U / m l (range 0.752.07). Serum laminin exceeded the normal range in only five patients (three IMF; one T M D ; one CML). However, the median serum laminin concentration in myelofibrosis (MF) patients with acute disease (n = 7) was significantly higher than in the healthy control group (1.58 U/ml; (range 1.15-2.07)vs 1.13 U / m l (range 0.75-1.67;) p = 0.00015). The serum laminin concentration in MF-patients with acute disease (n = 7) was also significantly higher than in those with chronic disease (n = 11) (median 1.02
625
U / m l ; range 0.75-1.76; p = 0.012). No significant difference in serum laminin was found between MF patients with chronic stable disease (n = 5) as compared with chronic active disease (n = 7) [28]. Although serum laminin was within the normal range in the majority of patients, the serum values were found to covariate closely with the serum concentration of PIIINP, the leucocyte count and plasma lactic dehydrogenase in individual patients who were followed by serial measurements (n = 25). Figures 1 and 2 illustrate the covariation between the above parameters in two representative patients. When assessed for the whole series of patients no significant correlation was recorded between serum laminin and serum PIIINP (p = 0.24), between serum laminin and plasma lactic dehydrogenase ( p = 0 . 1 9 ) or between serum laminin and the leucocyte count (p = 0.56). The median serum laminin value in patients with a huge spleen (3+) was significantly lower than in the patient group with a normal spleen size/ previous splenectomy (1.03 U / m l (range 0.75-1.40) vs 1.35 (range 0.90-2.03); p = 0.027). Cytotoxic treatment was associated with declining serum laminin concentrations, which was particularly evident in those receiving intensive c h e m o t h e r a p y (n = 2) (Figs 1, 2). In individual patients (n = 7) the serum laminin concentration before cytotoxic treatment (median = 1.35; range 0.9-1.7) was significantly higher than after c h e m o t h e r a p y (median = 0.99; range 0.7-1.3; p = 0.03). Discussion The present study has shown that the serum concentration of laminin is within the normal range in most patients with IMF and related disorders. However, a comparison of patients with acute vs chronic disease showed that serum laminin was higher in the former subset. M o r e o v e r , a close covariation was found between the serum laminin concentration and serum PIIINP and between serum laminin and conventional markers of disease activity, including the leucocyte count and plasma L D H . This association was most evident in patients with blastic transformation, in whom intensive c h e m o t h e r a p y was followed by declining serum antigen levels, whereas rising serum laminin values were found in those with relapsing disease. Several issues should be addressed in the interpretation of these results. First, circulating laminin is heterogeneous consisting of intact as well as various proteinase-resistant fragments. The relationship of these various antigen forms and laminin metabolism still remains to be determined. It has been demonstrated recently, however, that two different elastase
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Serum lamininP1 in idiopathicmyelofibrosis resistant fragments are taken up/degraded by liver endothelial and liver parenchymal cells, respectively. Judged by conventional biochemical markers severe liver dysfunction was not prevalent in the patients under study. However, specific indicators of, for example, Kuppfer and endothelial cell function are not available. Small amounts of laminin are also taken up by the spleen [26]. It follows that the serum concentration of laminin-related antigen depends not only on the amount of laminin protein released to the circulation but also on the extent of uptake and degradation, particularly in the liver. The present finding of an inverse relationship between S-laminin and spleen size suggests an altered function of the enlarged spleen with respect to laminin uptake and degradation. A similar inverse relationship between plasma fibronectin and spleen size has been reported earlier [27]. It is not clear whether the putative splenic uptake of laminin and fibronectin is due to non-specific trapping in th~ enlarged spleen or to a receptor mechanism by endothelial cells [27]. The previously reported low hyaluronan concentrations in splenomegalic patients may reflect a similar mechanism [28]. Although the serum laminin values were found to covariate closely with the serum concentration of PIIINP, the leucocyte count and plasma lactic dehydrogenase in individual patients, following serial measurements no significant correlation existed between serum laminin and the above parameters when assessed for the whole series of patients. This discrepancy may be explained by the lower serum laminin levels in patients with large spleens. With the reservations given above the higher serum laminin levels in patients with active disease most reasonably reflects ongoing basement membrane neoformation. This notion is further supported by our previous findings of elevated serum concentration of type IV collagen antigen in a high proportion of MF-patients and a highly significant correlation between the serum concentrations of type IV collagen antigen (7-S collagen) and PIIINP [22]. Furthermore, our observation of a close covariation between serum laminin and serum PIIINP is in accordance with the current hypothesis for bone marrow fibrogenesis and angiogenesis in idiopathic myelofibrosis and related diseases, implying a common stimulus for both processes by the intramedullary release of growth factors from megakaryocytes (for example, platelet-derived growth factor and transforming growth factor-beta) [29-32]. In conclusion, our observations support the concept that the hyperactive myeloproliferation in IMF and related diseases is accompanied by synchronous changes in the metabolism of laminin and type III collagen.
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