Jun 12, 1983 - 22, 25]; head and neck carcinoma [23]; Hodgkin's disease [1, ... tumors [11]; malignant teratomas and gestational trophoblastic tumors [2].
LeukemiaResearchVoL7, No. 6, DI~.771-777,1983. Printedin GreatBritain.
0145-2126/83$3.00 + 0.00 © 1983PergamonPressLtd.
CIRCULATING IMMUNE COMPLEXES IN C H R O N I C MYELOID LEUKEMIA PATIENTS AT VARIOUS STAGES OF THE DISEASE DHANANJAYA SARANATH, SURESH ADVANI* a n d SUDHA GANGAL Immunology Division, Cancer Research Institute and *Tata Memorial Hospital, Tara Memorial Centre, Parel, Bombay-400 012, india
(Received 3 June 1983. Accepted 12 June 1983) Abstract--Circulating immune complexes (CICs) in sera from patients suffering from chronic myeloid leukemia (CML) at initial diagnosis, in 'remission', at relapse and in blastic crisis have been quantitated using fluid [12~l]Clq' binding assay in terms of per cent binding activity and ~g/ml aggregated human globulin (AHG) equivalents. The CIq binding acitivity (CIq-BA) has been compared within the groups of CML patients in different phases of the disease as well as with sera obtained from normal healthy donors. The results showed that the mean CIq-BA was significantly increased in CML patients at initial diagnosis (25.74 -t- 3.48, p < 0.001), in relapse (53.36 -t- 6.9, p < 0.001) and in blastic crisis (60.5 ± 8.7, p < 0.001) when compared to control sera. Sera of 'remission' patients showed significant decrease in CIq-BA when compared to sera collected in active phases of the disease, however, the values were still significantly higher (12.87 + 1.58, p < 0.02) than those of normal healthy donors. When the levels of CICs as assessed by C Iq-BA were compared with the WBC/blast counts of CML patients in chronic as well as blastic phase, it was noted that the variations in numbers of circulating leukemic cells do not correlate with the CIC levels. The significance of assessment of CIC levels in monitoring the disease in CML patients is discussed.
Key words: CICs, CML, CIq-BA.
INTRODUCTION IMMUNE complexes have been shown to play a pathogenic role in many infections and autoimmune diseases [8, 18]. A considerable interest has been shown recently in the measurement of circulating immune complexes (CICs) in human malignant disease, the view being that this may be of value as a diagnostic or prognostic indicator of the disease. Elevated levels of CICs have been demonstrated in breast cancer [22]; lung cancer [12, 21, 22, 25]; head and neck carcinoma [23]; Hodgkin's disease [1, 4]; gastrointestinal tract cancer [24, 28, 32]; genitourinary tract cancer [7, 14, 29, 32]; melanoma [16, 27]; bone tumors [11]; malignant teratomas and gestational trophoblastic tumors [2]. Studies on the detection and quantitation of immune complexes in human leukemias have also been reported [3, 5, 6, 15, 19, 20]. These have been mainly restricted to acute ieukemias. Carpentier et al. [5] and Mod et al. [20] have studied CICs in chronic myeloid leukemia (CML) in the chronic phase and in blastosis. However, serial measurements of CICs in the sera of patients with CML have not been reported so far. Recent advances in technology have provided a variety of methods for the detection of CICs. Immune complexes have been detected using physico-chemical as well as biological methods. Under the auspices of WHO, 18 methods for detecting CICs in serum were evaluated by Lambert et al. [17]. It is also documented that of more than 30 assays Abbreviations: CML, Chronic myeioid leukemia; CICs, circulating immune complexes; CIq-BA, 12Sl-labeiled C lq binding activity; PEG, polyethylene glycol; BgA, bovine serum albumin; EDTA, ethylenediaminetetracetic acid; AI.IG, heat aggregated human gamma globulin; LAP, leukocyte alkaline phosphatase; M:E, myeioid: erythroid ratio in bone marrow. Correspondence to: Dr. Sudha Gangal, Head, Immunology Division, Cancer Research Institute, Tara Memorial Centre, Parel, Bombay-400 012, India. 771
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available for the detection o f C I C s , C l q solid phase or fluid phase b i n d i n g tests, cong l u t i n i n assays, m o n o c l o n a l R F i n h i b i t i o n a n d assay using Raji cells have been f o u n d to be most acceptable in a recent W H O / I U I S c o l l a b o r a t i v e study. T h e present study has been carried o u t using the fluid C l q b i n d i n g assay, to d e t e r m i n e the C I C levels in the sera o f C M L p a t i e n t s at d i f f e r e n t stages o f the disease, such as at the initial diagnosis before t r e a t m e n t , in r e m i s s i o n when patients are devoid o f t h e r a p y or are o n m a i n t e n a n c e t h e r a p y with B u s u l f a n , at relapse before t r e a t m e n t a n d in blastic crisis. T h e C l q b i n d i n g activity is c o m p a r e d a m o n g s t three g r o u p s o f C M L patients as well as with that o f the sera o b t a i n e d f r o m n o r m a l healthy d o n o r s . PATIENTS AND METHODS Patients
Serum samples were obtained from 56 patients with CML at initial diagnosis, 35 patients at remission, 20 patients in relapse and 18 patients in blastic crisis. CML patients were diagnosed on the basis of hepatosplenomegaly and other clinical symptoms. Hematologiqally, the peripheral WBC count ranged between 80,000 and 250,000/ram3 with immature granulocytes in all stages in circulation and the M:E ratio ranged between 10 and 30. All patients had LAP scores less than 10. Blood samples from these patients were collected before the commencement of the treatment. The treatment of choice was generally Busulfan 4 rag/day. Treatment was continued for about 4-8 weeks with a frequent check on clinical symptoms and peripheral blood count. Remission was defined as disappearance of hepatnsplenomegaly and other clinical symptoms, normal WBC count without immature cells in periphery and < 5070blasts in the bone marrow. Thirty-five patients were tested either in first or subsequent remissions. Similarly the relapse patients tested were either in first or subsequent relapses. Relapse was defined as increase in peripheral blood WBC count to over 50,000/ram 3 with or without clinical symptoms. The patients were given another course of Busulfan to control the disease in relapse. Serum samples were collected before recommencement of the therapy. Blastic crisis was defined as appearance of more than 20°7oblasts in bone marrow. Collection and storage o f sera
Sera from all these patients were stored in small aliquots at --20°C without inactivation, until use. As controls, sera from 50 healthy blood donors/laboratory personnel were also collected. Radiolabelled C l q binding tests
CIq was isolated and purifed according to the method of Zubler et al. [33]. The purity of Clq was checked by immunodiffusion using human anti-Clq serum and anti-total human serum. The purifed Clq was iodinated by the Lactoperoxidase method, according to Heusser et al. [131. The iodinated CIq was aliquoted into 200 ~i and stored at --20"C. On the day of the test, 200 I~ialiquot was diluted in 3-5 ml veronal buffer saline pH 7.4, with Ca2+ and Mg2+, containing 1070inactivated human AB group serum and 0.5% BSA. It was then centrifuged at 18,000 g for 40 rain to remove aggregated Clq and the supernatant was used in the test. The undiluted native serum samples and a 1:5 dilution of samples were each tested for Clq binding activity (CIq-BA) in duplicate. In Biogamma tubes, 50 ~i of test serum was mixed with 2 vol. of 0.2 M EDTA pH 7.5, and incubated at 37"C for 30 rain. Then 50 I~lof [125I]Clq and 1.0 ml of 3070(w/v) polyethylene glycol (tool. wt 6000) solution in borate buffer pH 8.3, with 0.05070Tween 20, were added to the mixture. The tubes were left in an ice bath for 60 rain and centrifuged at 1500 g at 40C for 20 rain. The supernatant was discarded and the precipitated immune complexes washed with the PEG solution. The radioactivity in the precipitate was then measured. In each test run, heat aggregated human gamma globulin (AHG) at different concentrations in normal human serum were used as positive controls. The AHG was stabilized by the addition of 0.5070 BSA to the aggregates. WHO supplied AHG standards kindly given by Dr. U. Nydegger (WHO Immunology Research and Training Centre, Geneva), were used for provisory reference curve. Results were expressed as per cent of [1251]Clq precipitated when compared with the total protein bound activity obtained by 2007o (w/v) trichloroacetic acid precipitation of 150 ~1 of inactivated human serum mixed with 50 ~1 of [1251]Clq, and as I~g/ml AHG equivalent. They were corrected for the nonspecific sticking of the iodinated C lq to the Biogamma tubes. RESULTS T h e C I q - B A o f sera f r o m 56 C M L patients at initial diagnosis, 35 patients in remission, 20 patients in relapse a n d 18 patients in blastic crisis is s u m m a r i z e d in T a b l e 1. As compared with sera f r o m 50 n o r m a l healthy i n d i v i d u a l s , whose per cent C l q - B A was 8.8 ± 0.82, the g r o u p m e a n values o f s e r u m C l q - B A was significantly increased in patients at initial diagnosis (25.74 5= 3.48 p < 0.001), in relapse (53.36 5= 6.9, p < 0.001), a n d in blastic crisis (60.5 5= 8.7, p < 0.001). S e r u m samples o b t a i n e d f r o m ' r e m i s s i o n ' patients, a l t h o u g h s h o w i n g higher values o f C l q - B A t h a n n o r m a l i n d i v i d u a l s (12.87 5= 1.58, p
