Methods. G. R. Pilkington, N. Kraft, V. Murdolo, G. T. H. Lee, S. V. Hunter, R. C. Atkins, and D.G.Jose 2

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1 Serological Typing of Acute Leukemia Using the Monoclonal Antibodies PHM 1, 2, 3, 6, CIKM5, and the Rabbit Antisera RARC2a (Ad) and RAALLP50 1 G. R. Pilkington, N. Kraft, V. Murdolo, G. T. H. Lee, S. V. Hunter, R. C. Atkins, and D.G.Jose 2 Acute lymphocytic leukaemia (ALL), can be subclassified on the basis of existing markers into T-ALL, B-ALL and non-b/t-all. Non-BIT-ALL can be further subdivided into null-all and common ALL (call) using the marker common ALL-associated antigen (CALLA) first described by Greaves et al. [1]. The call patients have better prognosis, longer remission duration and survival, and require less intensive chemotherapy for remission induction than do null-all patients [2, 3]. However, there are no markers specific for null-all, the leukaemic cells being characterized by a lack of markers. The null-all subgroup is clinically important, since it is moderately frequent, has a bad prognosis and is sometimes difficult to distinguish from myeloid leukaemia (especially when the myeloid blasts are primitive and cytochemistry is not definitive). The identification of surface antigens specific for null-all would improve the typing of ALL and its discrimination from AML, and might provide a potential antigen for treatment of null-all patients. Myeloid leukaemia represents a more diverse group than ALL on morphological classification. The demonstration of serologically identifiable antigens may recognise prognostic subgroups in this form of leukaemia which are not at present identifiable using morphology and cytochemistry, or may facilitate the identification of primitive myeloid cells where morphology and cytochemistry are inconclusive. The aims of this work were to produce antisera/antibodies which identified null-all (CALLA-, non-b/t-all) and myeloid cells or subgroups of myeloid leukaemias having different prognoses. Methods Patients' Cells Mononuclear cells enriched for blasts were separated from heparinized blood and marrow by single-step density gradient centrifugation on Ficoll-Hypaque (1.077 g/cm 3, 400 gx 30 min 20 C). Microcytotoxicity Test Complement-mediated lysis of patients' target cells was measured using a modification of the cytotoxicity test of Amos et al. [4] as previously described [5]. 1 This work was supported by a grant from the Research Committee, Cancer Institute, Melbourne 2 The authors wish to thank L. Bradley, J. Quirk and G. White for excellent technical assistance 588 A. Bernard et al. (eds.), Leucocyte Typing Springer-Verlag Berlin Heidelberg 1984

2 Flow Cytofluorometry The binding of mouse monoclonal antibodies was detected using a F(ab'h fragment of goat anti-mouse Ig (affinity purified) FITC (Cat. No l1, Cappel Laboratories, Cochranville, Pennsylvania). Binding of F( ab'h fragments of rabbit antisera was detected using a F( ab')2 fragment of goat anti-rabbit Ig (affinity purified) FITC (Cat. No , Cappel Laboratories). Indirect immunofluorescence was measured using an Ortho System 50 cell sorter coupled to a 2150 computer (Ortho Instruments, Westwood, Massachusetts), or using an Ortho FC200/4800A cytofluorograph coupled to an ND100 multichannel analyser (Nuclear Data, Chicago, Illinois). Cell Markers All mononuclear cell fractions were tested for the established markers, E-rosette receptor (E-RFC) both 4 oc and 37 oc, surface lg (Sig), myeloperoxidase and a-naphthylbutyrate esterase. Rabbit Antisera Rabbit antisera RARC2a (Ad) to adherent cells from myelomonocytic cell line RC2a and RAALLP50 (to a dalton membrane antigen on fresh blood leukaemic cells from a child with null-all) used in this study (Table 1) have been previously described [5, 6]. F(ab') 2 fragments were prepared according to the method of Goding [7]. Mouse Monoclonal Antibodies Monoclonal antibodies PHM1, 2 and 3 (Table 2) have been previously described [8]; PHM6 and CIKM5 (Table 1) have not. Typing of Cells from ALL Patients Serological typing of cells from ALL patients was performed using the cell markers above, as well as additional rabbit antisera to B cells (RAKB) and CALLA (RAN-1, rabbit antiserum to the call-derived cell line NALM-1) as previously reported [5] and standard monoclonal antibodies to HLA (W6/32), Ia (7.2) and CALLA (15, BA3 and PHM6). 589

3 Table 1. Antisera/ antibodies used in study Designation Source Immunogen Antigen Specificity (mol. wt.) PHM1 Monoclonal Blood 55K Leucocyte common (mouse IgG1) leucocytes (gp)a PHM2 Monoclonal RC2a 52K Monocyte, 30% MOb, (mouse IgGl) (AMML Cell line) (gp) 5% T cell, myeloblast endothelium PHM3 Monoclonal Thymocytes 50K Cortical thymocytes, (mouse IgG2a) (Juvenile, 5yr) (gp) 80% monocyte, MO (Peritoneal + + +) CIKM5 Monoclonal K562-L? Monoblast, mono- (mouse IgG1) (CML-BC cell line) cyte ( ± ), myelocyte RARC2a(Ad) Rabbit RC2a 50,70 K Myeloid, primitive (Abs BLCL)c (AMML cell line) (gp) T cell, 40% call PHM6 Monoclonal NALM-6? CALLA-like (mouse IgG2b) (call cell line) RAALLP50 Rabbit p50 50K 50% null-all, 40% T-ALL (Abs AB+d) (Null-ALL) (p)< <6% call a Glycoprotein b Macrophage (tissue and peritoneal) c Absorbed with B-lymphoblastoid cell lines (15lines including the autologous B-cellline to RC2a) and human AB, Rh + erythrocytes d Absorbed with human AB, Rh + erythrocytes e Protein Results Reactivity of RAALLP50 with Leukaemic Cells Results of cytotoxicity testing of cells from 87 children with ALL and 13 adults with ALL are listed in Table 2. Cells from these patients were typed using the markers Slg, E-RFC, rabbit antisera (RAKB (B-cell) and RAN-1 (CALLA) and monoclonal antibodies to Ia antigen (7.2) and CALLA (15, BA3 and PHM6). Table 2. Testing of ALL cells with RAALLP50 by cytotoxicity Null call T-ALL B-ALL Children RAALLP50 positive* per total no. of patients 6/12 4/69 2/5 0/3 Adult RAALLP50 positive per total no. of patients 0/3 0/6 1/3 0/1 * ;;. 20% cells killed/lysed by RAALLP50 after background subtraction (lysis of cells by complement alone) 590

4 Cells from six of 12 (50%) children with null-all and two of five (40%) T ALL patients reacted with RAALLP50, whereas cells from less than 6% of call patients (four of 69) reacted with RAALLP50. Two of these four positive call patients presented with white cell counts (WCC) over /j.Ll. Cells from the B-ALL patients did not react with RAALLP50. Cells from only one of the three adults with T-ALL reacted with RAALLP50. Cells from 62 patients with other haemopoietic malignancies did not react with RAALLP50 (Table 3). Cells from 20 children with ALL were tested by cytofluorometry with RAALLP50 (Table 4), with positive reactions in four of seven null-all, two of two T-ALL, none often call and neither of two B-ALL. Reactivity of RAALLP50 with Leukaemic Cell Lines Three of eight cell lines derived from T-ALL were positive with RAALLP50 by cytofluorometry. The myeloid leukaemia cell lines K562-L (from Dr. B. B. Lozzio), K562-N (from Dr. H.Zola, glycophorin positive), RC2a [9] (AMML derived), HL60, U937 and THP-1, were tested by cytofluorometry with RAALLP50. Only HL60 and K562-L were positive (50% and 21% of cells staining respectively). Table 3. Testing of other leukaemic cells with RAALLP50 by cytotoxicity Non-BIT B-CLL/ Hairy cell lymphoma lymphoma leukaemia RAALLP50 positive per total no. Ob/5 0/6 0/1 of patients T-CLLI lymphoma 0/12 CML AML AMML RAALLP50 positive per total no. oc/10 0/ of patients a ;;;.. 20% cells killed/lysed by RAALLP50 after background subtraction b Includes 3 x histiocytic lymphoma (esterase positive) c Includes 2 x CML-BC (myeloid) and 1 x CML-BC (lymphoid) AMoL 017 AUL 0/4 Table 4. Testing of cells from children with ALL with RAALLP50" by cytofluorometry Null-ALL call T-ALL B-ALL RAALLP50 positiveb per total no. of patients 4/7 0/10 2/2 012 a F( ab')2 fragment h ;;;.. 20% cells stained after background subtraction (staining with normal mouse Ig and conjugate) 591

5 Reactivity of RAALLP50 with Normal Haemopoetic Cells Normal peripheralleucocytes (eosinophil, neutrophil, monocyte, lymphocyte), normal marrow mononuclear cells and reactive T cell populations (20 samples) were negative by cytotoxicity with RAALLP50. Lymphocyte populations from tonsil were negative, 30%-40% ofthymocytes were positive when tested by cytofluorometry. Survival of RAALLP50-Positive Patients Null-ALL and call patients (Table 2) were divided into RAALLP50-positive (P50+) and -negative (P50-) groups. The 2-year survival of the null-all, P50 + group was < 40% (three of eight) compared with > 80% (five of six) for the null-all, P50- group (Table 5). Table 5. Childhood ALL: RAALLP50 survival (2 years) Survival Null-ALL P50+ <40% (3/8) >80% (5/6) call P % (3/3) PS0- PS0- >80% (21/26) Serological Subgrouping of Myeloid Leukaemias Results of serological typing of cells from 23 myeloid leukaemia patients with the monoclonal antibodies PHM1, 2 and 3 and the rabbit antiserum RARC2a(Ad) are depicted in Table 6. Reactivity with these monoclonal antibodies and rabbit antiserum results in seven different patterns (groups A-G). The 1-year survival for group D is three of eight, group B, five of seven and groups A, B, C, E, F, G (pooled), 12 of 15. Groups Band D were the two larger groups of myeloid leukaemia patients. Reactivity of CIKM5 with Leukemic Cells The reaction of monoclonal antibody CIKM5 with leukaemic cells by cytofluorometry is shown in Table 7. Cells from only one of 41lymphoid malignancies (one of nine null-all) tested were positive, but cells from patients with AMoL (two of three), AMML (one of three), CML-BC (one of two) and occasionally CML (one of six), were positive with CIKM5. Table 8 lists the results of testing haemopoietic cell lines with CIKM5 by cytofluorometry. The immunising cell K562-L reacted strongly, and K562-N, which has erythroid properties, and the cell line NALM-6 were both weakly positive with CIKM5. 592

6 Table 6. Serological subgrouping of myeloid leukaemia patients with PHM1, 2, 3 and RARC2a (Ad) by cytofluorometry Group Leukaemia Patient/no. Monoclonal antibody Antiserum PHM1 PHM2 PHM3 RARC2a(Ad) A CML S684 + CML S950 + B CML-BC(M) S AMoLb S CML-BC(L) T1 + + CML T AMMLb T AMML T CML T c CMMLb S AMML S D AMML P AML S AMMLh S AMLb T AMoLh T AMoLb T callb T CMoL< T CMML T E AML T ± F CML-BC(L) T G call T4 + CML T559 + call T798 + Thy ALL T816 + CML T827 + call T831 + F (ab')z fragment b Patients known to have survived less than 1 year post diagnosis due to leukemic relapse c Patient currently in relapse Table7. Reactivity ofcikms with leukaemic cells by cytofluorometry No. patients positive per no. tested Lymphoid leukaemia/lymphoma B CLL!B-lymph call Null-ALL T-ALL/lymph B-ALL 017 0/17 1/9 0/6 0/2 No. patients positive per no. tested Myeloid leukaemia AMoL AMML AML 2 /3 1/3 Both patients > 80% monoblast b Specimen differential count- 70% myelocyte- band 0/4 CML CML-BC 1/2 593

7 TableS. Reactivity ofcikm5 with myeloid leukaemia cell lines by cytofluorometry Cell line Derivation K562-L CML-BC K562-N CML-BC HL60 APML RC2a AMML U937 Histiocytic lymphoma THP-1 AMoL B-ee!! EBV transformed B-ee!! Burkitt's lymphoma T-cell T-ALL call call a ;; X background b ± = 2-3 x background c NALM-6 Type Primitive myeloid Primitive erythroid Promyelocytic Myelomonocytic Macrophage Monocytic Lymphoblastoid Lymphoblastoid Lymphoblastoid Lymphoblastoid 0/6 0/ Cf5 Reactivity of CIKMS with Normal Haemopoietic Cells Normal monocytes (five of five) reacted weakly (10%-20% cells reacted 2-3 times background), but peripheral lymphocytes (n=5), granulocytes (n=5), erythrocytes (n= 2) and thymocytes (n= 5), tonsilar lymphocytes (n= 4), splenic lymphocytes (n=2), marrow mononuclear cells (n=7) and foetal liver erythroblasts ( n = 2) did not react with CIKMS by cytofluorometry (data not shown). Discussion Results of testing cells from 87 children with ALL, 13 adults with ALL and 62 other leukaemia patients and 18 normal cell populations by cytotoxicity, along with results of testing cells from 20 children with ALL and 20 leukaemic cell lines by cytofluorometry have suggested the antigen(s) recognized by RAALLPSO to be restricted to subgroups of childhood null-all (50%) and T-ALL (40%), and rarely expressed on call ( <6% patients). The antigen detected by RAALLPSO was expressed on the HL60 and K562-L cell lines although this antigen was not detected on myeloid leukaemia cells by cytotoxicity. Four of the B/CALLA protocol reagents (FMC8, BA2, DuALL1 and SJ9A4) - those reacting with a 24,000-dalton antigen on call, null-all, B cells and myeloid cells (Pilkington et al., this volume) and therefore not null ALL specific - reacted with cells from null-all patients. Three of the monocyte/granulocyte protocol antibodies (75B10, MOP15 and T2) reacted with cells from null-all patients (Pilkington et al., this volume) but were also reactive with a wider range of cells, including myeloid cells. Therefore, from this group of 88 international antibodies, none were specific for cells from null-all patients, suggesting RAALLPSO to be of unique specificity. The significance of the subdivision of childhood null-all by the antigen(s) recognized by RAALLPSO is unclear; however, preliminary survival data suggest that this subdivision of the childhood null-all group into PSO + and PSO- subgroups may be of clinical importance. 594

8 Typing of cells from myeloid leukaemia patients with three monoclonal antibodies (PHM1, 2 and 3) and a rabbit antiserum RARC2a(Ad) has subdivided these leukaemia patients into seven groups not overtly related to the morphological classification (Table 6). Thus, although these antisera/ antibodies are not highly specific for a single lineage of cells (Table 1 ), they can be used together to define serological subgroups. The 1-year data for these patients suggests that group D has the poorer survival when compared with group B (the next largest group) or the other groups pooled together. PHM2 may have potential for treatment of myeloid leukaemia patients since it is present on cells from group 0 patients. Results presented for CIKM5 suggest this monoclonal antibody reacts strongly with monoblasts, weakly with monocytes, and weakly with cells from CML (including CML-BC) patients who have large numbers of myelocytes, and rarely with ALL (one of 26non-B/T-ALL). Future data will indicate whether the use of this antibody, which seems similar in specificity to Workshop monoclonals M13, M36, M39 and M42 (Pilkington et al., this volume), could help in identifying early cells of monocyte lineage. References 1. Greaves MF, Brown G, Rapson N, Lister TA (1975) Antisera to acute lymphoblastic leukemia cells. Clin Immunol Immunopathol4: Pinkel D (1979) 9th Annual David Kamofsky Lecture: Treatment of acute lymphocytic leukemia. Cancer43: Brouet JC, Seligmann M (1978) The immunological classification of acute lymphoblastic leukaemias. Cancer 42: Amos DB, Bashir H, Boyle W, MacQueen M, Tiilikainen A (1969) A simple microcytotoxicity test. Transplantation 7: Pilkington GR, Lee GTH, O'Keefe D, Plain M, Wilson FC, Jose DG (1980) Classification of childhood acute lymphocytic leukaemia using rabbit antisera to leukaemia cells and lymphoblastoid cell lines. Aust J Exp Bioi Med Sci 58: Pilkington GR, Jacobs D, Mackenzie S, Bradley TR, HunterS, Jose DG (1982) Rabbit antisera to cell lines RC2a and U937: Antigens expressed on human leukaemic cells of myeloid, monocyte and T-lymphocyte lineage. Aust J Exp Bioi Med Sci 60: Goding JW (1976) Conjugation of antibodies with fluorochromes: Modifications to the standard methods. J Immunol Methods 13: Becker GJ, Hancock WW, Kraft N, Lanyon HC, Atkins RC (1981) Monoclonal antibodies to human macrophage and leucocyte common antigens. Pathology 13: Bradley TR, Pilkington GR, Garson M, Hodgson GS, Kraft N (1982) Cell lines derived from a human myelomonocytic leukaemia. Br J Haematol 51: