Leukemia Research, Vol. 2, No. I, pp. 105-114. 0145-2126178/0301-0105502.00/O @ Pergamon Press Ltd. 1978. Printed in Great Britain

ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL) ASSOCIATED ANTIGEN--I. EXPRESSION IN

DIFFERENT HAEMATOPOIETIC MALIGNANCIES

MARION ROBERTS, MELVYN GREAVE$, GEORGE JANO~Y, ROBERT SUTHERLAND a n d CAROL PAIN

Membrane Immunology Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London (Received 20 December 1977. Accepted 3 January 1978).

Abstract--The cellular specificity of antisera to non-T, non-B ALL has been assessed in a series of 545 patients (adults and children) either presenting with untreated acute or chronic leukaemia or in relapse. The 'ALL associated antigen' was detected and evaluated by indirect immunofluorescence using standard microscopy and with the Fluorescence Activated Cell Sorter. Anti-ALL serum reacts with cells from a variable proportion of patients in the following groups: non-T, non-B ALL; Ph 1 positive acute leukaemia or CML in blast crisis; AUL; and a few lymphomas. A weak but definite expression of the ALL antigen was also detected in ! 0 % of ALLs with a thymic phenotype (Thy-ALL). Cross-absorbtion studies indicate that a single antigen or antigenic complex is shared by these various cell types. These observations are interpreted to suggest that several clinically and karyotypicaily distinct leukaemias involve a similar lymphoid or pre-lymphoid cell type and that the ALL associated antigen is most likely a normal gene product of that cell type or lineage involved in the disease.

INTRODUCTION

THE PRODUCTION and testing of rabbit antisera to acute lymphoblastic ieukaemia of the common or non-T, non-B subclass has been described previously [1-3]. After appropriate extensive absorption these sera were able to distinguish, by immunofluorescent binding criteria, ALL cells from both mature

Correspondence to: Dr. M. F. Greaves, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX.

Abbreviations: ALL, Acute lymphoblastic ieukaemia; AUL, Acute undifferentiated ieukaemia; AML, Acute myeloblastic leukaemia; CML, Chronic myeloid leukaemia; CML bc, Chronic myeioid leukaemia in blast crisis; CLL, Chronic lymphocytic leukaemia; Ph 1, The Philadelphia chromosome [t(22q-, 9q+)]; T, Thymus derived cell; B, Bursa-equivalent derived cell; Thy-ALL, ALL with a thymic phenotype; c-ALL, ALL with the major or common phenotype (ALL antigen positive); B-ALL, ALL with a B cell phenotype; TdT, Terminal deoxynucleotidyl transferase; Hex I, Hexosaminidase intermediate peak isoenzyme; lg, Immunogiobulin; Smlg, Surface membrane immunogiobulin; HuTLA, Human T lymphocyte antigen(s); HuMA, Human myeloid-monocytic antigen(s); la, Membrane antigens coded for in mice by genes of the H-2 linked I (Immune response gene) region/loci. Called Ia or 'Ia-like' in man by analogy. Antisera to these human antigens are referred to as anti-'Ia' or anti-(g)p28,33 (the B cell glycoproteins commonly used to produce the reagents); E, Erythrocyte (sheep) rosettes; A.Phos, Acid phospha- tase; PAS, Periodic acid Schiff; FACS, Fluorescence Activated Cell Sorter.

haemopoietic cells and other leukaemias. An early suggestion that anti-ALL sera might react also with some chronic myeloid leukaemias (CMLs) in blast crisis [1] has now been fully substantiated [4, 5]i In this paper we document more precisely the expression of the 'ALL associated' antigen in a large series of

• i different leukaemias and show that a single common antigen or antigenic complex is found on ithe majority of non-T, non-B ALL, some AUL I a proportion of Philadelphia chromosome posilive CML in blast crisis and in some lymphomas. Preliminary analysis of these data has been pUb- lished previously [6].

MATERIALS AND METHODS

Patients

Blood and bone marrow samples from patients Were received from clinicians throughout the U.K. as we~! as from major leukaemia centres in London (Hospital[ for Sick Children, St. Bartholomew's Hospital, R_~yal Marsden Hospital, Hammersmith Hospital). Most samples were from newly presenting untreated cas¢~ of acute leukaernia (se¢ Results). Approximately 10~o Were from relapsed cases and these are analysed separably. Most samples were from patients entering the MRC UKALL therapy trials; the relationship of immUno- logical phenotype to the prognosis of the disease is b~ing assessed in this trial and will be published later.

Cells

Lymphocytes and blast cells were prepared by Ficoll- Isopaque density gradient cvntrifugation from hepar- inised samples of peripheral blood and bone marrow.

105

106 MARXON ROBERTS et al.

Cells from solid tissues were prepared by teasing and filtration through nylon mesh.

E-rosette test

Sheep erythrocyte rosettes were used as a standard T cell marker [7, 8]. 25 ~1 of lymphocytes (at 10~/ml) were mixed with 25 ~1 foetal calf serum (absorbed with sheep red blood cells) and 50 al of 2 % sheep red blood cells (pretreated with 15 U/ml neuraminidase at 37 ° for 30 min). The mixture was centrifuged for 5 rain at 400 g (av) and left 1 h at room temperature before gentle resuspen- sion, dilution and counting.

Immunofluorescence

The binding of antisera were assessed by indirect immunofluorescence with fluoresceinated goat anti-rabbit IgG or by direct immunofluorescence in the case of anti- Ig. Fluorescence of cells was evaluated using a Zeiss photomicroscope with incident illumination and also with the Fluorescence Activated Cell Sorter (FACS) (Becton Dickinson, Mountain View, California) as previously described [9].

Antisera

Anti-ALL sera were raised in rabbits by 2 or 3 weekly intravenous injections of 2 x 10' viable ALL cells coated with anti-lymphocyte serum as previously described [I, 2]. The sera were absorbed with red blood cells, normal lymphocytes (tonsils), normal bone marrow, A M L and/or CML cells and ultracentrifuged at 90,000 g for I h before use. Some sera have required further absorption with thymus cells. Over the past 4 years l0 such sera have been prepared and have given essentially identical results with only slight differences in titre.

Goat anti-human immunoglobulin was a directly fluoresceinated F(ab') 2 fragment of IgG (kindly supplied by Dr. I. Chantler, Wellcome Research Labs, Beckenham).

Anti-Ia serum [10] was prepared by immunising rabbits with precipitin lines formed in agarose gels between a detergent extract of human spleen and an anti-la (p28,33) serum which had itself been previously raised against isolated B cell glycoproteins (see refs. [5, I I] for further details).

Anti,T cell serum was raised in rabbits against monkey thymocytes and absorbed with B cell lines and CLLcells as previously described [! 2].

Anti-myeloid/monocytic serum was raised in a rabbit against acute myelomonocytic and acute monocytic ieukaemia [13] and was absorbed with red blood cells and monocyte-free lymphoid cells (tonsils). The pepsin digest F(ab') I fragment of IgG was used in testing.

Absorption of antisera or F(ab') 2 antibody fragments was performed by incubating viable packed cells with the antiserum in a 1:3 ratio (of volumes) for I h at 4°C.

RESULTS

Blood and/or bone marrow cell suspensions from 545 patients have been tested with membrane

markers including anti-ALL serum. These represent all samples received up until 30 December, 1976 in which there were considered to be an assessable level of leukaemic cells (see below) and on which we have received a final diagnosis. The haematologicai diagnosis was made by the haematoiogists of the referring hospital and included assessment of PAS, peroxidase or Sudan Black cytochemistry [22]. The diagnosis was made without knowledge of the results of the immunological typing. The cases tested were divided into adults (more than 15 years old) and children. Most of the cases tested (416) were untreated patients presenting with acute leukaemia; the samples tested being taken before therapy was started.' Forty-three samples (8 °/) were from patients in relapse of acute leukaemia and 86 were from patients with other haemopoietic malignancies (e.g. chronic leukaemias and lymphomas).

All cases were phenotyped with anti-ALL, anti- Smlg, and E-rosettes. Approximately 100 cases were also typed with anti-T serum, anti-myelo-monocytic (M) serum and anti- 'Ia-like' (p28,33) serum. The detailed membrane and enzyme marker analysis of some of these cases has been presented previously [5]. Since normal blood and marrow have less than 1% strongly positive cells reactive with anti-ALL serum [2, 14, 15] a confident identification of leukaemic cells could be made if the malignant cells constituted 5% or more of the cell suspension. Other membrane markers used are, however, reactive with normal cells present at moderate to high frequencies in blood and/or marrow and there- fore positive phenotyping of leukaemic cells is only possible when both the proportion and absolute count of leukaemic cells is known to be high, i.e. blasts >30 °/ in blood and/or marrow (whichever / O

is available). Patients with blast cell percentages below 300/0 were therefore tested with anti-ALL serum but are only included in the analysis when positive with anti-ALL serum. This screening pro- cedure results in some bias in the proportion of ALL cases ascribed to different immunologically defined subgroups since patients with minimal disease (if anti-ALL negative) are excluded.

Adul ts

134 adults with acute leukaemia were tested prior to treatment (Table 1). 65 patients with a diagnosis of ALL were tested and of these 31 (48%) were positive with anti-ALL sera. Five ALL patients had a thymic phenotype and these were negative with anti-ALL. Two of the E- ALL had a B cell pheno- type (Smlg +) and these were both anti-ALL negative.

Cells from three out of six AUL cases were

ALL antigen expression

TABLE 1. REACTIVITY OF ANTI-ALL SERUM WITH DIFFERENT HUMAN HAEMATOLOGICAL MALIGNANCIES

107

ALL Ph ~ pos. leukaemia Patients E + ALL E- ALL AML AUL AL/BCt CML Lymphoma CLL Others§

Adults 5 60 50 6 40 17 17 9 (~:~) (5:0) (46:i4) (22:28) (4:2) (18:22)

anti-ALL positive: 0 31 1 3 18 1 :l: 4 0

18

0

Children 31 192 31 12 5 2 6 1 (3:'~) (25:6) (105:87) (17: 14) (4:8)

anti-ALL positive: 0 156 2* 5 4 0 2 0

Relapse cases are not included; see text for results on these 43 patients. * See text for reservations on these two cases. "t Presenting with Ph ~ positive acute leukaemia or blast crisis in patients with typical prior CML (see text). :~ CML in accelerated phase (early blast crisis?--see text). § See text for details.

positive with ant i -ALL: all were negative with T and B cell markers. Of the 50 with a haematoiogical diagnosis of A M L only one was positive with anti- ALL serum. 17 o / ( o f 36 tested) were positive with anti-human immunoglobuiln; however, this reac- tivity was polyclonal (i.e. kappa plus lambda light chain) and when the cells were tested after incuba- tion at 37 ° for 1 h the staining was usually con- siderably diminished or absent.

For ty adults with Ph ~ positive leukaemia in the acute phase were tested. Some (13) presented in blast crisis and were diagnosed by the presence of the Philadelphia chromosome and others (27) had had a phase of classical Philadelphia positive chronic myeloid leukaemia before development of the acute blast crisis [5, 16, 17]. Eighteen (45 %) were positive with the ant i -ALL serum and twelve of the 24 assessable (50 %) were positive with the anti-myeloid serum. These positive cases include two in which the blasts were positive with both the anti-myeloid and ant i -ALL serum.

Children

269 children with acute leukaemia have been tested prior to therapy; 223 were diagnosed haema- tologically as ALL and 154 (70%) of these were positive with the anti-ALL serum and negative for T and B cell markers; 31 (14.1%) were Thy-ALLs by the E-rosette test, and 34 (15%) were negative with both these markers and the anti-human im- munoglobulin. Two E- ALL had a B cell phenotype (Smlg ÷) and were ant i -ALL negative.

Thirty-one were diagnosed as having A M L and two were positive with the anti-ALL serum. In one case the result with the ant i -ALL serum was equivo- cal as the bone marrow contained 95 % blast cells

but the antiserum stained only 16% of the cells and the staining was faint and Iocalised on pseudopt~dia of the cells. The other positive case was a child ~vith a hypoplastic marrow with gradual increase of blasts. HaematologicaUy these were considered myelo-monocytoid.

Five children with blast crisis of CML were tested and in four the blasts reacted with the anti-ALL serum and in the other one with the anti-my¢loid serum. Clinical aspects of these cases are being reported in detail elsewhere [18].

In both adults and children with anti-ALL positive cells the proport ion of reactive cells was invariably higher in marrow than in blood although in iboth sites the same range of intensity of stalnmgi was usually demonstrable by FACS analysis (see Fig. 1).

In the ALL patient series described above the ant i -ALL positive subgroup was quite distinct ifrom the Thy-ALL group. This distinction was maintained (in approx. 100 patients studied) when addiiional markers (anti-HuTLA, anti-Ia, p23,30) werg in- cluded [5, 6]. In a more recent series (excluded!from Table 1), however, we have detected a Thy!ALL subgroup with weak but detectable expression Of the ALL antigen. To date (September 1977) nine of such cases have been detected out of a total of 90 T lineage malignancies examined. All nine had ~ Thy- ALL phenotype (i.e. HuTLA +, E +, SmIg-~ Ia- , TdT+). In some respects, however, these were not typical Thy-ALL cases: Two (out of three tested) differed from Thy-ALL in being acid phosphatase negative [19, 20], two (out of two tested) differed from 90% of non-Thy, ALL antigen positive ALLs in lacking the hexosaminidase intermediate peak isoenzyme [21], and three of the nine formed only a small percentage ( ~ 10) of E-rosettes. These obser-

108 MARION ROBERTS ¢t aL

vations suggest that this small subgroup may have an intermediate phenotyl~ between that of Thy-ALL and common ALL.

Relapses

Of the 17 adults tested in relapse of ALL, eight (47 %) were positive with anti-ALL serum and of 21 children in relapse 16 (76%) were positive. These are similar percentages observed at presentation. Four of the positive relapsed cases in children were central nervous system relapse, the cells tested being obtained from cerebrospinal fluid. A single case of ant i -ALL positive testictilar relapse was confirmed on a biopsy sample.

Phenotypic shifts

During the time of this study we have examined 18 patients at onset of acute disease and again at relapse. Eleven retained the same phenotype at relapse (eight were anti-ALL positive and three negative) and seven showed an altered phenotype. The seven with altered phenotype included two cases of CML plesenting in blast crisis (or 'Pht positive acute leukaemia) and two cases of ALL with anti-ALL negative blasts in the bone marrow at presentation and relapse. These four cases had anti- ALL positive blast cells in the cerebrospinal fluid on central nervous system relapse. There were also two cases of ALL which were negative on presentation and positive on marrow relapse and one case of A UL which was anti-ALL positive on presentation but negative at relapse.

Chronic leukaemias

Nine cases of CLL in adults and one case in a child were tested and all were negative with anti- ALL serum. Nineteen cases of CML were tested (two of these in children) and in one adult 8 % cells were positive with ant i -ALL serum. This case was in the accelerated phase with 11% blasts and 38% promyelocytes in the marrow when tested.

L ymphomas

Samples of tissue or blood containing malignant cells from 23 cases of lymphoma (17 adults and 6 children) were tested. In ten cases bone marrow was tested, in four cases lymph node, and in seven cases blood, in one case cerebrospinai fluid and another pleural effusion. In all these cases the tested sample was shown to be involved in the disease process by haematological or histopathological assessment.

As the samples derived from several different hospitals the pathological diagnosis and classifica- tion were not consistent. Because of this a detailed

correlation of histological type of lymphoma and immunological surface marker results was not undertaken in this series. However, the majority of the lymphomas were negative with the anti-ALL serum (74%). Six positive cases were observed and these consisted of two leukaemic transformations of non-Hodgkin's lymphoma (data on type of lympho- ma not available), two cases of poorly differentiated diffuse lymphocytic lymphoma (with marrow in- volvement of 80 and 99 ~o malignant cells), one case of blastic lymphoid malignancy which later became frankly leukaemic, and one case of non-Hodgkin's lymphoma (with 81% malignant cells in the marrow). One case of Hodgkin's ieukaemia was found to be negative with anti-ALL serum and one other case of leukaemic transformation of non-Hodgkin's lymphoma was negative.

Amongst the negative cases were three of poorly differentiated diffuse lymphocytic iymphoma, two of blastic lymphoid malignancy, one of histiocytic lymphoma, one of immunoblastic lymphoma, one of follicular lymphoma and two of lymphosarcoma. In five other cases the data on the type of lymphoma was not available.

Other haematological malignancies

No detectable expression of the ALL antigen occurred in myeloma (two cases), erythroleukaemia (three cases), 'hairy cell' leukaemia (four cases), Hodgkin's disease--Reed Sternberg Cell Leukaemia (one case), Sezary syndrome (five cases) and pro- lymphocytic leukaemia (three cases).

Bone marrow samples heavily infiltrated with non-haematological malignant cel ls- - rhabdomyo- sarcoma (one case), neuroblastoma (two c a s e s ) - were also unreactive with anti-ALL serum.

Cross absorption studies

Different aliquots of anti-ALL serum were absorbed with lymphoblasts from four patients with non-T, non-B ALL which were diagnosed on classical morphological and cytochemical charac- teristics [22] and were positive with the antiserum. These absorbed antisera were then used to test other cases of ALL, AUL or Ph ~ positive CML in blast crisis which had reacted with the original ant i-ALL serum (Fig. 2). In all cases the activity of the serum tested against different leukaemic cells was completely removed. One aliquot of the anti- ALL serum was absorbed with cells of a haema- tologically diagnosed undifferentiated leukaemia (AUL) which reacted positively with the anti-ALL serum. This absorbed aliquot also showed no residual activity against anti-ALL positive ALLs.

109

FIo. 1. Fluorescence Activated Cell Sorter profiles of ALL cells stained with anti-ALL serum. Legend--A,B,C: Vertical axis: relative fluorescence intensity;

Horizontal axis: relative cell size. A: Anti-ALL stained bone marrow cells (ALL patient) 85 ~ cells positive.

B: Anti-ALL stained blood mononuclear cells (same ALL patient) 3 0 ~ cells positive. C: Normal rabbit serum control on bone marrow cells of same patient.

In these spot diagrams (Polaroid photographs of FACS oscilloscope screen) cloud patterns are generated by fluorescent signals of individual cells. D: Comparative fluorescence histogrammVertical axis: relative cell number; Horizontal axis: relative fluorescence intensity. Upper curve: bone marrow cells with anti-ALL (cf. A); Intermediate curve: blood cells with anti-ALL serum (cf. B); Lower curve, on baseline: blood and bone marrow cells with normal serum control.

110

ALL T

(I~i-ALL ob=.

- bc Thy - ALL C

cALL

TP¢-AL.L

CML-bc

Fro. 2. Analysis of antibody binding to leukaemic cells using the Fluorescence Activated Cell Sorter (FACS-I).

Legend: Each of the 18 pictures is a polaroid photograph taken of the oscilloscope screen of the FACS following accumulative analysis of 10,000-20,000 cells. The white areas represent 'fingerprints' of the three ieukaemic cell populations listed on left when tested with the six different antisera given above. In the fingerprint each white dot corresponds to the signal emitted from an individual cell. Vertical axes: relative fluorescence intensity: Horizontal axes: relative light scattering capacity

(approximates to cell size). For leukaemia and antisera abbreviations see text. The 4th and 5th columns from the left indicate

the lack of staining of all three anti-ALL positive leukaemic types when the anti-ALL serum was pre-absorhed with either Pht positive CML in blast crisis (bc) or one of the rare (10~) cases of Thy- ALL with a weak anti*ALL reactivity.

C is normal rabbit serum absorbed with human lymphocytes (tonsils). For results of other crces-absorptiom see text. Standard FACS settings for the experiments illustrated above were: Laser 200 roW, PMT 600 V,

Fluorescence Gain 16/I, Scatter Gain 2/1.

ALL antigen expression 111

Other aliquots of anti-ALL sera were absorbed with cells from two cases of Thy-ALL with weak anti-ALL binding (see above) or with cells from two patients with Philadelphia chromosome positive CML-BC which had a lymphoid morphology. One of these latter cases presented in blast crisis with only slight abnormalities in the mature neutrophils present. The other case presented with typical CML and after 18 months blast crisis occurred and was of lymphoid morphology. Both had the Philadelphia chromosome throughout and both were reactive with anti-ALL serum. These absorbed aliquots of serum had no remaining activity against Ph 1 negative ALL cells tested or against cells from another ALL antigen positive case of CML in blast crisis (Fig. 2).

These cross absorption studies which were not designed to reveal quantitative differences in ALL antigen expression clearly indicate that the s a m e

ALL antigen or antigenic complex is expressed on Ph ~ negative and Ph ~ positive leukaemias.

DISCUSSION

These results confirm and extend earlier studies which demonstrated the expression of a unique membrane antigen in non-T, non-B acute lympho- blastic leukaemia [1-3, reviewed in 14, 23]. Several other laboratories have now produced similar if not identical anti-ALL sera which react with non-T ALL [24, 25] (J. M inowada personal communication, H-G. Thiel personal communication).

The observations reported here establish that a single antigen or antigenic complex is shared by leukaemic cases diagnosed as ALL, AUL, Ph 1 ALL or Ph 1 positive CML in blast crisis and some iymphomas. Studies on leukaemic cell lines derived from these ieukaemias also show that a single ALL antigen is shared by these cell types [26-28] and that it is a stable gene product of these cells.

Within Ph 1 negative ALL, 70% (in children), and 48 % (in adults) 'of cases are ALL antigen positive and all of these fell into the non-T, non-B category. Parallel studies incorporating enzyme markers have indicated that this major subclass of ALL has the overall phenotype: ALL ~, la +, HuTLA-, E-, Smlg-, HuMA-, TdT-, Hex I ÷, A.Phos- [5, 6, 21, 23, 29]*. Anti-ALL serum therefore identifies and subdivides a major group of acute leukaemias pre- viously defined by negative criteria (i.e. 'null', non-T, non-B or O-ALL). We provisionally refer to this

*See list of abbrevations on page 105.

group as common (c) ALL. Although all ALL cases reacting strongly with anti-ALL are also positive with anti-gp28,33 or la-like antigen [5, 6] it is important to appreciate the differences in these two reagents. They react with quite distinct and separate membrane glycoproteins (see following paper) and have quite a different spectrum of cellular specificlty, In contrast to the ALL antigen the la-like antigel~ is present on ALL antigen negative non-T, noh-B ALLs [5, 6, 23], AML cells [10, 30, 31], normal myeloid precursors [29, 32-34], B cells [35] and a subset of peripheral T cells [23, 36].

Previous studies have suggested that in children, patients with Thy-ALL phenotype have a con- siderably poorer prognosis than 'null" cell ALL [~7- 39]. In a recent study of 91 children treated ;~t a single centre over a 4 y period we have been ablg to demonstrate that anti-ALL serum identifes m~ore precisely the subgroup of ALL with the best pt~og- nosis [40].

Cases with a thymic phenotype (Thy-ALL: HuTLA +, E +, la-, Smlg-) constituted 14% of children and 8 % of adults and showed a distinct I~ias in the male to female sex ratio as previously repottep by others [37, 38]. We have now (September 1977) seen a total of 90 Thy-ALLs and other T leukaemias and the overall ~':$ ratio is 6.5:1. 90% of thesg T lineage malignancies have no demonstrable react- ivity with anti-ALL; however, approximately 1D% (9/90) of Thy-ALL cases tested to date (and not included in the survey summarised in Table 1) had a weak but definite expression of the ALL antilgen and when tested were capable of absorbing all fthe anti-ALL binding activity (see Fig. 2). All cases v~ere Thymic-ALLs as judged by their HuTLA+/Ia-/ TdT + phenotype; however, it is interesting that three had weak E-rosetting capacity. Most had other phenotyping features (see Results) which seem to suggest that an infrequent subgroup of ALLs exists with a phenotype intermediate between ihat of the common form of ALL and the Thy-ALL type. These cases have not been followed sufficiently l~ong to determine whether their prognosis falls into that of the Thy-ALL or common ALL groups [40]. It is interesting to note that of six Thy-ALL cell I!nes tested four have a weak membrane expression of~ the ALL antigen [28, 41] and one (MOLT-4) wit h no cell surface reactivity secretes a glycoprotein into the culture medium which binds to anti-ALL serum [and has the same molecular properties as the antigen purified from non-T, non-B or common ALL cell lines (see following paper).

Eight (out of 18) cases diagnosed as AUL were reactive with anti-ALL serum. When tested with

112 MARION ROBERTS et al.

additional markers these cases had the typical overall ALL phenotype.

Similarly, in 49 % of Pht positive CML in blast crisis ant i -ALL binding was demonstrated and the same unique, identical immunological and enzy- matic phenotype established. These positive cases usually reciprocated with cases of blast crisis which had an AML-l ike phenotype: ALL- , HuTLA- , E-, Ia +, H u M A +, TdT- (this study and refs. [5, 29]). In some cases, however, a mixed population of im- mature lymphoid (ALL-like) and myeloid (AML- like) was found to co-exist [42].

Very few other ieukaemias were reactive with anti- ALL serum. Those that were included two children diagnosed as A M L and six diagnosed as primary lymphomas. However, in only one case so far have we identified ALL positive cells in the lymph node biopsy of an unequivocal case of lymphoma. Chronic lymphocytic and prolymphocytic leukae- mias were invariably negative. Ph ~ positive chronic myeloid leukaemia cells were, in contrast to many CMLs in blast crisis, unreactive with anti-ALL serum (but reactive with anti-myeioid serum).

Stable expression of the ALL antigen is usually maintained by leukacmic clones in vivo since cases which were ant i -ALL positive at presentation relapsed subsequently with the same phenotype. In a few cases, however, a phenotype shift occurred resulting in loss or acquisition of the ALL antigen (see Results). Shifts in membrane phenotypes were far more frequent and dramatic in Ph ~ positive CML (with blast crisis) reflecting the substantial intraclonal evolution and competition which can occur in this disease [17, 23, 42].

These results suggest that Ph ~ negative and Ph ~ positive lymphoblastic, and 'undifferentiated' acute leukaemias can share an identical leukaemia asso- ciated antigen, and have an overall phenotype which

is also qualitatively identical with respect to the markers so far available. Although the karyotypic data imply these diseases have distinct aetiologies we suggest that a common phenotype probably reflects an origin from the same cell type or closely related cells. The observations on CML in blast crisis suggest that this cell may be a common pro- genitor of the lymphoid and myeloid l ineages-- equivalent to the so-called pluripotential stem cell (or CFUs) in rodents [43]. The important corollary of this interpretation is that the ALL antigen itself (and the other components of the 'common' pheno- type) is a reflection of normal gene activity of the target cel l - -as opposed to being a ieukaemia specific alteration (i.e. it is a progenitor or stem cell antigen). Thig interpretation is supported by a lack of cross- reactivity of ant i-ALL and oncogenic viruses [3] and more specifically by the demonstration of infrequent non-T, non-B ' lymphoid ' cells with the ALL+/Ia ÷ phenotype in non-leukaemic individuals [15, 23].

Acknowledgements--We would like to acknowledge the essential help and interest of many clinicians and haematologists who participated in this study: Dr. H. Kay (MRC UKALL Trials Office and Royal Marsden Hospital, London); Dr. G. Prentice (Royal Marsden Hospital, London); Drs. A. Lister, R. Woodruff, M. Beard, A. Paxton, S. Johnson and J. Maipas (St. Bar- tholomew's Hospital, Medical Oncoiogy Unit and Haematoiogy Department); Drs. J. Chesselis, N. Rapson and Professor R. Hardisty (Dept. Haematology, Institute of Child Health and Hospital for Sick Children, London); Dr. D. Catovsky and Prof. D. Galton (Hammersmith Hospital, London); Dr. A. Goldstone (University College Hospital, London); Prof. V. Hoffbrand and Dr. K. Ganeshaguru (for TdT assays, Dept. Haematology, Royal Free Hospital, London) and Dr. M. Pippard (Radcliffe Infirmary, Oxford).

We are very grateful to D. Capellaro and D. Delia for experiments using the FACS machine.

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ALL antigen expression 113

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