All masquerading as aul

LeukemiaResearchVol.7. No. 6, pp. 735-746, 1983 Printedin Great Britain.

0145-2126/8353.00 + .00 ¢) 1983PergamonPres,, lad.

ALL MASQUERADING

AS A U L

M. F. GREAVES,* R. BELL,~ J. AMESS§ and T. A. LISTER~ *Membrane Immunology Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K., :~iCRF Department of Medical Oncology, St. Bartholomew's Hospital, West Smithfield, London ECIA 7BE, U.K. and §Department of Haematology, St. Bartholomew's Hospital, West Smithfield, London ECIA 7BE, U.K.

(Received 25 April 1983, Revision accepted 2 May 1983) Abstract--Of 597 cases of acute leukaemia in adults (>16 years) seen at St. Bartholomew's Hospital, London, between May 1973 and January 1982, 412 were diagnosed as AML, 103 as ALL and 58 as Philadelphia chromosome positive blast crisis of CML (13 presenting as acute ieukaemia and 45 having a prior chronic phase). The remaining 24 cases were considered to be acute undifferentiated leukaemia. Twenty-one of the latter were investigated using a panel of immunological markers at diagnosis and/or retrospectively using frozen cell suspensions. Eighteen out of 21 were shown to have a predominantly 'lymphoid' phenotype which comprised 12 cases of common ALL (two of whom were Ph~ positive), three cases of null-ALL, one case with a probable early thymic phenotype, and two cases with a monoclonal B lymphoblast phenotype. One 'common ALL' and one 'null-ALL' had a significant proportion of pre-B (cytoplasmic p chain + ) cells. One other case reacted with anti-myeloid sera. Leukaemic blasts from two patients were unreactive with all markers tested. No cases of glycophorin positive erythroleukaemia or anti-platelet (glycoprotein I) positive leukaemia were detected. These observations suggest that the overwhelming majority of acute leukaemias have an identifiable affiliation to the lymphoid or myeloid lineages and that patients diagnosed haematologicaily as 'AUL' might benefit by therapy appropriate for their ieukaemic cell type.

Key words: Differentiation, monoclonal antibody, leukaemia.

INTRODUCTION A SMALL proportion (5-15°70) of acute leukaemias in adults and children have been difficult to classify by available morphological and cytochemical methods [3]. These cases are variably referred to as 'unclassifiable' or 'undifferentiated' leukaemias, their negative marker status being considered to reflect either an origin from haemopoietic stem cells (which are presumed to be similarly 'marker-less') or a loss of features associated with malignant progression. The development of immunological and enzymatic markers has resulted in a more precise approach to cell identity in which a composite phenotyoe may more reliably place the dominant leukaemic clone in relation to its normal counterparts [19, 14, 18]. Although no definitive marker for pluripotential stem cells is yet available it might be anticipated that these newer probes would identify some unequivocal lymphoid or myeloid phenotypes associated with the 'undifferentiated' morphology. This view is supported by the observation that cells from some cases of AUL react with antisera to the common (non-T, non-B) subclass of ALL [33]. Similarly, some AUL may be TdT positive [4, 23]. These data indicate a possible lymphoid origin of some AUL although this interpretation must be considered with some caution in the light of reports that occasional myeloid leukaemias may also be TdT positive [5]. Cells from some cases of AUL may also be reactive with antisera considered to be specific for myeloid cells [35]. Abbreviations: ALL, acute lymphoblastic leukaemia; A UL, acute undifferentiated leukaemia; CML, chronic myeloid leukaemia; CGL, chronic granulocytic leukaemia; AML, acute myeloblastic leukaemia; TdT, terminal deoxynucleotidal transferase; lg, immunoglobulin; E, erythrocyte; FACS, fluorescence activated cell sorter. tCurrent address: Section of Medical Oncology, University Hospital, 75 E. Newton Street, Boston, MA 02118, U.S.A. 735

736

M . F . GREAVESet al.

We have investigated the cell type affiliations of leukaemic cells from a series of patients diagnosed and treated at a single centre and whom were considered to have AUL both initially and on review by one of the authors (J.A.). In addition to TdT we used a panel of monoclonal antibodies that are considered to react selectively with cells of different lineages (Table 2). The results suggest that 'undifferentiated' leukaemia may not exist.

MATERIALS AND METHODS Patients Between May 1973 and January 1982 597 adult patients (i.e. >16 years) with acute leukaemia were diagnosed at St. Bartholomew's Hospital, London. O f these cases, 411 were AML and 103 ALL. Fifty-eight cases had a pht chromosome (22q--) and were either presenting as acute leukaemia (No. 13, see ref. [2]) or were blast crises evolving from a prior, clinically detected chronic phase o f CGL (No. 45). Twenty-four o f the remaining 25 cases were considered upon review o f the slides (by J.A.) to be haematoiogically undifferentiated by virtue o f their morphological features on Romanowsky staining and lack of Periodic Acid Schiff or Sudan Black staining. One case initially diagnosed as AUL was re-classified as AML. Presenting features o f these AUL patients are listed in Table 1. They were treated with a variety of chemotherapeutic regimes as also indicated in Table 5 (see Results). The treatment policy for A U L changed during the period o f this study. Initially, young patients with lymphadenopathy and splenomegaly received treatment appropriate for ALL (OPAL, ref. [25]; H E A V ' D = modified OPAL); other patients received A M L directed therapy (Barb I-V, ref. [9]; Barts VIII, ref. [26]). Subsequently the policy changed so that all patients received ALL treatment.

TABLE I. AUL PATIENTS*

% Blasts No.

I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Sex

~ ~ ~ Q ~ 9 Q ~ o" a" o" ce ~ o" Q ~ o" ~ o" o" ~ o" Q ~

Age

16 39 41 15 31 40 21 19 60 56 30 25 54

53 36 15 50 66 62 25 62 30 74 74

W BC × 109/I

BM

Blood

49.8 11.8 1.9 12.8 4.6 6.9 18.3 4.4 3.0 3.2 102.4 15.1 0.9 5.5 78.4 25.4 13.7 16.5 2.1 14.9 1.1 3.7 3.5 1.9

91 95 65 95 95 70 95 95 90 95 99 99 95 71 90 90 70 95 80 85 95 47 95 90

84 80 26 78 21 27 67 16 33 25 94

54 13 71 88 74 68 18 41 50 16 3 25 0

*All patients were white Caucasians except No. 1 who was black. Cells Blood and bone marrow from all but two o f these 24 AUL cases were tested with a standard battery o f immunological markers as part o f a routine immuno-diagnostic service (see refs. [20, 13]). For the purpose of this study employing a more extensive marker analysis, frozen blood leukaemic blasts kept from diagnosis in a liquid nitrogen store were used. Sufficient stored cells were available in 21 of the 24 patients. Results with standard or

Undifferentiated leukaemia

737

routine markers: anti-cALL, anti-DR, anti-lg, E rosettes and TdT (enzymatic assay 1976-1978, see ref. [22]; immunofluorescence 1979-1982, see ref. [4]) were concordant on fresh and frozen material. Following thawing and washing (twice) cells were centrifuged in ficolt-isopaque (1.099 g/cm3; 400 g, 20°C, 40 min). L e u k a e m i c cell markers

A panel of cell surface markers including monoclonal antibodies was used as listed in Table 2. The preparation, application and specificity of these markers are discussed in the references given. TdT was detected by indirect immunofluorescent staining of fixed cytospin preparations using an affinity purified goat antibody to TdT [4]. Binding of antisera was revealed by fluorescein isothiocyanate labelled anti-rabbit, goat or mouse lg and evaluated using a Standard 16 Zeiss photomicrc~scope with incident illumination as well as with the Fluorescence Activated Cell Sorter (FACS). Sheep anti-human lg antibodies used for detecting cell surface lg were fluorescein labelled F(ab')2 preparations (Kailestad Labs). Staining for cytoplasmic immunoglobulins (/~, y, x, ~.) was carried out on cytospin preparations fixed in 95°70 ethanol-5% acetic acid using affinity purified and fluorochrome labelled antibodies specific for human immunoglobulin heavy or light chains. All antisera and antibodies had been titred to give saturated staining on known positive cells using the FACS or, in the case of intracellular staining reagents, on appropriate positive cells using fluorescence microscopy.

TABLE2. CELLMARKERSUSED Ref. 1. Conventional antisera: R. anti-cALL (gpl00) R. anti-monocyte (M l ) R. anti-myeloid (M2) G. anti-Ig (Ig, x, ~.) R. anti-TdT 2. Monoclonal antibodies: anti-erythroid (glycophorin) anti-platelet (gly.coprotein l) anti-cALL (gpl00) anti-HLA-ABC anti-HLA-DR anti-T:

17 1

32 4

LICR.LON.RI0

10

AN51 J5 W6.32 DA2

28 31 7 "7

I.

Pan T

WT1 Leu-1 (L17F12)

37 11

2. 3. 4.

Thymic Mature T Thymic + mature T subset

NA134 % OKT3

27

5. 6.

E rosette receptor Transferrin receptor

30 OKT4 OKT8 OKTI 1

38

OKT9

36

3. Others: Sheep erythrocyte rosettes

RESULTS B l o o d l e u k a e m i c b l a s t cell p o p u l a t i o n s w e r e a v a i l a b l e f r o m 21 p a t i e n t s d i a g n o s e d as A U L . T h e s e w e r e i n v e s t i g a t e d b y a n e x t e n s i v e p a n e l o f i m m u n o l o g i c a l m a r k e r s (see T a b l e 2). P o s i t i v e r e s u l t s (e.g. p o s i t i v i t y w i t h a n t i - c A L L ) w e r e c o n f i r m e d b y r e p e a t testing.

85 >90

55 82

95 79

9

13 14 15

10 11 12

70 90

95 60 95

6 7 8

75 55 55

50 >90 85

75 90 80 >95

85 91 56 >95 29

1 2 3 4 5

TdT

cALL/ gpi00

Patient No.

25

25 -------

-----

cyt. ~ut

D

m

m

Smlg,

Lymphoid markers (070)

h

D

n

m

m

T§

T A B L E

3. C E L L

D

D

ER

M A R K E R

m

i

MI

R E S U L T S

A

U

L

B

M2

m

PI

m

m

m

i

Glyco

'Myeloid' markers (07e)*

IN

90 90 >95

>95 90 95 64 90 44 76 80 84

80

87

HLA-DR

>95 >95 >95

>95

>95 90 >95 >95 >95

85

95

HLA-ABC

Others (07e)

t~

<

m >

K

.

. .

19

20 21

.

. .

--

--

60

TdT

.

. .

.

--

--

--

cyt. /tt

. .

.

(x)

79(g)

69

20

Smlg

.

. .

--

.

25

T§

. .

. --

30

ER

. .

.

. .

+

MI

.

. .

.

+

M2

.

. .

_

PI

. .

'Myeloid' markers (%)*

--

--

Glyco

--

90

45

12

HLA-DR

>95 90

>95

>95

>95

>95

HLA-ABC

Others (%)

glycophorin.

*See Table 2. ]'Cytoplasmic IJ chain. :l:Surface membrane immunoglobulin. §Leu-! and NAI34. - - = <5e/0, blank = not assessed. P l = monoclonal (AN51); anti-platelet glycoprotein I. Glyco = monoclonal ( L I C R . L O N . R I 0 ) anti-

--

--

17

--

16

18

cALL/ gpl00

Patient No.

Lymphoid markers (%)

TABLE 3, (CONrINUEI))

1"0

g

M . F . GREAVESetal.

740

The results are summarised in Table 3. Sixteen cases (Nos. 1-16) were positive for TdT (Fig. 1). Six of these had been originally (at the time of diagnosis) assayed enzymatically for TdT and gave positive values of between 18.8 and 212 units/10 s cells. These TdT positive AUL could be further divided into those that were reactive (see Fig. 2) with rabbit and monoclonal (JS) anti-cALL (gpl00 antigen) (i.e. common ALL phenotype; cases 1-12) and those that were not (i.e. null-ALL phenotype; cases 13-15). Cases 11 and 12 were found to have the Philadelphia chromosome and one of these (case 1I) subsequently developed a stable Ph' positive chronic granulocytic leukaemia (i.e. reverted to a chronic phase). These lymphoid cases were unreactive with anti-myeloid reagents (see Table 3). Two cases (Nos. 17 and 18) expressed cell surface immunoglobulin and were both x light chain restricted (Fig. 2). In one case (No. 17) the same B cell phenotype was demonstrated in a subsequent relapse (Fig. 2). One case (No. 16) was provisionally typed as null-ALL (TdT +, c A L L - ) but, in contrast to the other three such cases in this series, and null-ALL in general [13], was DR-. This observation raised the possibility that this patient might have an immature T/thymic A L L even though the cells were only minimally reactive with the anti-T monoclonal L17F12 (Leu-l). Cells from this patient were retested with a battery of anti-T cell monoclonals (see Table 4). The pan-T cell monoclonal WT1 was positive on the majority of blast cells. NAI34 (cf. OKT6) was positive on a relatively small proportion (12%); however, double staining revealed that these were TdT + leukaemic cells.

TABLE 4. T CELL ANTIGENS EXPRESSED BY LEUKAEMIC CELLS IN

A U L rATIENT NO. 16

Monoclonal antibody

07o + ve cells

Leu-I OKT3 OKT4 NAI34 OKT8 OKT9 OKTI 1A WTI

25 10 30 12" 5 60 30 88

TdT Blasts

60 74

* N A I 3 4 + cells were TdT + .

All cases were tested for cytoplasmic lg/a chains, i.e. pre-B phenotype (cf. refs. [39, 21]). Two cases had a significant proportion of positive cells (20-25070), patient No. 6 with a common A L L phenotype and patient No. 5 with a null-ALL phenotype. Neither of these cases expressed cytoplasmic light chains or cell surface lg. One case (No. 19) was reactive with anti-myeloid sera but negative for other markers including those identifying lymphoblastic leukaemia. The two remaining cases were of uncertain phenotype. One (No. 20) was found on more recent re-testing to be reactive with monoclonal antibody OKM-I which usually reacts selectively with relatively mature myeloid ceils [6]. This was an unexpected result since the same cells were unreactive with the more broad spectrum rabbit anti-myeloid sera.

it

]=IG. 1. Immunological markers in 'AUL'. A,B: Phase contrast and fluorescence of 'AUL' cells stained with anti-TdT (case No. 9). C: Fluorescence Activated Cell Sorter analysis (FACS) of AUL cells stained with monoclonal J5 anti-cALL versus control (c) ( = patient no. 11). Vertical axis: relative cell number; horizontal axis: relative cell size (light scatter). D: FACS analysis of ' A U L ' with a monocional (~) B cell phenotype (patient No. 17 in relapse), x: stained with anti-kaplm light chain; ~: control with anti-larnbda light chain. 741

743

Undifferentiated leukaemia

T h e c l i n i c a l c o u r s e o f this g r o u p o f 24 A U L is s u m m a r i s e d in T a b l e 5. N i n e o f t h e twelve patients including the two that were Pht positive achieved a complete remission. Seven of those nine remitters, and two of the three non-remitters, had received therapy a p p r o p r i a t e f o r A L L ( O P A L o r H E A V ' D ) . O f t h e r e m a i n i n g 12 p a t i e n t s o n l y t w o a c h i e v e d c o m p l e t e r e m i s s i o n . O n e p a t i e n t ( N o . 22) w a s n o t e v a l u a t e d w i t h m a r k e r s , the o t h e r t y p e d as a B - A L L a n d h a d a r e m i s s i o n l a s t i n g 15 m o n t h s . T h e t h r e e p a t i e n t s w h o t y p e d as n o n - A L L ( N o s . 19, 20, 21) all r e c e i v e d t h e r a p y a p p r o p r i a t e f o r A L L ( O P A L ) , b u t all t h r e e f a i l e d to r e m i t . TABLE5. CLINICALASSESSMENT Patient No.

CR

Rm. DuE (days)

Survival (days)

Therapy schedule

1 2 3 4 5 6 7 8 9 l0

No No Yes Yes Yes No Yes Yes Yes Yes

--99 39 878 -281 634 > >

112 16 434 630 >1440 14 331 >670 > >

OPAL HEAV'D B VIII BV OPAL B VIII HEAV'D HEAV'D HEAV'D HEAV'D

Ph I + cALL: 11 12

Yes Yes

>268 ! >25

>2938 >175

OPAL HEAV'D

13 14

No No

---

>116 326

15

No

--

18

HEAV'D B VIII B VIII

16

No

--

540

17

Yes

18

No

--

>1449 3

19

No

--

86

OPAL

20 21

No No

---

65 66

OPAL OPAL

Untested: 22 23 24

Yes No No

419 ---

cALL:

Null-ALL:

T-ALL: OPAL

B-ALL: 469

OPAL No therapy

Myeloid: Uncertain:

1127 92 11

B VIII No therapy HEAV'D

OPAL = Vincristine, Prednisolone, Adriamycin, L-aslmraginase. HEAV'D = OPAL + Cyclophosphamide and dexamethasone instead of Prednisolone. B V = Daunorubicin + Cytosine arabinoside. B Vlll = Adriamycin, Vincristine, Prednisolone, Cytosine arabinoside. DISCUSSION T h e t e r m ' u n d i f f e r e n t i a t e d ' is u s e d in r e l a t i o n t o l e u k a e m i a t o d e n o t e m a l i g n a n t cells whose morphological and cytochemical features provide no evidence for maturation or

744

M.F. GREA'VESetal.

cell type identity. This is generally taken to reflect either an origin (and presumed maturation arrest) from normal cells that are similarly undifferentiated or alternatively the loss of recognisable features as a consequence of malignancy (i.e. anaplasia). The former concept is recognised also in the term 'stem cell leukaemia' which is sometimes applied. These terms and concepts require re-evaluation in the light of advances in our understanding of the biology of differentiation and in the recognition of individual cell types by selective immunological and enzymatic markers. The term 'undifferentiated', although possibly having some empirical value and clinical correlates, is a misnomer since all cells, including stem cells, in the bone marrow and other tissues (e.g. epithelia) are differentiated, i.e. highly restricted in terms of their developmental or cell lineage options. This genetic commitment is not clearly reflected in morphological features but will have some distinctive molecular markers. Stem cells, progenitor cells and malignant cells arising at and arrested in similar developmental positions can be expected to express similar features of immature cells. Acute leukaemias and probably many other cancers (e.g. epithelial carcinomas) probably arise from progenitor or stem cell populations and retain phenotypic features indicative of such an origin. Lymphoid leukaemias and to a lesser extent non-lymphoid leukaemias can now be subdivided into groups whose detailed cell surface phenotype and pattern of enzyme expression reflects an apparent fidelity to lineage or cell type and identifies an approximate cell lineage position at which the majority of the leukaemic cells appear to be arrested [l 8, 15]. Lymphoblastic leukaemias are not necessarily completely or irreversibly frozen in such early developmental positions and, significantly, marker phenotype may not directly identify the actual target cells for the leukaemia (e.g. lymphoblastic phenotype in Ph ~ positive CGL originating in pluripotential stem cells; ref. [16]). The majority of acute leukaemias can be classified as lymphoblastic or myelogenous by morphological and enzymatic criteria. Extensive immunological, biochemical and molecular analysis of the former group (ALL) has established that these leukaemic cells have the features of precursor cells or immature cells in the T or B lymphocyte lineages [30, 13]. Myeloid leukaemias similarly express features of immature cells with, in different categories, varying degrees of maturation. Although the precise haematological criteria for identifying acute leukaemia as lymphoid or myeloid differ between different centres, most haematologists recognise that a significant proportion of cases are mixed or of equivocal cell type. The proportion of such cases is between 2 and 15o70 in various centres [3] and in our own series reported here was 4o70..The strategy for dealing with such leukaemias varies. They may be termed 'stem cell', 'undifferentiated' (i.e. AUL) or 'nonlymphoid'. Their treatment varies and may be selected on the basis of the age bias of lymphoid versus myeloid leukaemias, i.e. young AUL treated as ALL and older patients treated as AML. In the group of patients reported here, 19 or 22 could be affiliated with lymphoid or myeloid lineages. Twelve had a common ALL phenotype (Nos. 1-12, Table I), confirming our previous report based on a multi-centre study [33]. Two of these twelve had a Philadelphia chromosome and one of these (No. 11) subsequently reverted to chronic granulocytic leukaemia. We [2] and others [29, 34] have previously reported patients presenting with Ph ~ positive ALL or (less frequently) AML. It is known that in some instances Ph ~ALL may convert into a chronic granulocytic leukaemia [8]. These data therefore indicate that AUL is heterogeneous and that in most cases can be identified as lymphoid and, more rarely, myeloid. This is a phenotypic grouping and does not prove unequivocally that these cells are truly equivalent to lymphoid restricted or myeloid restricted precursors. This would require knowledge of pluripotential stem cell phenotype, induction of maturation along the lymphoid or myeloid axis or molecular analysis. Since a substantial proportion of ALL cases or lymphoblasts synthesise

Undifferentiated leukaemia

745

immunoglobulin heavy chain [39, 21] and the majority have re-arranged light chain and/ or heavy chain immunoglobulin genes [24],. it is likely that most non-T ALL, including common ALL, are equivalent to B cell precursors. We have recently found that patient No. 11 does have re-arranged ta chain genes in his Ph 1+ AUL phase but has a germ line configuration in his subsequent phase of CGL [12]. The identification of a substantial proportion of AUL as ALL is in part supported by the subsequent clinical course observed in this relatively small group of patients and indicates that ALL therapy may be appropriate for AUL patients whose cells have ALL markers. Acknowledgements--We are grateful to Barbara Kirk for chromosome studies, Gita Hariri, Winston Verbi and Jean Robinson for cell marker tests, and Drs. J. Ritz, G. Goldstein, W. Tax, A. McMichael, W. Bodmer, R. Levy and P. Edwards for kindly supplying monoclonal antibody reagents. We thank Jackie Needham for typing the manuscript.

REFERENCES 1. BAKERM., FALK R. E., FALK J. & GREAVES M. F. (1976) Detection of monocyte specific antigen on human acute leukaemia cells. Br. J. Haemat. 32, 13. 2. BEARD M. E. J., DURRANT J., CATOVSKY D., WILTSHAW E., AMESS J. L., BREARLEY R. L., KIRK B., WRIGLEY P. F. M., JANOSSY G., GREAVESM. F. & GALTON D. A. G. (1976) Blast crisis of chronic myeloid leukemia (CML) - - I. Presentation simulating acute lymphoid leukaemia (ALL). Br. d. Haemat. 34, 167. 3. BESSlSM. & BRECHER G. (Eds.) (1975) UnclassifiableLeukemias. Springer-Verlag, Berlin. 4. BOLLUMF. (1979) Terminal deoxynucleotidyl transferase as a hemopoietic cell marker. Blood.~4, 1203. 5. BRADSTOCK K. F., HOFFBRAND A. V., GANESHAGURO K., LLEWELLIN P., PATTERSON K., WONKE B., PRENTICE A. G., BENNETT i . , PIZZOLO G., BOLLUM F. J. & JANOSSYG. (1981) Terminal deoxynucleotidyl transferase expression in acute non-lymphoid leukaemia: an analysis by immunofluorescence. Br. J. Haemat. 47, 133. 6. BREARO J. M., REINHERZ E. L., KUNG P. C., GOLDSTEIN G. & SCHLOSSMANS. (1980) A monoclonal antibody reactive with human peripheral blood monocytes. J. lmmun. 124, 1943. 7. BRODSKYF. M., PARHAM P., BARNSTABLEC. J., CRUMPTON M. J. & BODMERW. F. (1979) Hybrid myeloma monoclonal antibodies against MHC products. Immunol. Rev. 47, 3. 8. CATOVSKYD. (1979) Phi-positive acute leukaemia and chronic granulocytic leukaemia: one or two diseases? Br. J. Haemat. 42,493. 9. CROWTHER D., POWLES R. L., BATEMAN C. J. T., BEARD M. E. J., GAUCI C. L., WRIGLEY P. F. M., MALPAS J. S., HAMILTON-FAIRLEYG. & BODLEY-SCO'I-I"R. (1973) Management of adult acute myelogenous leukaemia. Br. reed. J. 1,131. 10. EDWARDS P. A. W. (1980) Monoclonal antibodies that bind to the human erythrocyte-membrane glycoproteins glycophorin A and Band 3. Biochem. Soc. Trans. 8,334. 11. ENGELMANE. G., WARNKE R., FOX R. I., DILLEY J., BENIKEC. J. & LEVY R. (1981) Immunologic studies of a human T lymphocyte antigen recognised by a monoclonal antibody. Proc. natn. Acad. Sci. U.S.A. 78, 1791. 12. FORD A. M., MOLGAARDH. V., GREAVESM. F. & GOULD H. J. (1983) Immunoglobulin gene organisation and expression in haemopoietic stem cell leukaemia. EMBO Journal 2, 997. 13. GREAVESM. F. (1981a) Analysis of the clinical and biological significance of lymphoid phenotypes in acute leukemia. CancerRes. 41, 4752. 14. GREAVESM. F. (1981b) Monoclonal antibodies as probes for leukaemic heterogeneity and haemopoietic differentiation. In Leukemia Markers (KNAPP W., Ed.), p. 19. Academic Press, New York. 15. GREAVES M. F. (1982a) 'Target' cells, cellular phenotypes and lineage fidelity in acute leukaemia. J. cell. PhysioL suppl. 1,113. 16. GREAVES M. F. (1982b) 'Target' cells, differentiation and clonal evolution in chronic granulocytic leukaemia: A 'model' for understanding the biology of malignancy. In Chronic Granulocytic Leukaemia (SHAw M. T., Ed.), p. 15. Praeger, New York. 17. GREAVES M. F., BROWN G., RAPSON N. & LISTER T. A. (1975) Antisera to acute lymphoblastic leukaemia cells. Clin. Immunol. Immunopath. 4, 67. 18. GREAVES M. F., DELIA D., ROBINSON J., SUTHERLAND R. & NEWMAN R. (1981a) Exploitation of monoclonal antibodies: A 'Who's who' of haemopoietic malignancy. Blood Cells 7, 257. 19. GREAVES i . F. & JANOSSY G. (1978) Patterns of gene expression and the cellular origins of human leukaemia. Biochim. biophys. Acta 516, 193. 20. GREAVES M. F., JANOSSY G., PETO J. & KAY H. (1981b) lmmunologically defined subclasses of acute lymphoblastic leukaemia in children: their relationship to presentation features and prognosis. Br. J. Haemat. 48, 179. 21. GREAVESM. F., VERBI W., VOGLER L., COOPER M., ELLIS R., GANESHAGURUK., HOFFBRAND V., JANOSSY G. & BOLLUMF. J. (1979) Antigenic and enzymatic phenotypes of the pre-B subclass of acute lymphoblastic leukaemia. Leukemia Res. 3, 353.

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