Hybrid acute leukemia

LeukemiaResearchVOL8, No. 6. pp. 929-936, 1984. Printedin GreatBritain.

0145-2126/84$3.00+0.00 © 1984PergamonPressLtd.

EDITORIAL HYBRID

ACUTE

LEUKEMIA

ISAAC BEN-BASSAT* a n d ROBERT PETER GALEi" *Insitute of Hematology, Chaim Sheba Medical Center TeI-Hashomer and the Sackler School of Medicine, TeI-Aviv University, Israel and tTransplantation Biology Unit and the Department of Medicine (Hematology and Oncology), UCLA School of Medicine, Los Angeles, California, U.S.A. (Received 9 January 1984. Accepted 2 February 1984) Key words: Leukemia, biphenotypic, biclonal, hybrid leukemia.

INTRODUCTION THE ACUTE leukemias, acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML), are characterized by the proliferation of immature lymphoid and myeloid cells, respectively. Most data, including chromosome analyses, studies of glucose-6-phosphate dehydrogenase (G-6-PD) isoenzymes and immunoglobulin (Ig) alloand idiotype expression, and analysis of Ig gene rearrangements, indicate a clonal origin of most cases of acute leukemia. It recent studies 5 0 - > 9 0 % of AML and a lower proportion of those of ALL, have been demonstrated to have clonal chromosome abnormalities [44, 45, 53]. Similarly, clonality of AML has been demonstrated by analysis of G-6-PD expression in G-6-PD heterozgotes [17, 18, 50,]. Finally, most cases of B-ALL, particularly the Burkitt subtype, express only a single lg heavy or light chain allotype [37]. More detailed analyses using Ig gene probes indicate clonal Ig gene rearrangement [10, 24, 25]. Although the clonality of acute leukemia is reasonably well documented, it has been more difficult to define precisely the level of stem cell differentiation at which malignant transformation has occurred. In typical cases of ALL and AML, malignant transformation is postulated to occur at the level of committed stem cells which give rise to either lymphoid or myeloid progeny. In some young patients with AML, malignant transformation may occur at the level of a relatively mature (committed) stem cell such that the malignant phenotype is restricted to granulocytes and monocytes/macrophages; red blood cells and megakaryocytes are not involved [17, 18]. In other patients, malignant transformation appears to occur in a relatively immature (uncommitted) stem cell such that all myeloid elements including RBC arise from the malignant clone [6]. There have also been detailed studies of clonality and of the level of malignant transformation in chronic myelogenous leukemia (CML), a closely related disorder [15]. In CML a specific chromosome translocation, the Ph ~chromosome [t(9;22)], is present in myeloid cells and B lymphocytes [27, 49]. These data as well as studies of G-6-PD expression [16], suggest malignant transformation at the level of a stem cell committed to myeloid and B-, but not T-, lymphocyte development during the chronic phase. In the acute phase some recent studies suggest that, in rare instances, T-lymphocytes may also be involved; this is unusual occurring in less than 1070 of cases [19, 21].

Abbreviations: G-6-PD, glucose-6-phosphate dehydrogenase; CFU-S, colony-forming units-spleen; CML, chronic myelogenous leukemia; AP, acid phophatase; TdT, terminal deoxynucleotidal transferase; MPO, myeloperoxidase: SB, Sudan black; CAE, chloracetate esterase; NSE, non-specific esterase; CALLA, common ALL antigen; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia. Correspondence to." Dr. Robert P. Gale, Department of Medicine (Hematology and Ontology), UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. 929

930

ISAACBEN-BASSATand ROBERT PETER GALE

In mice it is possible to identify relatively uncommitted stem cells, referred to as colonyforming units-spleen (CFU-S), which give rise to both lymphoid and myeloid progeny [46]. Data supporting the presence of a similar pluripotent stem cell in man comes from the aforementioned lymphoid and myeloid involvement in patients with CML. Recently a pluripotent stem cell capable of giving rise to lymphoid and myeloid progeny has been identified in in vitro studies using human cells [14, 26, 30]. As previously described, there are multiple potential sites for malignant transformation in acute leukemia. One would anticipate that in some patients with acute leukemia, malignant transformation might occur at the level of a pluripotent stem cell. This would result in hybrid leukemia with both lymphoid and myeloid features. Because of the potential inportance of these hybrid leukemias to our understanding of the biology of acute leukemia, we critically reviewed reported cases of either biclonal or biphenotypic acute leukemias.

Definitions of biphenotypic and biclonal leukemias We selected the term 'hybrid acute leukemia" to describe leukemias which demonstrated malignant involvement of both myeloid and lymphoid cells. When the hybrid leukemia was characterized by a relatively homogeneous population of leukemia cells each of which demonstrated features of lymphoid and myeloid cells, it was referred to as biphenotypic. If, in contrast, the leukemic population was heterogeneous with some cells demonstrating lymphoid features whereas others demonstrated myeloid features, it was referred to as biclonal. Use of the term 'biclonal' is not intended to imply that the leukemia arose from distinct clones at the stem cell level but rather that its phenotypic expression was biclonal. Most data, in fact, indicate that these biclonal leukemias arise from a single malignant clone. In order for a patient to be characterized as having biclonal leukemia, the two leukemia phenotypes had to be concurrent or occur within six months of one another. This time limit was incorporated to prevent inclusion of cases of AML developing as a consequence of treatment of ALL cytotoxic chemotherapy or radiation, i.e. therapyinduced leukemia. The term 'simultaneous leukemias' was not used since the two forms of leukemia were not always concurrent. Likewise, the term 'mixed leukemia' was avoided since it is imprecise and can iml~ly either biphenotypic or biclonal leukemia. Cases of acute leukemia developing in patients with chronic lymphocytic leukemia, CML, lymphomas, multiple myeloma or other myeloproliferative disorders have been reviewed elsewhere and were excluded from this analysis. We likewise excluded all cases in which the Ph ~ chromosome [t(9;22)} was reported.

Criteria for lymphoid and myeloid involvement Several criteria were used by the original investigators to define the involvement of lymphoid cells in the leukemic process. Generally these included: (l) morphologic features determined by light and electron microscopy; (2) cytochemical staining with periodic acid-Schiff (PAS) or focal staining for acid phosphatase (AP); (3) the presence of terminal deoxynucleotidyl transferase (TdT) activity and (4) immunological markers such. as rosette formation with sheep red blood cells (E-rosettes), or the presence of T- or B-lymphocyte-related antigens detected by reactivity with polyclonal or monoclonal antibodies. Reagents utilized for these studies included antibodies to HLA-DR (Ia), common ALL antigen (CALLA), BA-1, BA-2, B-l, B-2, human T-lymphocyte (HuTLA) antigens and the OKT3, OKT4, OKTS/8, OKT6, OKTI1, T101 monoclonal antibodies. Other, less well defined antibodies, were also used in some cases. Criteria used to indicate involvement of myeloid cells included: (1) morphologic features using light and electron microscopy specifically the presence of cytoplasmic granules and Auer rods; (2) cytochemical stains including myeloperoxidase (MPO); Sudan black (SB); chloracetate (CAE) and non-specific esterase (NSE). In rare cases myeloid involvement was defined by reactivity with antibodies to purported granulocyte or monocyte/macrophage specific antigens such as VIM-DS, OKMI, My-l, MCS-1, MCS-2

Hybrid acute leukemia

93 l

and anti-GEA. In one study antibodies to spectrin or to factor-VIII were used to define erythroid and megakaryocytic involvement respectively [39]. D A T A ANALYSIS The biomedical literature was searched for reported cases of biphenotypic or biclonal leukemia using two overlapping programs of the National Library o f Medicine, Medline and Cancernet. The search was restricted to the time periods and journals for which there was computer access capability ending in January 1984. We also reviewed articles referred to in the above citations. Using this data base, 57 cases of hybrid acute leukemia were identified [3, 4, 7, 8, !3, 22, 23, 28, 29, 31,33-35, 41,43, 48]. Eleven additional cases have recently been reported in abstracts [1, 12, 42]. A critical analysis of these 68 cases revealed that in many instances criteria for indicating lymphoid or myeloid involvement were not convincing. For example, there are several reports of biphenotypic or biclonal leukemia in which lymphoid involvement is based solely on the demonstration of T d T in otherwise typical AML. There are, however, data indicating that T d T reactivity may not be specific to lymphoid cells. In a recent review high levels of TdT were reported in 24 o f 310 cases of AML [1 I]. Similar reservations apply to the use of cytochemical reactivity with PAS or focal A P staining to define lymphoid involvement. Least convincing are data using purported lymphocyte specific antibodies. For example, H L A - D R antigens are present on immature myeloid cells such as CFU-C and monocytes [5, 51]. Normal granulocytes and fibroblasts express C A L L A antigen [9]. Other immunologic tests such as E-rosette formation or cytoplasmic or surface Ig reactivity are more convincing. Documentation of myeloid involvement is less problematic. Although cytoplasmic granules and inclusions [20, 52] including SB positive granules [47] can occur in lymphoid cells, Auer rods and cytochemical reactivity with M P O , CAE, SB arid NSE are reasonably convincing of myeloid involvement. Recently, antibodies have been used to define myeloid involvement; this area is less well-developed and it is likely that some purported anti-myeloid antibodies may not be specific. In order to identify clear-cut cases for more detailed analysis, we utilized relatively restrictive criteria for study inclusion. Lymphoid involvement was defined by the presence of one or more of the foilowing: (1) E-rosette formation; (2) cytoplasmic or surface Is; (3) cytochemical reactivity (AP or PAS) and reactivity with antilymphoid antibodies (CALLA, BA-1, BA-2, etc.) or (4) T d T reactivity with either cytochemical or immune lymphoid characteristics. Myeloid involvement was defined by: (1) cytochemical reactivity (MPO, SB, CAE or NSE), or (2) the presence of Auer rods. Reactivity with purported antilymphoid or antimyeloid antibodies or increased T d T activity alone were considered insufficient criteria for study entry. Eighteen of the 68 reported cases satisfied these more stringent on-study criteria [7, 8, 13, 22, 23, 29, 31, 33-35, 41, 43, 48]. Five cases were biphenotypic, 12 biclonal and one undefined; these cases are reviewed in Table 1. There were eight males and nine females; median age was 16 years (range: 2 months-66 years). In one case neither sex nor age was indicated. Most patients were adults except for patients with the t(4; 1 I) translocation many of whom were children. In 12 patients with biclonal leukemia, seven were simultaneous, four sequential and one undefined. Lymphoid involvement was documented by elevated levels of TdT in 14 cases, PAS reactivity in 11 cases, AP in one case and PAS and A P in three cases. E-rosettes were p~esent in two cases, reactivity with anti-T antibodies in four cases, anti-B antibodies in six cases, anti-CALLA in one case and surface and cytoplasmic Ig in one case. Myeloid involvement was documented by the presence of Auer rods in four cases. M P O was present in five cases; NSE in five cases and SB in one case. Six cases had M P O and other activity including CAE (two cases) NSE (five cases) and SB (one case). Chromosome studies were

+

7

18

M

M

F

+

+

+

4-

+

+

Lymphoid*

PAS,AP

PAS,AP

PAS

PAS

PAS

PAS

PAS

PAS N

PAS

PAS

AP

PAS,AP

PAS

PAS

PAS

SB

Cytochem

ERFC

ERFC, auti-T

BA-I ,BA-2

BA-2

BA-I,BA-2

CALLA

BA-2

Auer rods

Erythroblasts

MPO,NSE

MPO

MPO,SB

MPO,NSE

NSE

**

NSE

MPO,NSE

NSE

NSE

BA-2

BA-2

MPO,CAE,NSE,SB

NSE

MPO(:t:)

MPO(-t-)

MPO

MPO,CAE,NSE

Myeloidt

MPO

Auer rods

Phagocytosis

Auer rods

Auer rods

Morphology

Anti-T

Anti-T

Clg,Slg

Anti-T

Cytochem Immune

No Phj

NI

t(4;I 1)

t(4;l I)

t(4; I 1)

t(4; 11)

t(4;I I)

t(4; 1 l)

No Ph ~

Abnormal

NI

t(4;l I)

NI

NI~

Chromosome

+

+

+

¶

+

+

+

+

+

+

+

+

4,

4.

4-

4

+

+

48

43

41

35

34

34

34

34

34

34

33

3I

29

27

22

13

8

7

l0

8

7

6

3

2

3

2

§

I

2

Ref. Patient

Hybrid leukemia Biphenotypic Biclonal

*TdT, terminal deoxynucleotidal transferase; Cytochem. cytochemistry; PAS, periodic acid-Schiff; AP, acid phosphatase; cig, cytoplasmic Ig; SIg, surface lg; BA-I, BA-2, CALLA, anti-T, polyclonal or monoclonal antibodies; E-RFC, sheep erythrocyte rosette-farming cells. ~'Cytochem, cytochemist~y; SB, Sudan black; MPO, myeloperoxidase; NSE, non-specific esterase; CAE, chloracetate esterase. ~/Nl, normal. §This case is identical to [23], patient I. IWeak to moderate PAS in cases 9-14. ¶Type of hybrid leukemia could not be determined from available data. **Pseudoperoxidase activity o f hemoglobin.

64

17

F

5

28

15

16

F

2/12

14

F

M

2/i 2

3/12

F

12

33

11

F

13

7

30

9

l0

M

M

4.

+

6

7

F

+

4,

+

+

TdT

+

33

6

F

M

M

M

F

Sex

8

16

55

5

2/12

3

4

35

66

1

2

Age

Case

TABLE 1.

g

"~

O

t~

z

). t%

b.I

Hybrid acute leukemia

933

reported in 14 cases; seven had t(4;11) and four had normal karyotypes. One karyotype was reported as abnormal and two as having no Ph'-chromosome. By study design no cases with the Ph' chromosome were included. DISCUSSION Most investigators believe that hybrid leukemias, biphenotypic or biclonal, can occur in patients with acute leukemia. We were surprised to find only 68 reports of hybrid leukemia in the biomedical literature, only 18 o f which satisfied our prospective on-study criteria for both lymphoid and myeloid involvement. The actual incidence may even be lower since only 14 cases had chromosome studies performed and the four unstudied cases might have had the Ph' chromosome. Interestingly, seven o f the cases studied had a specific chromosome translocation, t(4;l 1). Classification of these cases is problematic and some authors initially reported these patients as having non-B, non-T A L L [2]. More recently several groups have concluded that these cases represented leukemia, of an 'early myeloid progenitor cell' [32, 34]. Using our criteria seven patients had evidence of both lymphoid and myeloid involvement (Table 1) and were classified as hybrid leukemias. It is interesting that in patients with the t(4; 11) transiocation as well as in a substantial proportion of the other cases of hybrid leukemia in this study, myeloid involvement was predominantly monocytic rather than granulocytic. This may suggest a closer relationship between lymphoid and monocyte progenitors or, more likely, that NSE is not an adequate discriminator between the lymphoid and myeloid lineages. A patient with acute leukemia with two concurrent clones, one diploid and one tetraploid, was recently briefly reported [1]. The diploid cells had low levels o f RNA and were TdT-positive whereas the tetraploid cells had high levels of RNA and were SBreactive. The authors concluded that the patient had a biclonal leukemia with lymphoid and myeloid involvement. Although this case failed to meet our on-study criteria it is one o f the few instances in which purportedly distinct clones were identified on the level of DNA content. Given the frequent development of biphenotypic and biclonal leukemias in C M L and its occasional occurrence in other hematologic malignancies, why are hybrid acute leukemias seemingly so rare? One possibility is that hybrid leukemias occur more frequently but are undetected. This would not be surprising since once the diagnosis of either A L L or A M L is established it is uncommon to perform detailed leukemia marker analyses of the excluded phenotype. Similarly, when only a proportion o f the leukemia cells express a specific marker such as M P O , lack of reactivity of the residual leukemic population is typically ascribed to immaturity of the cells or to insensitivity of the test. Thus, in order to accurately diagnose hybrid leukemias one need be aware of this possibility and use definitive discriminatory tests of both phenotypes. The latter requirement is problematic and it is frequently necessary to utilize a battery of tests, no one o f which is definitive. Therefore, a substantial number of cases of hybrid leukemias may be currently undiagnosed. A similar situation existed in CML where, until recently, the development o f lymphoid transformation in up to one third of the cases was unrecognized. A second potential explanation for the apparent rarity o f hybrid acute leukemias compared to CML is that the relatively brief survival of patients with acute leukemia restricts the likelihood of clonal de-evolution and a subsequent switch in phenotypic expression. Thus, although patients with CML may survive with active leukemia for 3--4 years, prolonged survival of patients with active acute leukemia is not possible; patients either achieve a remission or die of uncontrolled disease. Another possible explanation of this disparity in the incidence of hybrid leukemias is a difference in the level o f stern cell commitment at which malignant transformation occurs in CML vs acute leukemia. It is also possible that we have been too strict in the on-study criteria for defining hybrid leukemias;

934

ISAACBEN-BASSATand ROBERTPETER GALE

we felt it, however, judicious to be critical in view of the important biologic implications of these cases. Clearly the hybrid acute leukemias are an important area for future investigation. It will require additional data to determine if they are rare or whether increased awareness and the availability of more discriminative leukemia markers will permit more accurate identification of these interesting cases Acknowledgements--Part of this study was performed while Robert Peter Gale was the Meyerhoff Visiting Scientist at the Department of Cell Biology, Weizmann Institute of Science. Shaun Taylor prepared the manuscript and Dr. C. N. Muller-B&at kindly reviewed the manuscript. Note added in proof: Recently two additional cases have been reported as hybrid leukemia. One (EMAMI A. et al. (1983) A m J. Ped. HematoL/Oncol. 5, 341) did not meet our on-study criteria. A second (UEDA T. et al. (1984) Leukemia Res. 8, 63) qualifies as a biclonal simultaneous case.

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ISAAC BEN-BASSATand ROBERT PETER GALE

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