Childhood biphenotypic leukemia: Detection of mixed lymphoid and myeloid populations in bone marrow specimens

Childhood Biphenotypic Leukemia: Detection of Mixed Lymphoid and Myeloid Populations in Bone Marrow Specimens A SCHMII-r-GRAFF, MD,* H, JORGENS, MD,t A REIFENHAUSER,MD,t

D. SCHWAMBORN,MD,t AND U. GOBEL, MDt In a retrospective study, consecutive bone marrow biopsies and smears from 104 children with leukemia were analyzed for expression of lymphoid and myeloid lineage-associated features.

MATERIALS AND METHODS

Eighty-six cases were diagnosed as acute lymphoblastic leukemia (ALL), 14 cases as acute non-lymphocytic leukemia (ANLL), and one case as chronic myelogenous leukemia (CML). Finally, three children were classified as biphenotypic leukemia demonstrating mixed populations of lymphoid and myeloid blast cells from the onset of the disease. Thus, leukemia with a dual phenotype was assessed in 2.9% of all cases examined. The recognition of bilineage origin even by conventional methods such as morphology, cytochemistry and marker studies may be important for the selection of an effective treatment. HuM PATHOL 19:651--656. 9 1988 by W.B. Saunders Company.

Smears from peripheral blood and bone marrow were stained with May-Gr~nwald-Giemsa (MGG) and tested by published procedures as follows: naphtholAS-D c h l o r o a c e t a t e esterase, m y e l o p e r o x i d a s e (MPO), alpha-naphthyl acetate esterase, acid phosphatase, and the periodic acid-Schiff (PAS) reaction. Decalcified paraffin-embedded bone marrow biopsies were stained with hematoxilin eosin, Giemsa, Gomori's reticulin stain. Cytochemical reactions for naphthol-AS-D chloroacetate esterase, acid phosphatase with tartrate inhibition, and PAS were performed. Fresh samples of peripheral blood and bone marrow were assayed for terminal desoxynucleotidyl transferase (TdT; Bethesda Research Laboratory, Bethesda, MD) and by a panel of commercially available monoclonal antibodies including OKT 3, OKT 4, OKT 8, OKT 11, OKM 1 (Ortho Pharmaceutical, Raritan, NJ), Leu-4, Leu-12, Leu-M 1, common acute lymphoblastic leukemia antigen (CALLA; BectonDickinson), and BA1 (Hybritech, San Diego, CA). An indirect immunofluorescent staining was used with a fluorescein-conjugated goat anti-mouse Ig antiserum (Dakopatts). In addition, immunologic phenotype was studied by microcytotoxicity assay at the Department of Immunogenetics, University of Essen. For electron microscopy, mononuclear cells were fixed in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, postfixed in 1% osmium tetroxie, embedded in epon and stained with uranyl acetate and lead citrate. Chromosome analysis was performed at the Cytogenetic Laboratory, Pediatric Hospital, University of GieBen. Karyotyping with the trypsin-Giemsa banding technique was performed on bone marrow aspirates obtained at the time of diagnosis. The ceils were cultured for 24 hours in RPMJ 1640 medium supplemented with 20% fetal calf serum. Methotrexate and thymidine were added to the cultures for 17 and 4.20 hours, respectively. Cells were then exposed to colcemid for 10 minutes. Populations were not separated by FACS according to size or surface antigens prior to cytogenetic analysis.

Most acute leukemias can be classified as lymphoblastic or myelogenous by morphologic, cytochemical, or i m m u n o l o g i c m a r k e r studies. T h e F r e n c h American-British (FAB) Cooperative Group classification scheme is based on the morphologic similarity of the leukemic cells to normal counterpart cells, t In acute myeloid leukemias, the morphologic variants reflect the degree of maturation. It has generally been agreed that leukemic cells are phenotypically similar to normal hematopoietic progenitors or display minimal deviation or asynchrony of maturation. 2 Evidence was obtained indicating that most leukemias are of clonal origin 3 and may result from an uncoupling between the ability to proliferate and to differentiate. 4 However, at least some cases of acute leukemia obviously show deviation f r o m n o r m a l hematopoietic differentiation programs. 5 Considerable interest has recently been directed to acute leukemias displaying both lymphoid and myeloid features. 6'7 This prompted us to review consecutive bone marrow biopsies and smears from 104 children, with the objective of demonstrating cellular heterogeneity of leukemic blast cell populations in individual patients. Here, we report morphologic, immunologic, and clinical features of three cases in which cells of two lineages coexisting simultaneously at initial diagnosis could be delineated. From the University of D/isseldorf, *Departments of Pathology and tPediatric Hematology and Oncology, DUsseldorf, Federal Republic of Germany. Revision accepted for publication 30 July 1987. Address correspondence and reprint requests to Dr SchmittGr~iff: Department of Pathology, University of D~isseldorf, MoorenstraBe 5, 4000 DUsseldorf 1, Federal Republic of Germany.

Morphologic Studies

RESULTSAND CLINICAL COURSE Between 1980 and 1985, classification studies were performed on blood and bone marrow smears

9 1988 by W.B. Saunders Company. 0046-8177/88/1906-000355.00/0

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3b). Only rare normal hematopoietic precursors were observed. Immunological marker studies on leukemic cells at initial diagnosis are summarized in Table 2. Expression of markers of more than one lineage in the same cell could not be demonstrated. Chromosome analysis of 10 metaphases from patient 1 revealed a mixture of normal and clonally abnormal cells. Four cells had the following chromosomal rearrangement: 46, XY, 11 q - , - 6 , - 8 , + t (6;8). Six cells exhibited a normal male karyotype (46, XY). Fourteen cells from patient 1 had no cytogenetic abnormalities. In patient 3, the bone marrow cytogenetic study demonstrated Philadelphia chromosome translocation in all 16 metaphases examined. The peripheral blood and bone marrow findings, including cytochemistry, ultrastructure, and immunologic surface markers, seemed consistent with leukemia of two different lineages. The occurrence of myeloid blast forms, along with lymphoblasts, in the peripheral blood was of special diagnostic value, since a small number of immature myeloid cells in bone marrow specimens from patients with ALL may be interpreted as residual normal elements. Diagnosis of acute bephenotypic leukemia with dual lymphoblastic and myelomonocytic populations was made in patients 1 and 2. The Philadelphia chromosome in patient 3 suggested either a mixed blast crisis of CML without a clinically preexisting chronic phase or an acute phi-positive bilineage leukemia. A chemotherapy treatment protocol was begun according to the COALL-82 study s for high-risk ALL pediatric patients. The induction therapy consisted of vincristine, prednisone, adriamycin, and e-asparaginase followed directly by a consolidation phase with intermediate dose methotraxate/citrovorum factor r e s c u e , c y t o s i n e a r a b i n o s i d e , a n d 6 - m e r captopurine. This treatment resulted in a shift from the initial predominance of lymphoblasts to an expansion of myeloblasts and monocytoid blasts (Fig 4) in all three children. Therapy was then altered to an acute myelogenous leukemia (AML) regimen consisting of continous infusion cytosine arabinoside in combination with daunomycin and VP16 according to the BFM-AML 83 protocol. 9 Following this induction, a consolidation phase was added with vincristine, prednisone, daunomycin, cytosine arabinoside, and cyclophosphamide in combination with central nervous system (CNS) prophylaxis. 9 On this protocol, patient 1 entered complete remission. Patient 3 achieved a partial remission with persistence of the Philadelphia

and on bone marrow biopsies from 104 consecutive children with leukemia. Eighty-six cases were classified as aute lymphoblastic leukemia (ALL), 14 as acute non-lymphocytic leukemia (ANLL), and one as chronic myelogenous leukemia (CML). The remaining three cases were difficult to classify within the FAB framework. They were termed biphenotypic leukemia because of a cytological, cytochemical, and immunological pattern of a mixed lymphoid and myeloid phenotype. Table 1 illustrates clinical features of the three children at the onset of the disease: All three children presented with a high white blood cell count above 90 • 10~ and extramedullary tissue infiltration indicating a large leukemic cell mass. Peripheral-blood smears revealed a predominance of nongranular blasts with a high nuclear to cytoplasmic ratio and lymphoid morphologic features. However, cytochemical stains of the blood smears disclosed a second population of immature myeloid ceils positive for peroxidase (Fig la). A small percentage of blast forms from patients 1 and 2 consisted of myelomonocytic cells that were alpha-naphthyl acetate esterase positive. The typical ultrastructural appearance of lymphoblasts and myeloid blast forms occurring synchronously in the blood is shown in Figure lb. At initial diagnosis, hypercellular bone marrow specimens yielded about 90% of blasts. Two different types of blast ceils could be distinguished; the prominent type, accounting for about 80% of leukemic cells, consisted of blast cells showing lymphoid morphology (Figs 2 and 3). Lymphoblasts were positive for acid phosphatase. Cells from cases 1 and 3 displayed numerous fine granules scattered in the cytoplasm. Lymphoblasts from case 2 stained predominantly in single cytoplasmic dots (Fig 2b). The PAS reaction was positive in cytoplasm granules in 80% of the blasts from case 3 and in scattered blasts from case 1. Positivity for MPO was noted in about 15% to 20% of the blasts from cases 2 and 3 (Fig 2a) and in <5% of the blasts from case 1. Auer rods were observed in rare blast cells from cases 1 and 3. About 5% to 10% of the blasts from cases 1 and 2 were diffusely positive for alpha-naphthyl acetate esterase. T d T positivity was found in about 50% of blasts from case 1. Bone marrow biopsies showed a diffuse infiltration of marrow spaces by blast cells that varied in size from about 10 to 25~m. The larger type of cells, which accounted for a minority of blasts, were positive for naphthol AS-D chloroacetate esterase (Fig

TABLE 1. Clinical Summary

Age at diagnosis Sex WBC (• 109) at diagnosis Extramedullary involvement at diagnosis Survival (mo)

Patient No. 1

Patient No. 2

Patient No. 3

10 mo M 222 Kidney infiltration 17

10 yr, 1 mo M 92.9 Nodal disease 10

8 yr F 350 CNS leukemia 16

ABBREVIATIONS:too, month; yr, year; WBC, white blood cell count. 652

CHILDHOOD BIPHENOTYPICLEUKEMIA(Schmitt-GrOff et al) W

Q

9

P

ql m

a FIGURE 1. Peripheral blood cells from patient 3 unmasking the heterogeneity of leukemic cells. Negative reaction for myeloperoxidase in small lymphoid blast cells, as contrasted with larger myeloid cells positively stained [a, approximate magnification xS00]. UItrastructure of lymphoid and myeloid appearing cells, L, blast celt with moderate degree of chromatin condensation, scarce cytoplasmic granules, and numerous mitochondria suggestive of lymphoid morphology; M, leukemic promyelocyte with abundant electron dense priman/granules [b. approximate magnification x5,300].

chromosome. In patient 2, therapy for AML again permitted a conversion to a predominately lymphoid blast phenotype. He did not achieve a continous hematologic remission. Bone marrow transplantation, following total body irradiation and administration of cyclophosphamide, was carried out in cases 1 and 3. These children subsequently presented with bone marrow relapses again. In these relapses, blast cells only expressed lymphoid phenotype indicating a shift to a single lineage. All three patients died of recurrent disease within 17 months after initial diagnosis. Postmortem examinations were not performed. DISCUSSION Based on conventional morphological and cytochemical studies, three of the 104 children we examined presented evidence of two leukemic cell lineages coexisting simultaneously at the onset of the disease. These results agreed with phenotypic characterization by monoclonal antibodies unmasking the heterogeneity of blast cells. Cytochemical and immunological analysis did not provide consistent indication of simultaneous expression of lymphoid and myeloid characteristics on individual blasts in our patients. T h e terms "acute mixed leukemias" or "acute biphenotypic leukemias" have been associated with two different situations: leukemias with a mixture of two

distinct populations of blasts with lymphoid and myeloid phenotypes have to be distinguished from leukemias in which individual blasts display lymphoid and myeloid characteristics. This latter situation in which individual blasts have phenotypic and genotypic marker expression of different lineages has been considered as lineage infidelity of leukemic cells, l~ The genotypic and phenotypic coexpression of characteristics normally restricted to a single lineage supports the idea that there may be a misprogramming of differentiation in some leukemic blasts representing highly atypical cells with aberrant gene expression. 5 The model of lineage infidelity challenges the view that leukemic cells are phenotypically similar to normal hematopoietic progenitors. Conversely, verifiable examples of lineage infidelity may not reflect genetic misprogramming but rather the existence of a transient phase of limited promiscuity of gene expression occurring in normal progenitors and preserved in some leukemic populations. 2 Pui et al 6 reported three children with acute leukemia yielding cytochemical features of ANLL but an immunological phenotype typical o f ALL. These findings warranted the designation "acute leukemia with a mixed lymphoid-m,,yeloid phenotype" or "acute mixed lineage leukemia (AMLL). 7 The prevalence of cases with AMLL in the series of Mirro et al 7 was at least 20% (25 of 123) among children with acute leu653

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FIGURE 2. Bone marrow smears from patient 2 at the time of diagnosis. Myeloperoxidase~strong activity in some myeloid cells whereas blast cells showing lymphoid morphology are uniformly negative [a, approximate magnification x800]. Acid phosphatase~lymphoid cells show cytoplasm granules most often in a dotlike pattern [b, approximate magnification xl,250].

kemia. It is noteworthy that none of the 25 cases with childhood mixed lineage leukemia yielded dual lymphoid-myeloid populations by standard diagnostic criteria, such as morphology and cytochemistry. O f particular interest in our study was the demonstrated pattern of two distinct blast cell populations that could be identified by conventional methods such as morphology and cytochemistry in three of the 104 children with leukemia. The findings in our three patients are consistent with biphenotypic leukemias making up ceils of two lineages coexisting simultaneously within the same samples. These biphenotypic features presumably do not violate the concept of lineage fidelity. The dual morphologic and immunologic phenotype observed in our cases strengthens the concept that malignant transformation at the level of a very early lymphohematopoietic precursor, which is capable of differentiating into both lymphoid and rnyeloid cells, may be involved in the genesis of this type of leukemia. In this context, mixed blast crisis of CML may be mentioned. Griffin et a112 identified populations of both lymphoid and myeloid blasts in one of 30 CML cases in blastic transformation. In a patient with a

FIGURE 3. Bone marrow specimens from patient I at initial diagnosis. May-Gr0nwald-Giemsa staining of bone marrow smears reveals a mixed population of small lymphoblasts [L] and large myeloid cells [M] with large nucleoli and cytoplasmic granules [a, approximate magnification xi,250]. NaphthoI-AS-D chloroacetate esterase staining [arrows] of the bone marrow biopsy demonstrates the myeloid nature of a minority of immature bone marrow cells [arrows] [b, approximate magnification x800].

mixed blast crisis of CML examined by Ha et al, 13 the blasts consisted of two different myeloid and lymphoid populations. T h e CALLA + cells retained a germline configuration of Ig genes suggesting an emergence prior to commitment to B lineage. The presence of Phachromosome in some of the rare acute leukemias with mixed cell populations has been TABLE 2.

ImmunologicPhenolypeat Diagnosis

Reactivity of MoAbs used

Patient No. 1

Patient No. 2

Patient No. 3

Pan T Hel T Sup T Pan B CALLA Pan Mono Pan Mono/Granulo

+ + + +

* + + * + +

+ * + +

ABBREVIATIONS: M o A b s , m o n o c l o n a l a n t i b o d i e s . * E x p r e s s i o n o f t h e m a r k e r b y > / 6 0 % o f t h e b l a s t cell p o p u l a tion. + M a r k e r p o s i t i v i t y o f a m i n o r i t y o f b l a s t cells.

654

CHILDHOOD BIPHENOTYPIC LEUKEMIA (Schmitt-GrOff et al)

FIGURE4. Bone marrow and peripheral blood cellsfrom patient I at the time of firstrelapse. Naphthol-AS-D chloroacetate esterase stain distinguishes some granulocytic cells [arrows] a m o n g abundant negative monocytoid precursors in the core biopsy [Q, approximate magnification xi,250). May-Gr0nwald-Giemsa staining of blood smears yields a small percentage of Iymphoid blasts (arrow] and a prevalence of leukemic cellswith a monocytoid morphology [b, approximate magnification x800].

emphasized. In the two cases reported by Marie et al, 1~ as well as in two other patients with a mixture of lymphoid and myeloid blasts reported by Janossy et a/,l~ Phlchromosome was present. Den Ottolander et a116 o b s e r v e d a case o f a c u t e l e u k e m i a with

apeutic programs directed to blast cells of a single lineage led to striking changes in the proportions of the two leukemic populations, providing an obvious growth advantage to the other lineage. To our knowledge, no data concerning the incidence of biphenotypic leukemia with distinct blast cell populations are available. In the literature, only rare cases with this type of leukemia have been described. In addition to the cases already mentioned above, Mertelsman et a119 and Ueda et al 2~ documented two adult patients with distinct lymphocytic and monocytic populations. The case o f mixed leukemia, lymphatic and myelomonocytic, reported by Hull et al, 21 seems not to fit in the subgroup of acute biphenotypic leukemia, since this patient had chronic lymphocytic leukemia and myelomonocytic leukemia. The children with biphenotypic leukemia in our study presented with features associated with a poor prognosis: high white blood cell counts, lymphadenopathy, and CNS involvement. Bulk disease at initial diagnosis indicated a large tumor-cell burden. Their disease seemed to be less responsive to the treatments commonly used for ALL and AML. The children with biphenotypic leukemia o f our series, as well as the child reported by Creutzig et al, 17 fared poorly; none of these children achieved continuous complete remission. All succumbed to their disease despite aggressive chemotherapy and bone marrow transplantation in two cases. Survival was < 18 months from the first diagnosis of leukemia. However, two adult cases of acute biphenotypic leukemia achieved complete remission of long duration.Xg'2~ Biphenotypic leukemia in childhood seems to be a biologic entity more aggressive than other forms of lymphoblastic or non-lymphocytic leukemia. Morphologic, cytochemical, and immunologic identification of the cellular lineages involved in this unusual type of leukemia are clinically important for the selection of an appropriate treatment because this unfavorable phenotype predicts a poor response to conventional chemotherapy protocols. REFERENCES

phtchromosome and monosomy 7 in which the peripheral blood contained three types of blasts indicating a trilineage leukemia. The classical translocation t(9q + ; 2 2 q - ) in case 3 of our study may correspond to a mixed blastic phase of CML without a diagnosed preceding chronic phase or to a phi-positive acute biphenotypic leukemia. Two children with biphenotypic leukemia reported by Creutzig et a117 and Perentesis et al xs initially showed characteristics of ALL that converted within a few days to AML during induction chemotherapy. According to Perentesis et al, 18 this observation favors the hypothesis that biphenotypic leukemia may arise in a stem cell capable of d i f f e r e n t i a t i o n into both l y m p h o i d and myeloid clones that are both independently and selectively sensitive to specific chemotherapeutic regimens. In the biphenotypic leukemias of our study, chemother655

1. BennettJM, CatovskyD, Daniel MT, et al: Proposals for the classification of the acute leukemias. Brit J Haematol 33:451, 1976 2. GreavesMF, Chan LC, FurleyAJW,et al: Lineagepromiscuity in hemopoietic differentiation and leukemia. Blood 67:1, 1986 3. FialkowPJ, SingerJW, AdamsonJW, et al: Acute nonlymphocytic leukemia: Heterogeneity of stem cell origin. Blood 57:1068, 1981 4. SachsL: Constitutiveuncouplingof pathwaysof gene expression that control growth and differentiationin myeloidleukemia: A model for the origin and progression of malignancy. Proc Natl Acad Sci USA 77:6152, 1980 5. McCullochEA: Stemcellsin normaland leukemichemopoiesis (Henry Stratton Lecture, 1982). Blood 62:1, 1983 6. Pui CH, Dahl GV, MelvinS, et al: Acuteleukemiawith a mixed lymphoidand myeloidphenotype. Brit J Haematol56:121, 1984 7. MirroJ, ZipfTF, Pui CH, et al: Acute mixedlineageleukemia: Clinicopathologic correlations and prognostic significance. Blood 66:1115, 1985

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8. Janka GE, Winkler K. J/irgens H, et al: Akute lymphoblastische Leuk~imie im Kindesalter: Die COALL-Studien. Klin P/idiat 198:171, 1986 9. Creutzig U, Ritter J, Budde M, et ah Aktuelle Ergebnisse der kooperativen AML-Therapiestudien bei Kindern: BFM-78 und-83. Klin P~diat 198:183, 1986 10. Smith LJ, Curtis JE, Messner HA, et al: Lineage infidelity in acute leukemia. Blood 61:1138, 1983 11. Palumbo A, MinowadaJ, Erikson J, et al: Lineage infidelity of a human myelogenous leukemia cell line. Blood 64:1059, 1984 12. Griffin JD, Todd III RF, Ritz J, et al: Differentiation patterns in the blastic phase of chronic myeloid leukemia. Blood 61:85, 1983 13. Ha K, Freedman MH, Hrincu A, et ah Separation of lymphoid and myeloid blasts in the mixed blast crisis of chronic myelogenous leukemia: No evidence for Ig gene rearrangement in CALLA positive blasts. Blood 66:1404, 1985 14. Marie JP, PerrotJY, Boucheix C, et al: Determination of ultrastructural peroxidase and immunologic membrane markers in the diagnosis of acute leukemia. Blood 59:270, 1982 15. Janossy G, Woodruff RK, Paxton A, et al: Membrane marker

656

16. 17.

18.

19.

20. 21.

and cell separation studies in Phi-positive leukemia. Blood 51:861, 1978 den Ottolander GJ, Brederoo P, Geraedts JPM, et al: Trilineage acute leukemia in combined Phi-chromosome positivity and monosomy 7. Acta Haemat 73:129, 1985 Creutzig U, Eschenbach C, Ritter J, et al: Akute Leuk~imie bei einem 13-j~hrigen Jungen mit gleichzeitigem Auftreten von Lymphoblasten und Monoblasten. Klin P~idiat 193:162, 1981 Perentesis J, Ramsay NKC, Brunning R, et ah Biphenotypic leukemia: immunologic and morphologic evidence for a common lymphoid-myeloid progenitor in humans. J Pediatr 102:63, 1983 Mertelsmann R, Koziner B, Ralph P, et al: Evidence for distinct lymphocytic and monocytic populations in a patient with terminal transferase-positive acute leukemia. Blood 51:1051, 1978 Ueda T, Kita K, Kagawa D, et al: Acute leukemia with two cell populations of lymphoblasts and monoblasts. Leuk Res 8:63, 1984 Hull MT, Griep JA: Mixed leukemia, lymphatic and myelomonocytic. Am J Clin Pathol 74:473, 1980