Diagnostic criteria for hypocellular acute leukemia: A clinical entity distinct from overt acute leukemia and myelodysplastic syndrome

Lademio Research Vol. 20, No. 7, pp. 563-574, 1996. Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0145-2126/96 $15.00 + 0.00

Pergamon 0145-2126(95)00136-O

DIAGNOSTIC CRITERIA FOR HYPOCELLULAR ACUTE LEUKEMIA: A CLINICAL ENTITY DISTINCT FROM OVERT ACUTE LEUKEMIA AND MYELODYSPLASTIC SYNDROME Kazuhiro :Nagai*, Tomoko Kohno*, Yun-Xian Chen*, Hideki Tsushima*, Hiroyuki Mori*, Hideo Nakamura*, Itsuro Jinnait, Tatsuki Matsuo *, Kazutaka Kuriyama*, Masao Tomonaga* and John M. Bennett: *Department of Hematology, Atomic Disease Institute, Nagasaki IJniversity School of Medicine, Nagasaki, Japan; tFirst Department of Internal Medicine, Saitama Medical College, Saitama, Japan; and SUniversity of Rochester Cancer Center, Rochester, New York, U.S.A. (Received 28 March 1995. Revision accepted 9 September 1995) Abstract-In order to establish diagnostic criteria for hypocellular acute leukemia (HL), we have reviewed 32 cases selected on the basis of hypothetical 40% or less cellularity, by focusing on morphology, immunophenotype, karyotype and response to low dose Ara-C (LDAC) regimen and compared them with 40 cases of myelodysplastic syndrome (MDS) and 66 cases of overt acute myeloid leukemia (AML). The onset age ranged from 44 to 75 years (median 67 years). Bone marrow (BM) cellularity ranged from 12.4 to 39.8% (mean 29.8%) in HL, being significantly lower than in MDS (mean 80.7%) or AML (mean 86.4%) (P < 0.001). All reviewed cases characteristically showed smoldering clinical course, bi- or pancytopenia with rare leukemic blasts in the peripheral blood (PB), proliferation of type I leukemic blasts in the BM and markedly reduced background hematopoietic cells with some dysplastic changes in 12/32 cases (37.50/6). Blast percentage (blast %) in the BM ranged from 38.2 to 93.7% (mean 57.3%) in all nucleated cells (ANC). Although a considerable number of cases had blasts with negative or very low myeloperoxidase activity, immunophenotyping revealed that the leukemic blasts in HL had only myeloid markers. Karyotyping revealed non-random chromosome abnormalities in 30% of cases analyzed, which were considerably different from those seen in MDS. With LDAC regimen, a significantly higher CR rate (13/20 cases: 65.0%) was gained in HL than in RAEB/RAEB-t (0%) and overt AML in the elderly cases (27.3%) (P < 0.05). In CR, most cases showed recovery to normocellular BM with an apparent normalization of PB parameters. However, 12 CR cases relapsed 4-12 months later; most of which again showed hypocellular BM. These results indicate that HL is a distinct subtype of AML characterized by slow but distinct proliferation of immature myeloid blasts and by unique hematological features distinct from MDS or overt AML in the elderly. We propose the following diagnostic criteria: (I) pancytopenia with rare appearance of blasts in PB; (2) less than 40% BM hypocellularity; (3) more than 30% blasts in BM-ANC; and (4) myeloid phenotypes of leukemic blasts by MPO staining and/or immunophenotyping. Copyright 0 1996 Elsevier Science Ltd. Key worcfs: Hypocellular

acute leukemia,

diagnosis,

clinical

features.

clinical course, pancytopenia and increased numbers of blasts in hypocellular bone marrow (BM). However, its precise incidence and natural history are not yet established, since the diagnostic criteria for HL have not reached a uniform agreement among investigators [l-6]. More recently, there seems to be confusion in the

Introduction Hypocellular acute leukemia (HL) has been described as a subset of acute leukemia characterized by smoldering Abbreviations: AML, acute myeloid leukemia; ANC, all nucleated cells; BM, bone marrow; CR, complete remission; HL, hypocellular acute leukemia; LDAC, low dose cytarabine &a-C); MDS, myelodysplastic syndrome; MPO, myeloperoxidase; NEC, non-erythroid cells; PB, peripheral blood; RAEB, refractory anemia with excess blasts. Correspondence to: Kazuhiro Nagai, M.D., Department of Hematology, Atomic Disease Institute, Nagasaki University School of Medicine, l-12-4 Sakamoto, 852 Nagasaki, Japan.

differential

diagnosis between

HL and myelodysplastic

syndrome (MDS), especially refractory anemia with excess blasts (RAEB) or RAEB in transformation (MB-t). There is also an increasing number of reports of MDS with hypocellular BM (hypoMDS). We considered previously that the diagnosis of HL 563

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Table 1. Subtype distribution of acute leukemia diagnosed at Nagasaki University Hospital from 1965 to 1992 Subtype

Case number

AML ALL HL Others

305 (65) 103 (22) 47 (10) 15 (3)

was established when the “needle biopsy showed less than 50% cellular marrow and the blast % exceeds 15% of the all nucleated bone marrow cells” [6]. At our institute, HL cases accordingly accounted for 10% of all cases with acute leukemia (Table 1). Although the majority of patients were diagnosed as HL with myeloid feature, two were called lymphoid (blasts were myeloperoxidase (MPO) negative and TdT positive) and two were classified as ‘null’ (MPO negative, TdT negative). In order to achieve better agreement or concordance among investigators, and to provide a common language (taxonomy) to establish incidence and natural history, two morphologists (JMB and KN) recently reviewed independently 27 cases of HL previously diagnosed by another investigator (MT) according to the abovementioned criteria. We gained high concordance (24 out of 27 cases: 88.9%) between observers (KN/JMB), with only three cases re-classified as MDS, all of which showed BM cellularity between 40 and 50%. Based on this high concordance, we reviewed 32 cases in the present study, including 24 previous and eight new cases selected on the basis of hypothetical 40% or less BM cellularity, by focusing on their morphology, immunophenotype, karyotype and therapeutic response to low dose Ara-C (LDAC) regimen. In order to establish the diagnostic criteria of HL, we also compared the clinical and hematological features of HL with those of RAEB/RAEB-t and overt AML, especially of the elderly. Materials and Methods Cases We investigated 32 cases of HL who presented with smoldering clinical course, considerable cytopenia in more than two hematopoietic cell lineages and increased numbers of blasts in hypocellular BM. These cases were initially diagnosed and treated at Nagasaki University Hospital and some affiliated hospitals. Cytopenia in each lineage was defined as follows; Hb < 12 g/dl, WBC count < 3500/@ and/or neutrophil < lOOO/pl, platelet < lOO,OOO/pl.For comparison, 40 cases with MDS (RA: 22 cases; RAEB: six cases; RAEB-t: 10 cases; and RA with ringed sideroblasts: two cases) and 66 cases with overt acute myeloid leukemia (AML) were also

examined. The AML cases were divided into two groups: AML young (39 cases, age ~60 years) and AML elderly (27 cases, age >60 years). Morphology Hematoxylin and eosin (HE)-stained BM biopsy or clot sections and May-Giemsa (MG) and myeloperoxidase (MPO)-stained peripheral blood (PB) and BM smears were conventionally prepared. All examinations were performed by two observers (TK and KN) independently. For evaluation of BM cellularity, we employed point-counting eye pieces. This point-counting method was the same as described in previous reports [7-91. In short, after installation of this tool into a light microscope, we selected from six to 10 fields of a magnification of 400-1000 at random. Then, counting points on cellular marrow and fatty marrow separately and summing up, we calculated the ratio of cellular marrow to fatty marrow. Normal cellularity was based on the age grouping of patients as Hartsock et al. demonstrated [lo]. To evaluate the significance of the increase in blast % in HL BM, first we counted 500 consecutive nucleated BM cells and calculated the blast % in all nucleated BM cells, in all nucleated cells excluding lymphoid cells, in all nucleated cells excluding erythroid cells, and in all nucleated cells excluding both erythroid and lymphoid cells. The diagnosis of overt AML was established when blast % exceeded 30% in an apparent cellular marrow according to FAB classification. Immunophenotyping In the seven recent cases of HL, immunophenotyping of leukemic blasts was performed using flow cytometry (FCM) analysis in a standard conventional manner. Some cases were further examined by using immunocytochemistry (ICC) [ 111. Markers and monoclonal antibodies (in parentheses) were used as follows: CD13 (My7), CD33 (My9 and LeuM9 for ICC), CD14 (My4), CD34 (MylO). CD41 (P2), CD19 (B4), CD3 (OKT3 and Leu4 for ICC), CD7 (Leu9). Cytoplasmic MPO antigen (CLB-MPOl) was detected only by ICC staining. Karyotyping Cytogenetic analysis of leukemic blasts was performed by a synchronization technique with methotrexate and bromodeoxyuridine. Metaphases were analyzed in conventional Q- and G-banding methods. Chromosoma1 abnormalities were described according to the International System for Human Cytogenetic Nomenclature. In vitro colony assay In 21 HL cases, granulocyte/macrophage colony (CFU-GM) and erythropoietic burst (BFU-E) assays

Diagnosis of hypocellular acute leukemia

565

Chemotherapy Twenty cases were treated with LDAC regimen (0.2 mglkgJday/continuous i.v./2-3 weeks). Patients who obtained complete remission (CR) were given two or three courses of LDAC or conventional BHAC-DMP regimen (behenoyl Ara-C, daunorubicin, 6-MP and prednisolone) as consolidation. Complete remission was defined as follows: BM of normal cellularity with less than 5% myeloblasts, an apparent recovery of hematopoiesis, and no symptoms of leukemia. Twelve cases were treated with only supportive care such as blood transfusion and prophylaxis for infection and with no chemotherapy for leukemic cells, since their general condition was considered to be intolerable to antileukemic agents. Eight cases of RAEBRAEB-t and 11 cases of overt AML in the elderly treated with LDAC regimen were compared with the HL group, retrospectively.

cellularity(%) IOO-

fJO=

60.

40.

20=

00

[Al

PI

PI

1D

Fig. 1. Bone marrow cellularity of HL; comparison with MDS and overt AML. In every cases, BM cellularity was evaluated by the point-counting method described in ‘Materials and Methods’. The Mann Whitney U test was applied for statistical analysis. (A) Thirty-two reviewed cases with HL. (B) Forty cases with MDS. (C) Twenty-seven cases with overt AML in the elderly (>60 years old). (D) Thirty-nine cases with overt AML in patients ~60 years old.

were performed in methylcellulose culture using a modification of the method of Iscove et al [12]. In short, 1 x lo5 light-density and phagocyte-depleted mononuclear cells from BM were inoculated into 0.25 ml of Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, U.K.) containing 0.88% methylcellulose and 20% fetal calf serum (FCS) (Flow Laboratories, McLean, VA, U.S.A.). For stimulation of GM colonies. recombinant human granulocytic colony stimulating factor (rhG-CSF; Kirin, Japan), and recombinant human granulomonocytic colony stimulating factor (rhGMCSF; Kirin, Japan) were added to methylcellulose culture to obtain final concentrations of 250 pmolil and 10 @ml, respectively. For erythropoietic burst formation, 2 U/ml recombinant human erythropoietic (rhEP0; Kirin, Japan) was added. Culture was performed in 24well plates at 37°C in a humidified atmosphere of 5% CO2 in air and on days 7-10 and day 14, CFU-GM and BFU-E were counted, respectively. For comparison, colony assay studies were performed in 16 of RAEB/ RAEB-t cases and 20 of overt AML in the elderly cases with identical maneuver.

Statistical analysis The two-sample t-test, chi-squared analysis, Fisher’s exact probability test and the Mann-Whitney U test were used to assess the significance of difference between comparable groups. The generalized Wilcoxon and log rank methods were used for comparison of survival rate. Results BM cellularity In 32 HL cases, BM cellularity ranged from 12.4 to 39.8% (mean 29.8%), being significantly lower than in MDS (mean 80.7%) or overt AML cases (mean 86.4%) (P < 0.001) (Fig. 1). When compared with cellularity in overt AML cases in the elderly, which ranged from 36.0 to 100% (mean 79.6%) hypocellularity of BM in HL cases was significantly distinct (P< 0.001) (Fig. 1). Using the point-counting method, we could evaluate BM cellularity efficiently and reproducibly without discordant results between observers (data not shown). Blast % in BM The BM blast % changed in each case according to four different estimations (Fig. 2). Blast % in all nucleated BM cells including Iymphoid cells ranged from 20.8 to 71.0% (mean 33.7%) being less than 30% in 13 cases (40.6%). In all nucleated cells excluding lymphoid cells, blast % ranged from 38.2 to 93.7% (mean 57.3%). In all nucleated cells excluding erythroid cells, it ranged from 22.0 to 75.6% (mean 46.4%) and in

all nucleated cells excluding both erythroid and lymphoid cells, it ranged from 57.6 to 95.7% (mean 78.5%) respectively. As a result, counting in all nucleated

cells excluding

lymphoid

cells or excluding

K. Nagai et al.

566 blast count(%)

blast count(%)

80

60

60

40

40

20

20

0

.

.

.

[AI

WI

ICI

Chart

1

.

WI

il

;81 il rbr Chart

II

Fig. 2. Comparison of BM blast percentage in HL cases and MDS (RAEB, RAEB-t) cases according to four different methods. Each blast percentage was estimated in (A) all nucleated bone marrow cells; (B) all nucleated cells excluding lymphoid cells (so-called ‘ANC’ in the FAB criteria), (C) all nucleated cells excluding erythroid cells; and (D) all nucleated cells excluding erythroid and lymphoid cells (so-called ‘NEC’ in the FAB criteria). Chart I: 32 HL cases; Chart II: 16 RAEB/RAEB-t cases.

both erythroid and lymphoid cells, the blast % apparently exceeded 30% in all cases. On the other hand, 15 out of 16 cases of RAEB and RAEB-t showed less than 30% of blasts in BM by any of the four blastcounting methods. There was no case with the erythroid component exceeding 50% in the reviewed HL cases. The erythroblast % ranged from 0.4 to 37.5% with a median of 28.5%. PB findings

All 32 HL cases showed moderate to severe bi- or pancytopenia with relative lymphocytosis (Table 2). The percentage of blasts in HL ranged from 0 to 5.0% with a mean of 0.8% and a median of O%, being significantly less than that in RAEB/RAEB-t cases or in AML in the elderly (P-c 0.01) (Table 2). Pseudo-Pelger cells were identified in nine of 32 cases (28.1%). BM findings

The majority of the blasts were agranular [‘type I’ blasts; Fig. 3(A)]. Auer rods were observed in four cases of HL. The median MPO positive rate in blasts was 6.0% (range O-64.0%) and the average rate was 11.0% (Table 2). Twenty-two cases (68.8%) showed less than

3.0% positivity, and only one case showed a rate above 50% (Fig. 4). Non-specific esterase and chloroacetate esterase was negative in leukemic blasts in most cases (data not shown). In comparison with cases of overt AML, a majority of HL cases showed proliferation of immature myeloid blasts with minimal or no maturation. Conspicuously absent were morphological features of blasts analogous to FAB-M3, M5 and M7, although concomitant proliferation of monocytoid cells with leukemic blasts was observed in some cases. Background mature hematopoietic cells were markedly reduced in BM and the lymphocyte counts were significantly higher than those in RAEB/RAEB-t or overt AML in the elderly (P < 0.01) (Table 2). However, in such mature hemopoietic cells, dysgranulopoiesis (dys-G) was observed in 10 cases, accompanied by dysmegakaryocytopoiesis (dys-M) in one and dyserythropoiesis (dys-E) in four cases. The dys-M was observed separately in one case and dys-E in one instance. Tri-lineage dysplasia was observed in one case. Therefore, in 12/32 cases (37.5%), some dysplasia was recognized in the BM aspirates [Fig. 3 (B) and (C)l.

Diagnosis of hypocellular acute leukemia

(4

(E)

Fig. 3. Representative example of hypocellular leukemia. (A) Leukemic type 1 blasts observed in a HL case (May-GrunwaldGiemsa stain; x1000). (B) Dysmegakaryocytic change observed in a HL case (May-Gnmwald-Giemsa stain x1000). (C) Dysgranulocytic change observed in a HL case (May-Grunwald-Giemsa stain; x 1000). (D) Biopsy sample of hypocellular marrow on initial diagnosis (hematoxylin and eosin stain; x200). (E) Biopsy sample of hypocellular marrow of the same case on remission phase (hematoxylin eosin stain; x200). (F) Biopsy sample of hypocellular marrow of the same case on relapse phase (hematoxylin and eosin stain; x200).

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et al

Table 2. Comparison of clinical characteristics of HL, MDS (RAEB or RAEB-t) and overt AML in the elderly

(n = 16)

(n :L32)

Overt AML in the elderly (n = 27)

59 (3677) 6110

67 (44-75) 9123

68 (60-87) 12115

9.2 (6.1-14.0) 3.5 (0.8-7.3) 67.5 (10-312.0) 14/16 (87.5%) 3.9 (O-30) 37.0 (6.0-86.0)

P
8.1 (3.8-13.2) 1.45 (0.55-4.5) 52.5 (1.5-262.5) 12/32 (37.5%) 0.78 (O-5.0) 73.2 (15.1-94.0)

P
9.1 (4.7-13.4) 63.8 (0.6-353.8) 69.3 (0.6-300.0) 27/27 (100%) 51.2 (O-99) 22.5 (O-86)

16116 (100%) 15/16 (93.8%)

P
12/32 (37.5%) lo/32 (31.3%)

P
6127 (22.2%) 24127 (88.9%)

43.9 (o-100) 7.1 (4.0-51.0)

P
6.0 (o-64.0) 29.1 (7.6-47.2)

P
55.4 (t&100) 6.7 (&20)

RAEB, RAEB-t Characteristic Age (years; median) Sex (F/M) Peripheral blood (mean; range) Hb (g/dl) WBC (x 109/1) Platelets (x 109/1) Appearance of blasts (cases) Blast % Lymphocytes (%) Bone marrow (mean; range) With dysplastic changes Blast MPO positive (> 3%) cases MPO positive rate(%) Lymphocytes (%)

P
Table 3. Immunophenotype of leukemic blasts in HL cases (flow cytometric analysis)

Case SK MS MY CM TT yo+ KH

BM blast MPO positivity* (NLC; %) (%I 67.4 50.2 57.6 53.3 58.3 73.2 79.1

6.0 0.0 15.0 0.0 0.0 0.0 0.0

CD13 My7

CD33 My9

CD14 My4

CD34 My10

CD41 P2

CD19 B4

CD3 OKT3

CD7 Leu9

65.0 28.4 57.9 60.7 86.7 90.5 22.6

26.9 N.D. 10.1 46.0 53.9 70.7 9.1

6.4 N.D. 5.4 0.0 0.0 13.5 0.6

86.0 N.D. N.D. 52.0 89.6 84.9 85.3

3.8 0.8 12.1 1.8 2.1 7.1 0.0

2.8 0.0 N.D. 2.8 1.2 9.2 0.0

4.6 12.7 N.D. 0.0 1.3 6.7 0.0

0.4 1.4 3.8 7.6 3.4 11.3 N.D.

*Positive rates are indicated by percentage (%). Each value over 20% is in italics to indicate a ‘positive’ marker. tin this case, immunocytochemical detection of MPO was carried out; consequently, positive blasts of 16% were observed. In six other cases, this immunocytochemical study for MPO was not performed.

Table 4. Observed frequency of distribution of primary chromosomal abnormalities in cases with HL, AML in the elderly and MDS

No. of patients analyzed No. of patients having abnormal karyotype -515q-7/7qTrisomy 8 No. 11 abnormalities Phl t(8;21) t(15;17) inv(16) Others Complex abnormalities (more than three in one clone)

HL

AML in the elderly

MDS

16 5

20 12

40 22

0 0 2 0 0 0 0 1 2* l/5

0 0 2 3 1 1 2 0 3 4112

7 9 2 4 0 0 0 0 7t 10122

*One case had 45X0, -Y and the other had 51XX,?5p-, +[2,3, C, 16, 18, F, G] abnormalities. jSince -5/5q- and -7/7q- abnormalities overlapped in some cases with MDS, there were seven cases without any representative abnormalities such as those described in this table.

Diagnosisof hypocellularacuteleukemia

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P
MPOpositive rate(%) loo-

I1

80

60

40

20

0

HL(nd2)

overt AML in the ekterly(n=27)

Fig. 4. Distribution of MPO positive rate in HL and in overt AML in the elderly.

Immunophenotype of blasts in HL cases Immunophenotyping for the seven recent cases indicated that leukemic blasts in all of these cases expressed only myeloid markers in spite of negative or very low MPO positivity (Table 3). In one cytochemical MPO-negative case (case YO), ICC staining revealed apparent cytoplasmic MPO antigen. Karyotype of blasts in IIL cases In 16 (76.2%), karyotypes were gained. In five of them (31.3%), clonal chromosomal aberrations were detected (Table 4). Although, in some cases, these

abnormal karyotypes were similar to those commonly seen in MDS cases or in AML in the elderly cases (i.e. trisomy 8), we could not detect any specific non-random abnormalities for HL group because of their small number. Normal karyotypes were more frequent (68.7%) in HL than in MDS (45.2% in this series). In vitro colony assay in HL Poor growth of CFU-GM and BFU-E were observed in HL as well as in MDS and in AML cases. However, cluster fclrmation in HL was significantly less frequent than in RAEB/RAEB-t cases (P
570

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Table 5. Comparisonof in vitro colony assayof HL, MDS (RAEB, RAEB-t) and overt AML in the elderly Colony character CFU-GM (colony) (mean f SD; CFU/lO’ BMMNC) CFU-GM (cluster) (mean *SD; CFU/lO’ BMMNC) BFU-E (mean *SD; CFU/105 BMMNC)

RAEB, RAEB-t (n = 16)

(nHL21)

Overt AML in the elderly (n = 20)

4.688 k 7.033

6.805 k 7.118

0.652 + 1.191

3.333 k 6.475

2.043 k 4.039

7.988 f 15.30

1.448 k 1.975

40.99 + 44.951

P < 0.01

3.000 f 4.597

Normal control with identical conditions in our institute; CFU-GM lOO-200/10sBMMNC, BFU-E 4&70/105 BMMNC. Table 6. Treatment outcome of LDAC regimen in HL, RAEB/RAEB-t and overt AML in the elderly HL

CR PR NR

Dysplasia (-) (n = 11*>

Dysplasia (+) (n = 9)

Total (n = 20*)

RAEB/RAEB-t (n = 8)

Overt AML in elderly (n = 11)

8 (72.7) 0 2

5 (55.6) 0 4

13 (65.0) 0 6

0 (0) 2 6

3 (27.3) 1 7

Note: Each figure in parenthesesof the CR rank meansCR rate (%) in respectivesubgroup.The CR rate in HL was significantly higher than in RAEB/RAEB-t or in overt AML (P < 0.05). ‘Dysplasia’ meansmorphological dysplastic changeswhich are observed in initial BM slides, especially dysgranulocytic changesand dysmegakaryocyticchanges. *One casewas not evaluable becauseof early death due to asphyxia.

Occasionally, a few colonies with normal size were observed in HL. z

70

.z

60

5

50

Outcome of the LDAC regimen

a

40

x

30 20 10 0

z

70

.?

60

22

40 50

x

30 20 10 0

Fig. 5. Survival curve for HL cases(Kaplan-Meyer method). (A) Overall survival for 32 HL cases(1) comparedwith 16 RAEB/RAEB-t cases (2) and 27 overt AML cases in the elderly (3); (1) versus (3): P-co.05 (generalized Wilcoxon method). (B) Survival for HL cases according to treatment; treated with LDAC regimen (4); only supportive care (5).

Among 20 cases with HL who received LDAC treatment, 13 cases achieved CR (CR rate: 65.0%), significantly higher than those in RAEB/RAEB-t cases (0%) or overt AML cases in the elderly (27.3%) (Table 6). Cases of HL with morphological dysplasia also achieved CR, significantly higher than in ILAEB/RAEBt cases (P < 0.05). There was no significant difference in CR rate between HL cases with dysplastic changes and those without (55.6% and 72.7%, respectively). In spite of such a high response rate to the LDAC regimen, most CR cases relapsed early and eventually died. Consequently, in our retrospective analysis, there was no significant difference in disease-free survival (DFS) between HL cases and RAEB/RAEB-t or overt AML cases (Fig. 5). However, the last category contained a small fraction (20%) of long survivors. Nine out of 12 relapsed HL cases (75.0%) showed hypoplastic BM again, and three of them (25.0%) developed overt AML. A representative clinical course of a HL case treated with LDAC regimen is illustrated in Fig. 6. Bone marrow histology at diagnosis, in CR and at relapse is shown in Fig. 3 (D), (E) and (F), respectively.

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Discussion To recognize HL as a clinical entity of acute leukemia, evaluation of BM cellularity should be one of the essential determinants. Most previous reports defined the criterion for ‘hypocellular BM’ as below 40% [3] or 50% [4-61 or occasionally 30% [13,14]. However, since normal BM cellularities vary according to the ageing process [lo], these criteria have been considered controversial in clinical usage. In this context, Tuzuner et al. recently analyzed and compared histologically needle-biopsied BM specimens between AML, MDS and chronic myeloproliferative disorders (CMPD) 1151.When age-adjustment was undertaken to determine hypocellularity, there were relatively many cases of MDS showing hypocellularity irrespective of their age. In contrast, there were only a few cases of AML with true hypocellularity which was below the lower limit of normal range for each age group; all of them were elderly cases. All cases of CMPD exclusively had normo- to hypercellular BM irrespective of their age. In this AML series, however, they noted that about 10% of cases had relatively hypocellular BM which was below the median value for each age group and showed a relatively uniform clinical picture compatible with that we observed in our HL series. These HL cases were mostly elderly, with ages over 60 years. Taking account of low leukemic growth in HL which is characterized by severe BM failure in spite of a low leukemia burden and smoldering clinical course with persistent pancytopenia, it is reasonable to make a distinction between AML with hypocellular ( < 40%) BM and overt AML with cellular (>40%) BM. We recommend use of ‘hypocellular’ instead of ‘hypoplastic’ to avoid further debate on the establishment of ageadjusted criteria for the true histological hypoplasia. In this respect, we propose use of 40% as the discriminating point, irrespective of a patient’s age, since we can efficiently extract a homogeneous subgroup of elderly cases of AML with this criteria combined with 30% criteria for blast %. Tuzuner et al. also recommended this 40% criteria for distinguishing HL [15]. For objective and practical measurement of BM cellularity, we believe that a point-counting method is the most useful, presently [S, 91. Furthermore, Tuzuner et al. recently reported that comparing the biopsy specimens to a series of reference photographs of various degrees of cellularity was also recommended [16]. Previously, we confirmed that agreement between two independent observers, one using Tuzuner’s method and the other using the point-counting method, was excellent. The MRI technique is also efficient for more objective evaluation of the whole body BM cellularity. Accumulation of MRI data in HL as well as hypoMDS will greatly facilitate our understanding about distribu-

tion of hypocellular BM which cannot be identified by a single biopsy. In the present study, it appeared that in HL cases the blasts did not proliferate as rapidly compared to typical AML cases, and background mature hematopoietic cells were markedly reduced in number, resulting in the relative lymphocytosis. Different from overt leukemia as well as RAEBRAEB-t, therefore, the blast % might be markedly influenced by the high lymphocyte counts in HL, especially in oligoblastic cases. Thus, blast % of cases with hypocellular marrow should be evaluated in all nucleated cells excluding lymphocytes, defined as ‘all nucleated cells (ANC)’ in the FAB criteria [17]. Although there was no case whose erythroblast component in BM was beyond 50% in our series; blast % should be estimated in all nucleated cells excluding erythroid and lymphoid cells, defined as ‘non-erythroid cells (NEC)’ in the FAB criteria [17] in such cases. Using these methods of blast counting, we believe that all HL cases might be clearly defined as ‘acute leukemia’ with a definite proliferation of blasts showing maturation arrest. The ‘30% in ANC’ criteria for blast %, which practically corresponds to the FAB criteria for overt leukemia with cellular marrow, is most effective in diagnosing HL. Although there was a considerable number of cases with negative or low MPO positivity, immunophenotyping revealed that these blasts apparently expressed myeloid markers without any lymphoid marker expression. These observations suggest that blasts of HL tend to have immature myeloid nature analogous to those blasts of FAB-MO [18] and that immunophenotyping is useful and sometimes essential for the precise diagnosis. In our series of HL, significant dysplasia of mature hemopoietic cells was observed in as many cases as in de lzovo AML [19,20], suggesting that HL is essentially a particular type of AML. In spite of a smoldering course with persistent pancytopenia and some degree of morphological dysplastic changes in HL cases which suggest resemblance to MDS, the following observations indicate that HL is a clinical entity distinct from MDS: (1) markedly reduced hematopoietic cells and high lymphocyte count both in PB and BM were observed more frequently in HL than in MDS, indicating evident maturation arrest in HL blasts; (2) by in vitro colony assay, cluster formation of CFU-GM is less frequent in HL than in MDS. A few colonies with normal size were almost always observed in HL. In our previous cytogenetic study on these colonies, it was clarified that normal clones are persisting [21]; (3) we could not detect any non-random chromosomal abnormalities such as -7/7q- or 5q- which were commonly observed in MDS cases. Recently, some reports claimed the existence of hypoMDS as a clinical subcategory [13, 14, 22,231.

Diagnosis of hypocellular acute leukemia

Table 7. A proposalof diagnostic criteria for hypocellular AML 1. Cellularity of bone marrow < 40%; as determinedby core biopsy or clot section/confirmedby radiological evaluation for MRI 2. Blast percentagein bone marrow > 30% in ANC* 3. Cytopenia in peripheral blood more than in two blood cell lineages 4. Cytochemistry/immunophenotyping Myeloperoxidase (MPO) stain; positive blastsin BM 2396; when the blast MPO positivity is less than 3%, myeloid nature should be confirmed by immunophenotyping using specific myeloid markers (i.e. CD13, CD33, MPO-Ag) *ANC, all nucleated cells excluding lymphoid cells, as in FAB criteria.

However, its clinicopathological significance in comparison with typical MDS or HL is not yet established. Recent work by Tuzuner et al. demonstrated that there is no basic difference in clinical and hematological features between MDS with cellular marrow and hypoMDS with less than 30% cellularity [24]. It should be taken into account also that uneven distribution of cellular marrow may influence evaluation of marrow cellularity in MDS. Our recent observation with MRI began to provide such evidence of uneven distribution of cellular marrow in MDS. Furthermore, hypocellular marrow appears occasionally, observed in the clinical course of typical MDS cases with cellular marrow at diagnosis. We believe that our criteria for HL will shed some light on to this vague area between MDS and acute leukemia. In our HL cases, a significantly higher CR rate was gained with the LDAC regimen than in RAEBIRAEB-t and overt AML in the elderly. Although our comparison was retrospective in nature, this higher response rate might also indicate the difference in pathophysiology between HL and RAEB/RAEB-t or overt AML. Once CR was achieved, most HL cases showed full recovery of peripheral blood counts. This might indicate that normal pluripotent stem cells and the normal hematopoietic microenvironment are well conserved and able to reconstruct normal hematopoiesis. Furthermore, in the relapse phase, pancytopenia and hypocellular marrow with blast increase recurred again in a number of HL cases. This might indicate that such hypocellular BM in HL is a manifestation of unique pathophysiology which cannot be explained by age-oriented declining of BM cellularity. We suspect that leukemic blasts of HL cases have a strong growth-inhibitory effect on normal stem cells, but simultaneously show slow growth. These dual features can explain why severe hypocellularity ensues in spite of a low leukemic burden. Basic research is warranted for elucidation of such a mechanism. In conclusion, HL is a distinct subtype of AML occurring mainly in the elderly and comprising about

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10% of acute leukemia in Japan. Based on present data, we propose a practical criteria for the diagnosis of HL (Table 7). We believe that our criteria will effectively define HL cases from other categories, especially MDS. Prospective large-scale studies based on these criteria are needed for clarifying clinical and biological features of HL. New therapeutic approaches to AML in the elderly should be pursued separately for HL and overt leukemia. Acknowledgements-The authors are grateful to Dr Yoshiharu Yoshida (St Francesca Hospital, Nagasaki) and Dr Miyuki Kusano (Senju Hospital, Sasebo) for kindly providing clinical data of HL cases.

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