Acute Myeloid Leukemia With Minimal Differentiation

HEMATOPATHOLOGY Original Article

A c u t e

M y e l o i d

L e u k e m i a A Multiple

W i t h M i n i m a l Parameter

Differentiation

Study

PATTIL. COHEN, MD,1 JAMES D. HOYER, MD,1 PAUL J. KURTIN, MD,1 GORDON W. DEWALD, PhD,2 AND CURTIS A. HANSON, MD1 the blasts of 2 patients. Abnormal clonal karyotypes were found in 6 of 14 patients. Abnormalities of chromosomes 7 and 13 were the most common findings, most frequently manifested by monosomy 7 and trisomy 13. The median follow-up was 8 months. Eight patients died of their disease, three are alive with disease, and six are in first or second remission (including three patients treated with bone marrow transplantation). When narrowly defined, AMLMO appears to be a homogeneous entity that affects older and predominantly male patients. It has no single karyotypic abnormality, but complex karyotypes with monosomy 7 and trisomy 13 are commonly found. Acute myeloid leukemia with minimal differentiation is relatively resistant to chemotherapy; however, bone marrow transplantation may provide a better outcome for eligible patients. (Key words: Acute myeloid leukemia with minimal differentiation; Acute myeloid leukemia; Stem cell leukemia; Immunophenotyping) Am J Clin Pathol 1998;109:32-38.

Historically, the distinction between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) has been based on a combination of light microscopic morphology and cytochemical staining with myeloperoxidase (MPO) and Sudan black B (SBB) positivity used to define myeloid blasts. With the a d v e n t of i m m u n o p h e n o t y p i n g and electron microscopy, it was discovered that morphologically undifferentiated leukemic blasts could be myeloid in origin despite cytochemical negativity. 1 This type of leukemia was proposed as an entity by the National Cancer Institute in 1990, and the criteria for its recognition were proposed by the French-American-British Cooperative Group (FAB) in 1991. 2,3 It is now designated AML with minimal differentiation (AML-MO).

Few large s t u d i e s t h a t a n a l y z e this recently described entity are available. We undertook this study to better characterize AML-MO and for that purpose we reviewed the clinical records and the results of bone marrow morphology, cytochemistry, i m m u n o p h e n o t y p e , and cytogenetics in recently diagnosed cases of AML-MO at our institution. MATERIALS AND METHODS

All cases diagnosed as AML, FAB-MO at the Mayo Clinic between July 1993 and December 1995 were reviewed. The basic diagnostic criteria followed w e r e t h o s e of the FAB C o o p e r a t i v e G r o u p . 3 Specifically, the bone marrow aspirates contained at least 30% leukemic blasts that were negative or had fewer than 3% blasts positive with cytochemical MPO and immunophenotyping by flow cytometry From the Divisions of 1Hematopathology and ^-Laboratory Genetics, or immunohistochemistry demonstrated a myeloid Department of Laboratory Medicine and Pathology, Mayo Clinic, origin (positivity for CD13 a n d / o r CD33) of the Rochester, Minnesota. blasts. Lymphoid antigen positivity, in addition to Manuscript received March 7, 1997; revision accepted CD2 or CD7, did not lead to exclusion. WrightApril 23,1997. Giemsa-stained bone marrow preparations were Address reprint requests to Dr Hanson: Department of r e v i e w e d in all cases. Cytochemical stains perLaboratory Medicine and Pathology, Hilton 1020, Mayo Clinic, Rochester, MN 55905. formed included MPO, chloroacetate esterase, and 32

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Acute myeloid leukemia with minimal differentiation (AML-MO) is a recently described entity, and few large studies are available to characterize its clinical and pathologic features. We reviewed blood and bone marrow morphology, cytochemistry, immunophenotyping, and cytogenetics, as well as the clinical findings, of 17 patients with AML-MO. Most patients were male, with a median age of 62 years. Minimal background myelodysplastic features were identified in only 5 of 15 patients. Cytochemical stains for myeloperoxidase and a-naphthyl butyrate esterase were negative in the leukemic blasts of all specimens. Positivity for one or both myeloid-associated antigens, CD13 and CD33, was seen in all patients. Both CD34 and HLA-DR were positive in all tested cases. Lymphoid-associated antigens were identified in seven patients; these antigens were typically dim or weak in intensity. Terminal deoxynucleotidyltransferase positivity was seen in 14 of 14 tested patients; monoclonal anti-myeloperoxidase reactivity was seen in

COHEN ET AL Acute My d Leukemia

33

review, 18 p a t i e n t s w e r e e x c l u d e d from further analysis: Nine patients had a previous diagnosis of myelodysplastic s y n d r o m e . In three, the disease evolved from a previously diagnosed chronic myeloproliferative disorder. Three patients had insufficient characterization with myeloid markers by flow cytometric immunophenotyping or immunohistochemistry. In two, the disease was re-categorized, one as FAB-MI a n d the other as FAB-M2. Finally, one patient had increased monocytes and promonocytes that stained with a-naphthyl butyrate esterase, distinct from the c y t o c h e m i c a l l y n e g a t i v e b l a s t s . Although the findings were insufficient for a diagnosis of an acute myelomonocytic leukemia by FAB criteria, the increased monocytic component suggested that this was not an undifferentiated AML, and so this case was also excluded.

Flow cytometric immunophenotyping was performed in 16 cases on 13 bone marrow aspirates and 3 peripheral blood specimens on a FACScan Analyzer (Beckton-Dickinson) with the use of standard techniques. 6 Monoclonal antibodies directly labeled to a fluorochrome, including CD2, CD3, CD5, CD7, CD10, C D l l b , CD13, CD14, CD19, CD20, CD22, CD33, CD34, CD41, CD45, CD61, and HLA-DR (BectonDickinson), were used. Chromosome analysis was performed on bone marrow aspirates with both direct and unstimulated culture methods. Whenever possible, 20 metaphases were stained by either Giemsa stain with trypsin pretreatment (GTC banding) or fluorescent staining with quinacrine mustard (QFQ-banding). Representative metaphases were photographed, and karyotypes were written in accordance with the guidelines of the I n t e r n a t i o n a l System for H u m a n C y t o g e n e t i c Nomenclature. 7 Clinical information was obtained by review of the patient's clinical record, by direct contact with the patient's physician, or both.

Ten of the 17 patients were male, with a median age of 62 years (range, 19-86 years). Eleven patients were older than 60 years. Two patients had previous malignant neoplasms that were treated with chemotherapy. In one of these patients, multiple myeloma was diagnosed 11 years earlier and was treated with melphalan and prednisone. The other patient had non-Hodgkin's lymphoma 17 years earlier that was treated initially with partial gastrectomy; she subsequently received combination chemotherapy (CHOP) for stage III non-Hodgkin's lymphoma 3 years before her diagnosis of AML. Physical findings common to any acute leukemia were sometimes identified. Skin and mucosal lesions, including petechiae and oral apthous ulcers, were noted in seven patients; no cutaneous nodules were identified. S p l e n o m e g a l y w a s identified in t w o patients (4 and 10 cm below the left costal margin, respectively), and hepatomegaly was identified in one (16-18 cm). Palpable lymph nodes (up to 2 cm) were noted in three patients. One patient had leukemic blasts identified in the cerebrospinal fluid, and one patient had pulmonary leukemic infiltrates.

Clinical

Characteristics

RESULTS Blood Findings Identification

of Cases

O u r c o m p u t e r d a t a b a s e search i d e n t i f i e d 35 patients in w h o m AML, FAB-MO w a s d i a g n o s e d b e t w e e n July 1993 a n d D e c e m b e r 1995. In t w o patients, it was diagnosed at the time of relapse, 31 a n d 82 m o n t h s after their initial d i a g n o s i s . On Vol.

Most patients had peripheral blood findings characteristic of acute leukemia. The WBC was elevated in 5 of 17 patients, was within normal range in 2, and was low in 10 (range, 0.4-180.9 X 10 9 /L; median, 2.9 X 109). Only one patient had greater than 100 X 109 WBCs/L. Circulating leukemic blasts were identified •No. 1


a-naphthyl butyrate esterase in 15 of 17 cases; only MPO staining was available for 2 cases. 4 Paraffinembedded bone marrow trephine biopsy specimens were available in 14 cases. All were prepared in B-5 fixative, and a standard hematoxylin and eosin stain w a s u s e d . I m m u n o c h e m i c a l s t u d i e s u s i n g the labeled streptavidin biotin method were performed on bone marrow aspirate smears or bone marrow trephine biopsy specimens. 5 Antibodies from the following sources were used at the dilutions specified: MPO (DAKO, Carpinteria, Calif; monoclonal, from 1:50 to 1:400 in aspirate smears, and polyclonal, 1:5,000 in biopsy specimens) and terminal deoxynucleotidyltransferase (TdT) (Supertechs, Bethesda, Md; polyclonal, 1:50 in aspirate smears). Selected aspirate smears were stained with CD13 (DAKO; monoclonal, 1:50), CD19 (DAKO; monoclonal, 1:10), CD20 (DAKO; monoclonal, 1:75), CD33 (Coulter, Hialeah, Fla; monoclonal, 1:20), and CD34 (BectonDickinson, Franklin Lakes, NJ; monoclonal, 1:10).

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HEMATOPATHOLOGY Article

FIG 2. The cytoplasm of most acute myeloid leukemia with minimal differentiation, blasts was moderately basophilic. Small discrete cytoplasmic vacuoles were found in two thirds of cases (Wright-Giemsa, original magnification xl,000).

in 15 patients (range, 2% to 93%; median, 70%). Other than the circulating blasts, no significant or unique peripheral blood smear findings were noted. N o dimorphic RBC population, increased dacrocytes, dysplastic neutrophils, or atypical platelets were seen. Sixteen patients h a d anemia at presentation; the median hemoglobin was 8.9 g / d L (range, 4.7-14.3 g/dL). Thrombocytopenia was found in 12 patients; the median platelet count was 99 X 10 9 /L (range, 13-260 X 10 9 /L).

Myelodysplastic features were infrequent in these cases; only minimal dyserythropoiesis was identified in 5 of 15 cases. Overt dysplastic features were not seen in the granulocytic or megakaryocytic series, although in approximately one third of patients too few nonblastic cells remained for accurate evaluation. Of 10 cases available for review, the bone marrow trephine biopsy specimens were hypercellular in 9 and slightly hypocellular in 1. The biopsy findings confirmed the marrow aspirate features. No fibrosis was noted in the biopsy specimens.

Bone Marrow Findings

Cytochemistry

The bone marrow aspirate smears were hypercellular and clearly diagnostic of acute leukemia. The median bone marrow blast count was 85% (13/15 with > 70% blasts; range, 40%-90%). The blasts in all patients were i n t e r m e d i a t e in size and h a d high nuclear to cytoplasmic ratios. Nuclei were usually round to slightly irregular or indented; complex, folded nuclei were not seen. There was a fine chromatin pattern in all cases, and one to four usually prominent nucleoli were present in blasts from most cases (Fig 1). The scant c y t o p l a s m w a s u s u a l l y basophilic and agranular; no cytoplasmic budding was seen, but small cytoplasmic vacuoles could be identified in the blasts from 10 of 15 patients (Fig 2). No Auer rods were seen. The blasts did not have morphologic features suggesting granulocytic, monocytic, erythroid, or megakaryocytic differentiation.

In all cases, the cytochemical stains for MPO, chloroacetate esterase, a n d a - n a p h t h y l b u t y r a t e esterase were negative in the blasts. Background granulocytes and monocytes exhibited a n o r m a l staining pattern, thereby excluding a rare MPO or esterase deficiency and confirming an internal positive control reaction (Fig 3). Flow Cytometry and

Immunohistochemistry

Flow cytometric i m m u n o p h e n o t y p i n g was performed in 16 cases (Table 1). Positivity for one or both myeloid-associated antigens, CD13 and CD33, was seen in blasts from all patients. Specimens from 14 patients expressed both CD13 and CD33. One case expressed CD13 without CD33, and one had CD33 without CD13. CD33 expression was considered dim in 7 of these 16

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FIG 1. A characteristic hypercellular bone marrow aspirate in acute myeloid leukemia with minimal differentiation, with a high percentage of intermediate-sized blasts with round or slightly irregular nuclei and one or more prominent nucleoli (Wright-Giemsa, original magnification X1,000).

COHEN ET AL Acute Myeloid Leukemia

35

'#•i- - r v • -'

•***'", k

)

mM

'4 >

FIG 4. Immunocytochemical staining for terminal deoxynucleotidyltransferase was found in all tested cases. Moderate to strong nuclear staining is seen in this case (original magnification x 1,000).

specimens. CD14 expression was found in two cases, with dim expression in one. No specimens showed positivity for CD41 or CD61. Both CD34 and HLA-DR were positive in all 16 cases. Seven specimens demonstrated lymphoid-associated antigens; these were typically dim or weak in intensity. CD22 (present in 5 of 14 specimens) was the most frequently seen lymphoid antigen, foUowed by CD19 (4/16), CD20 (3/16), and CD7 (3/16). No specimen showed CD10 reactivity. In the single specimen in which only immunohistochemistry was used for immunophenotyping, CD13, CD33, and CD34 were positive, and CD19 and CD20 were negative. Specimens from all 14 studied cases were TdT positive by immunocytochemical staining. The degree of positivity ranged from moderate to strong, with nine specimens demonstrating strong positivity in approximately 90% of the blasts. The remaining specimens showed a positivity rate of 20% to 80%, varying in intensity from weakly positive in one case to moderately positive in the remaining cases (Fig 4). Immunochemical staining with anti-MPO antibody in the bone marrow aspirate (10 cases) or biopsy (2 cases) specimen was positive in 2 of 12 patients; in both, approximately 20% of blasts were MPO positive in the bone marrow aspirate specimen.

abnormal metaphases. Abnormalities of chromosomes 7 and 13 were the most common findings, each present in four patients; these abnormalities were most frequently manifested by monosomy 7 and trisomy 13. Two patients had chromosome 20 and 21 abnormalities.

Cytogenetics Abnormal clonal karyotypes were found in 6 of 14 patients (Table 2); three of these patients had no normal metaphases, and three had a mixture of normal and

Treatment and Survival Eleven of 17 patients were treated with curative intent. Within this g r o u p , 6 p a t i e n t s achieved a complete remission after first induction, 3 achieved TABLE 1. SUMMARY OF IMMUNOPHENOTYPIC RESULTS Antigen CD2 CD3

CD7 CD10 CD14 CD19 CD20 CD22 CD13 CD33 CD34 CD41 CD61 HLA-DR TdT Tdt = terminal deoxynucleotidyltransferase.

Vol. 109 • No. 1

No. Positive/No. Tested 1/16 1/16 5/16 (dim in 4) 0/16 2/16 (dim in 1) 3/17 (dim in 1) 0/17 6/15 (dim in 5) 16/17 16/17 (dim in 7) 16/16 (dim in 1) 0/16 0/16 16/16 (dim in 1) 14/14 (detailed in text)


FIG 3. Cytochemical myeloperoxidase stain in acute myeloid leukemia with minimal differentiation. Blasts were negative in all cases, with mature residual granulocytes as a positive internal control (lower center of photograph) (original magnification X 1,000).


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TABLE 2. SUMMARY OF CYTOGENETIC RESULTS Case No. 1 2* 3 4 5 6

Age/Sex 46/M 51/M 81/M 76/M 54/M 86/F

Karyotype 46,XY/inv(2)(pl?3q3?7)/t(7;13)(q34;ql4),add(15)(ql5)[4]/46,XY[16] 47,XY,+13[18] /48,XY,+9,+13[l ] /46,XY[3] 46/XY-12,+mar[2]/44,idem, -3, -7,idic(ll)(pll.l)[ll]/46,XY[7] 88-90,XXYY, -7,4-13x2, -17, -20, —21x2+mar[cp20] 45,XY,-17,der(20)t(17;20)(qll.2;qll.2),-21,+mar[14]/45,idem,-7,add(15)(q22)/del(15)(q22)/+mar[l] 47,XX,+13[8]/47,idem,i(13q)[2]/46,XX[10]

*Patient had a past history of multiple myeloma. Before the development of acute myeloid leukemia with minimal differentiation (AML-MO), a bone marrow specimen showing multiple myeloma had a karyotype of 46,XY,der(7)t(7;ll) (q32;ql3)[3]/46,XY[ll]. Cytogenetic study of a subsequent bone marrow specimen with residual AML-MO revealed persistence of the leukemic clone, with a karyotype of 47,XY,+13[5] /46,XY[3].

DISCUSSION In 1991, the FAB Cooperative Group defined AMLMO leukemia as a subtype in which fewer than 3% of blasts show cytochemical staining for MPO or SBB but express the pan-myeloid-associated antigens CD13 a n d / o r CD33 in the absence of lymphoid-specific markers. 3 Subsequent authors have sometimes modified this definition by including cases that had anti-MPO positive blasts by immunologic methods but failed to express any other myeloid marker, such as CD13 or CD33. 8 Other AML-MO studies included cases with blasts showing nonspecific esterase staining. 9 ' 10 We chose to exclude cases with any myeloid cytochemical staining in the blasts, because these cases would appear to represent AML with at least some cytochemical evidence of lineage maturation. This exclusion also adheres more closely to the FAB

Cooperative Group's original definition of AML-MO, in which only 1 of 10 cases demonstrated any MPO positive blasts, and this single case had fewer than 1% MPO positive blasts. 3 We believe that it is also important to exclude cases of myelodysplastic syndrome or chronic myeloproliferative disorder that have progressed to acute leukemia, because these cases may be biologically distinct from de novo AML-MO. It is difficult to define a diagnostic entity by the absence of a cytochemical enzyme stain. Laboratory-tolaboratory differences in specimen preparation, staining procedures, and enzyme substrates add a definite degree of laboratory variation. In addition, the interpretation of cytochemical stains lends itself to diagnostic subjectivity. In our series, three cases were excluded after careful review revealed rare cytochemical MPO-positive blasts or nonspecific esterase positivity in numerous non-blast cells. The number of more differentiated FAB cases originally classified as FAB-MO is higher in other series11; more frequently, this issue is not addressed. Anti-MPO-positive leukemic blasts by an immunocytochemical method were seen only rarely in our study. Certainly some studies have shown up to 100% of AML-MO cases with anti-MPO positivity, suggesting that an early form of the MPO enzyme can be detected before its cytochemical expression. 8-10 The absence of MPO, however, as detected by in situ hybridization for MPO messenger RNA has been noted in a high (50%) proportion of cases of AML-MO,12 raising questions about the validity of a high rate of anti-MPO detection in AML-MO. Some classification systems 13 and some studies have placed great weight on finding anti-MPO positivity in making a diagnosis of AML-MO, even to the point of relying on anti-MPO to classify cases as AML-MO in the absence of any other evidence of myeloid differentiation. 8 We would caution against overinterpretation of anti-MPO positivity in the absence of CD13 or CD33 antigen expression, because we and others have seen anti-MPO-positive blasts in

AJCP • January 1998


complete remission with further treatment, and 2 did not respond to the chemotherapeutic regimen. Four of these 11 patients underwent bone marrow t r a n s p l a n t a t i o n (two allogeneic, two autologous after first relapse); one patient w h o received an allogeneic transplant is currently in second remission. The median follow-up was 18 months (range, 5-107 months). Only the four patients who underw e n t bone m a r r o w transplantation have no evidence of disease; six patients died of their disease, and one is alive with disease. Median duration of survival a m o n g this treated group of patients is greater than 18 months. If the patients who received bone marrow transplant are excluded from consideration, the median survival was 9 months, with a median patient age of 62 years. If the patients who received low- or intermediate-dose chemotherapy (3 patients) or supportive care alone (3 patients) are included, 12 of 17 patients died of their disease after 1 to 34 months.

COHEN ET AL Acute Myelo id Leukemia

Although CD22 is generally considered a B-cell lineage-specific antibody, CD22 positivity has been previously described in cases of AML. 19 ' 20 We believe that the high rate of dim CD22 positivity seen in this series may represent a titering artifact in our laboratory. Vol.

Indeed, such occurrences must be considered in evaluating the significance of lymphoid antigen positivity in these cases. As was noted in a study of myeloid antigen-positive childhood acute lymphoblastic leukemias, both the use of fluorochrome and the use of different commercial antibody sources may significantly affect the percentage of antigen-positive cells.21 N o obvious correlation w a s seen in this study between lymphoid-associated antigen expression and TdT positivity; cases with a high percentage of TdT-positive blasts were not necessarily those with lymphoidassociated antigen expression. Previous studies of AMLMO have found a variable proportion of cases with TdT positivity, with rates of positivity between 14% and SO0/*.1' 3,9-11,22-26 W e believe that the high rate of TdT positivity in this study most likely reflects a uniformity in diagnosis and the immaturity of the leukemic blasts in this series. The TdT expression also complements the high rate of CD34 positivity found in our cases.27 Most previous studies of AML-MO have found the disease to be resistant to chemotherapy, with a worse prognosis than other AMLs. 10,11 ' 22 ' 24 ' 26 ' 28 In the current study, treatment of this disease varied considerably. Some patients received supportive care alone, whereas others received aggressive chemotherapy and bone marrow transplantation. With this variation in mind, we must nevertheless conclude that, in this series of patients, AML-MO did not confer a prognosis more dismal than other AMLs. Although it could be considered an acute leukemia relatively resistant to treatment, with more than one induction chemotherapy cycle required for a complete response, long-term remissions were seen in this series; however, even among patients who underwent bone marrow transp l a n t a t i o n , late relapses w e r e also seen. With a median survival of 9 months among patients w h o received treatment but did not undergo bone marrow t r a n s p l a n t a t i o n , we also m u s t consider that the median age of this group of patients was 62 years. This length of survival compares favorably with that found in a large retrospective study comparing survival among older and younger patients with AML. In that study, patients older than 60 years h a d a median survival of 4 months, whereas those younger than 60 years had a median survival of 12 months. 29 In summary, w h e n narrowly defined, AML-MO appears to be a rather homogeneous entity. Patients are usually older and more frequently male. No single karyotypic abnormality was found, but karyotypes were often complex, with a high proportion of cases with m o n o s o m y 7 a n d trisomy 13—chromosome abnormalities shared with other AMLs and previously • No. 1


otherwise classic cases of ALL. 14-16 This finding suggests that some lymphoblasts may contain rudimentary or precursor forms of MPO that cannot be modified to a functional e n z y m e . It w o u l d also seem possible that if, during the immunostaining procedure, cellular permeability occurs faster than protein/enzyme fixation, then there is a risk of cytoplasmic components, such as peroxidase-positive granules, leaching into accessible cells. If this were to occur in a case of ALL, a false-positive anti-MPO reaction would be observed. We and others have observed differences in anti-MPO reactivity as detected by flow cytometry when different fixation and permeabilization steps are used (unpublished results). In summary, anti-MPO appears to be of limited utility in the diagnosis of AML-MO, and we would not rely on its expression alone to diagnose this entity. In its original proposal for the recognition of AMLMO, the FAB Cooperative Group suggested excluding cases with any lymphoid antigen positivity, with the exception of CD7 and possibly CD2. 3 Other lymphoidassociated antigens, such as CD19, also should be included, as they have been described in otherwise classic cases of AML. 17 - 18 We concur with another recent report, in which a high incidence of lymphoid antigen positivity was found in AML-MO; we believe that these cases are best considered as minimally differentiated myeloid leukemias that share some lymphoid-associated antigen expression, rather than as acute biphenotypic leukemias. 1 0 Such cases in our series showed only one B- or T-lymphoid antigen, and rarely one B- and one T-lymphoid antigen in combination with the expression of myeloid antigens. Most frequently, lymphoid antigen expression was dim in contrast to usually bright myeloid antigen staining, as measured by flow cytometric immunophenotyping. No difficulty was found in distinguishing these cases from acute lymphocytic leukemias with myeloid antigen expression, because these cases show a converse pattern of strong (and usually multiple) lymphoid antigen expression and weak myeloid antigen expression. In addition, none of the cases of AML-MO exhibited the chromosomal abnormalities of t(9;22)(q34;qll) or llq23, which are associated with both lymphoid and myeloid leukemias and may represent true "stem cell" leukemias that are capable of differentiating along either lymphoid or myeloid lines. 18

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described as overrepresented in AML-MO.10' n' 24 The leukemic blasts are fairly uniform in both morphology and immunophenotype, with blasts in all tested cases positive for CD34, HLA-DR, and TdT. These findings appear to define an acute leukemia at the earliest recognizable stage of myeloid differentiation. REFERENCES

AJCP • January 1998


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