INTRODUCTION

Acute myeloid Leukemia is a heterogeneous disease. Therefore parameters are needed to classify this disease into biologic entities to understand its pathogenesis and develop specific treatment approaches.^{1, 2} As therapeutic advances were made, distinguishing the subtypes of acute leukemia became increasingly important.

Acute leukemias are classified on the basis of the presumed cell of origin.Concordance between experienced observers in the classification of acute leukemia increases from 70 to 99% when morphologic criteria are supplemented by cytochemical and immunphenotypic information.³

The modern era for classification of acute leukemias dates back to 1976, when international group of investigators from France, America and Britain developed a uniform classification system designated as French-American-British (FAB) classification⁴, which was subsequently revised in 1985.⁵

The FAB classification of AML divides cases into eight major groups with subtypes for three of them (table 1). The classification criteria are based on morphologic and cytochemical features; however for some of the categories, immunophenotyping is necessary.^{6, 7} It is lineage-based morphological classification that categorizes cases according to the degree of maturation of the leukemic cells and their lineage differentiation. The major advantage of the FAB classification system is its ease of use. The cytological criteria are well defined; they do not require high technology and can be applied in most laboratories through out the world^.
8Keeping in view these advantages, the FAB proposal was adopted internationally. It provided long needed standard terminology and was quickly accepted by most of the multi-institutional study groups for management plans and comparison of treatment results between morphologic subtypes for their prognostic significance.

Present study was done to determine the frequency of AML types in our population. As the patients were from all over Pakistan belonging to various ethnic groups, our study thus well represent AML subtypes of the entire country.

Although, it is a single institution based study, the number of cases studied is largest that is ever reported at national level. Hence, our study tends to establish a trend of AML subtypes in Pakistan.

Material and methods

This is descriptive case-control study done at hematology department of Aga Khan University Hospital. This is a tertiary care hospital receiving cases from all over the country. The study was conducted over a period of 2 years from January 1999 to December 2000.

Patients with history of previous hematological disorders like myelodysplasia, CML, aplastic anaemia and with history prior chemotherapy or radiotherapy were excluded from the study. The patients who were known cases of AML with or without treatment including relapsed cases were also not included.

Table-1: FAB Classification of AML

Myeloblastic leukemia minimally differentiated M0

Myeloblastic leukemia without maturation M1

Myeloblastic leukemia with maturation M2

Hypergranular promyelocytic leukemia M3

· Microgranular variant Myelomonocytic leukemia M4

· With bone marrow eosinophilia M4Eo

Monocytic Leukemia M5

· Poorly differentiated M5a

· Differentiated M5b

Erythroleukemia M6

Megakaryoblastic Leukemia M7

Based on this, a total of 116 subjects with newly diagnosed untreated denove AML were included in the study. It included patients of all age groups and both sexes.

The diagnosis of AML was established according to the standard practice, and was based on peripheral blood and bone marrow morphology and cytochemistry. Immunophenotyping was done where considered essential. Hematological parameters were done on Coulter counter (model stak-s).

Bone marrow aspiration was done from posterior iliac crest. A written consent was taken from patients or parents as appropriate. In every case, 6-8 smears were made; two of them with peripheral smears were stained by Leishman’s stain. In addition following cytochemical stains were carried out on peripheral blood and bone marrow smears in each case: Sudan blackB (SBB), periodic acid-Schiff (PAS), myeloperoxidase (MPO) and alpha naphthyl acetate esterase (ANAE) by commercially provided kits from Sigma Diagnostic according to manufacturer’s instructions. Immunophenotyping was carried out in some patients with diagnostic difficulties and was performed by 2 color flow cytometric analysis of bone marrow aspirate or peripheral blood specimens with a Becton Dickinson (Mountain View, Calif) FAC Scan instrument. After mononucleaer cell enrichment by centrifugation over Histopaque-1077 (Sigma, St. Louis Mo), samples were studied for surface antigen expression using a panel of 15 monoclonal antibodies supplied by (Becton Dickinson, San Jose, Calif and Dako Denmark).

All the cases were reviewed by first and third author independently. On the basis of morphology, cytochemistry and immunophenotyping, AML was classified into its various subtypes. (M0-M7) using FAB criteria. (Table-1)^8. The presence or absence of Auer rod in each bone marrow film was also noted.

RESULTS

Of 116 cases, 70 were males and 40 were females with male to female ratio1.5:1. The mean age was 32 years (range 6 months –85 years). 21 cases were up to the age of 15years comprising of 17 males and 4 females and their age ranges between 6 months- 15 years. Their haematological parameters are given in table 2. The CBC showed a wide range of variation in hemoglobin concentration and platelets ranging from subnormal to normal. Their Leukocyte count also showed variation from leucopenia to hyper leukocytosis.

In our study, we found AML (M4 FAB subtypes) to be the commonest comprising 42 out of 116 of total cases (36.25%). This also included 6 cases of M4Eo variant. The frequency of various AML subtypes according to FAB classification is given in Figure 1.

Figure 1: French –American –British (FAB) Sub types in 116 cases of AML

The cases were further divided in adult and children group. Both groups revealed M4 as a most common FAB type followed by M2. Relative frequencies of various subtypes for adults and children are illustrated in Table 3.

Auer rods were present in 36.2 %( 42 cases). They were more common in M3 subtype (9/12, 75%) followed by M2 (18/35, 51.4%), M4 (12/42, 28.5%), M1 (2/10, 20%) and M5 (1/7, 14.2%).The number of Auer rods per cell was consistently greater in M3 subtype. No Auer rods were observed in the M0 or M6 subtype.

Table-2: Haematological parameters in AML patients (n = 116)

	Mean	Range
Hemoglobin (gm/dl)	8.4	2.2-14.4
White Cell Count (10⁹/l	63.39	0.6-497.4
Platelet count (10⁹/l)	48.9	1.0-270.0

Table 3: FAB distribution of AML according to age group (n= 116)

Fab Group	< 15 Years n %	> 15 Years n %
M0	05 (23.8)	04 (4.2)
M1	01 (4.7)	09 (9.4)
M2	06 (28.5)	29 (30.5)
M3	00 (0)	12 (12.6)
M4	07 (33.3)	35 (36.8)
M5 a	00 (0)	04 (4.2)
M5 b	01 (4.7)	02 (2.1)
M6	01 (4.7)	00 (0)
M7	00 (0)	00 (0)
Total	21 (100)

DISCUSSION

With the introduction during the late 1960s and 1970s of increasingly effective therapy for acute leukemias, it became necessary to determine subgroups, which might require different treatment approaches. In 1976 FAB system ⁴ of classification was introduced, which was subsequently revised at various times to improve concordance.^{5, 6, 7} This system provided structured criteria for the diagnosis of various sub types of AML and is based mainly on morphological and cytochemical features; for some of the categories, immunophenotyping is necessary.^8,9 Since than it has been widely adopted nationally and internationally for classification of acute leukemias, although ambiguities still give rise to confusion and it has been the subject of recent criticism.¹⁰ So, in 1997 a revised WHO classification of AML was published which included cytogenetic studies as well.¹¹ However this classification is not practiced widely at national level because of financial constraints. The clinical and biological significance is also claimed for this system which accounts for distinct prognostic differences for various subtypes and their close association with chromosomal abnormalities. Although FAB proposals may be considered over simplification, but they do serve to provide the guidelines for both hematologists and non specialists and facilitate quick, consistent diagnosis and classification of these diseases. It is for these reasons that FAB is still favourite and popular among Pakistani hematologists.

The FAB distribution of AML has been extensively studied in the past decades at national and international levels.^9,12-19 In table 4 the adult results are compared with other series using FAB system. Most published data indicate the predominance of M2 as a most common subtype.^9.14-16,18Occurrence of this subtype is also common after primary malignancy.²⁰ However, two studies from Saudi Kingdom reported predominance of M4 and

M5 subtypes.^13,17 Nakase et al showed AML-M4 as common subtype in Australian population compared to Japanese, where AML-M2 is common.²¹ Present study also confirms M4 as the most common type followed by M2. This is in concordance with the previously published results from our institution by Kakepoto et al.¹⁹

Many of the differences in AML subtypes may be due to the subjectivity of morphologic diagnosis together with variable nature of acute myeloid leukemia subtypes, with no real demarcation. Some genetic factors may be responsible for a particular FAB subtype of AML in our population. Secondly most studies at national level have small number of patients and probably with underutilization of cytochemical stains. Moreover these studies were not subjected to immunophenotyping that may be because of error in diagnosis. The other reason for this discrepancy may be patients of different ethnic group and/or geographical variation.

Auer rods were seen in 36% of cases with highest frequency in M3. Spence et al ¹³ reported Auer rods in 40.6% of cases in their series. These results are consistent with present study.

Male to female ratio in present study is 1.5:1, which is in concordance with national and international studies.^14,16,17,22 The mean age (32 years) at presentation seems to be lower than the expected mean age reported in western countries where AML peaks in incidence after the 6^th decade of life.²³ However, this is similar to mean age reported in studies from Saudi Arabia ¹⁷and Pakistan.^16,19,24 The difference of mean age between present study and western studies is probably due to different geographical distribution.

Table 4: FAB Classification of AML in various centers in adults

Author/ Year/ No of Cases	M0%	M1%	M2%	M3%	M4%	M5%	M6%	M7%
Swirsky et al ¹²1986 (U.K) n= 619	00	30	25	5	23	13	2.4	00
Spence et al¹³1988 (KSA) n= 121	1.7	1.7	14.9	8.3	57.8	13.2	1.7	00
Raina et al ¹⁴1990 (Libya) n= 54	00	7	57	15	13	4	4	00
Hassan et al¹⁵1993(Pak) n= 62	1.6	22.5	32.2	9.1	22.5	8.6	1.6	1.6
Chaudry et al ¹⁶1993 (Pak) n=54	00	13	44.4	11.1	24	3.7	3.7	00
Harakati et al ¹⁷1998 (KSA) n=52	00	2	4	17	40	33	00	04
Khalidi et al ¹⁸ 1998 (USA) n=78	8.9	19.2	27.0	9.0	20.5	11.5	2.6	1.3
Kakepoto et al ¹⁹2002 (Pak.) n=74	00	8.1	16	15	46	9.5	00	2.7
Arber et al ⁹ 2003 (USA) n=255	7.0	19.2	28.67	8.7	26.7	4.8	2.5	2.4
Present Study n= 95	1.2	9.4	30.5	12.6	36.8	6.3	00	00

CONCLUSION

In conclusion the most common type observed in our study was Acute Myelomonocytic Leukemia (M4) followed by Acute Myeloblastic Leukemia with maturation (M2). Although demographic features is not the aim of our study but this is in accordance with other national studies.

A multi-institutional study with large sample size from other areas of Pakistan is needed to confirm our findings. It is desirable that more than one experienced observer should examine the material before a final diagnosis is made. A full range of cytochemical stains is essential with immunophenotyping studies where appropriate for proper identification of the acute leukemias.

ACKNOWLEDGEMENT

Authors thank Mr. Munir Gillani and Ms. Rozina Rehman for typing of manuscript and secretarial support and Dr. Bushra Moiz, Assistant Professor for the critical review of the same.

REFERENCES

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Keywords: French-American-British (FAB) classification, Acute myeloid Leukemia (AML), subtypes.

INTRODUCTION

Material and methods

DISCUSSION

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