acute myeloid leukaemia, the myelodysplastic syndromes and … marrow... · 2010. 1. 3. ·...

‘malignant’ characteristics may be manifested clini-cally as transformation to acute leukaemia. MDS maytherefore be regarded as a preleukaemic condition.

Acute myeloid leukaemia

AML is a heterogeneous disease. In different pati-ents the leukaemic clone shows differing patterns ofdifferentiation and maturation. From 1976 onwards,an international co-operative group, the French–American–British (FAB) group, published a seriesof papers on the classification of AML. The FABclassification [1,2] became widely accepted and wassubsequently incorporated into other systems ofclassification. It is based on the pattern of differenti-ation shown (for example: granulocytic, monocytic,erythroid, megakaryocytic) and the extent of mat-uration (for example: myeloblast, promyelocyte,granulocyte). Both differentiation and maturationare assessed and the predominant cell types inperipheral blood and bone marrow are determined.The FAB classification is summarized in Table 4.1.The FAB classification was incorporated into themorphologic, immunologic, cytogenic (MIC) classi-fication [3], the morphological, immunological,cytogenetic, molecular genetic (MIC-M) classification[4] and, most recently, the WHO classification [5].The WHO classification is summarized in Table 4.2.The major aim of this classification is to recognizesubtypes of AML that differ in their prognosis. Theclassification is hierarchical, so that therapy-relatedcases are first assigned to two specific categories.Cases falling into one of four cytogenetic subtypesare then categorized. Next, cases with multilineagedysplasia are assigned to a specific category. Finally,all remaining cases are categorized morphologic-ally by a modification of the FAB classification. Animportant difference from the FAB classification is

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ACUTE MYELOID LEUKAEMIA, THEMYELODYSPLASTIC SYNDROMESAND HISTIOCYTIC NEOPLASMS

Acute myeloid leukaemia (AML) is a disease resul-ting from the neoplastic proliferation of a clone ofmyeloid cells, characterized by uncoupling of prolif-eration and maturation. The leukaemic clone maybe derived from a pluripotent stem cell (capable ofgiving rise to both myeloid and lymphoid lineages),from a multipotent stem cell (capable of giving riseto more than one myeloid lineage) or from a com-mitted precursor cell (for example, one capable ofgiving rise only to cells of granulocyte and mono-cyte lineages). Normal haemopoietic marrow islargely replaced by immature myeloid cells, mainlyblast cells, which show a limited ability to differen-tiate into mature cells of the different myeloid lin-eages. Pancytopenia is common, as a result both ofthe replacement of normal bone marrow and of thedefective capacity for maturation of the leukaemicclone.

The myelodysplastic syndromes (MDS) resembleAML in that normal polyclonal haemopoietic bonemarrow is largely replaced by a neoplastic clone,usually derived from a multipotent stem cell. Theneoplastic clone is characterized by defective matu-ration so that haemopoiesis is usually both morpho-logically dysplastic and functionally ineffective. Inthe great majority of patients with MDS, the bonemarrow is hypercellular but there is increasedintramedullary death of haemopoietic precursorsleading to defective production of mature cells ofone or more haemopoietic lineages; this process,which leads to various combinations of anaemia,neutropenia and thrombocytopenia, is designatedineffective haemopoiesis. In MDS, as in AML, thereis imbalance between proliferation and maturationbut the degree of abnormality is less than in AML so that the proportion of blast cells is lower. Theneoplastic cells in MDS show a tendency to clonalevolution; emergence of a subclone with more

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Table 4.1 The FAB classification of acute myeloid leukaemia [1,2].

Criteria for FAB Criteria for classification as specific Equivalent namediagnosis of AML category FAB subtype of AML

M1 • Blasts ≥90% of bone marrow NEC Acute myeloblastic leukaemia (non-erythroid cells) without maturation

• ≥3% of blasts MPO- or SBB-positive• Maturing monocytic component in bone

marrow ≤10%• Maturing granulocytic component in bone

marrow ≤10%

M2 • Blasts 30–89% of BM NEC Acute myeloblastic leukaemia • BM maturing granulocytic with maturation

component >10% NEC• BM monocytic component <20% of NEC

and other criteria of M4 not met

M3 • Characteristic morphology Acute promyelocytic leukaemia

Blasts ≥30% M3v • Characteristic morphology Variant form of acute bone marrow cells* promyelocytic leukaemia

≥3% of blasts SBB- M4 • Blasts ≥30% of BM NEC Acute myelomonocytic or MPO-positive† • Granulocytic component ≥20% of BM NEC leukaemia

• Monocytic component ≥20% of BM NEC and either PB monocytes ≥5 × 109/l or BM like M2 but PB monocytes ≥5 × 109/l and cytochemical proof of monocytic differentiation

M5a • Blasts ≥30% of NEC Acute monoblastic leukaemia• BM monocytic component ≥80% of NEC • Monoblasts ≥80% of BM monocytic component

M5b • Monoblasts <80% of BM monocytic component Acute monocytic leukaemia• Blasts ≥30% of NEC BM monocytic component

≥80% of NEC

M6 • Erythroid cells ≥50% of BM cells Acute erythroleukaemia• BM blasts ≥30% of NEC

M7 • Blasts shown to be predominantly Acute megakaryoblastic megakaryoblasts leukaemia

M0 • <3% of blasts MPO- or SBB-positive Acute myeloid leukaemia• Lymphoid markers negative with minimal evidence of • Immunological or ultrastructural evidence of myeloid differentiation

myeloid differentiation

* Except in some M3 and some M6.† Except in M0 and some M5a.

that cases with between 20 and 30% of bone marrow blasts are classified as AML rather than asrefractory anaemia with excess of blasts in transfor-mation, a FAB category of poor prognosis MDS. Inaddition, cases with an even lower blast percentageare accepted as AML if they have one of the specific

cytogenetic abnormalities listedat(8;21), t(15;17),inv(16), t(16;16) or an 11q23 rearrangement. Sinceonly an outline of the WHO classification has so far been published, we shall discuss AML mainly interms of the FAB classification but will note wherethere are important differences from the WHO

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ACUTE MYELOID LEUKAEMIA 143

classification. We shall use a cut-off point of 20%blasts for categories of AML not defined by the FABgroup but shall retain the FAB definitions for FABcategories.

AML occurs at all ages but becomes increasinglycommon with advancing age. The incidence risesfrom one to 10/100 000/year between the ages of20 and 70 and is somewhat higher in men than inwomen.

The different categories of AML have many clin-ical features in common but some differences. Somedegree of hepatomegaly and splenomegaly is com-mon, particularly in those categories with a pro-minent monocytic component (M5 and, to a lesserextent, M4 AML). Lymphadenopathy and infiltra-tion of the skin, gums and tonsils are also morecommon in AML with a prominent monocytic com-ponent. As a consequence of pancytopenia, patientscommonly exhibit pallor and bruising and show

susceptibility to infection. A more marked bleedingtendency is usual in acute promyelocytic leukaemia(M3 AML), in which disseminated intravascularcoagulation (DIC) is a frequent feature.

The different categories of AML have certainhaematological features in common although the morphological features of the predominantleukaemic cells differ. Normocytic normochromicanaemia, neutropenia and thrombocytopenia arecommon. The total white cell count is usually elevated, as a result of the presence of circulatingleukaemic cells, but some patients have a normal orlow total count with few circulating immature cells.A normal or low count is most often observed in M3and M7 AML. In adults, M7 AML commonly pre-sents with the features of acute myelofibrosis, thatis with pancytopenia, few circulating immaturecells and a bone marrow which, as a consequence ofbone marrow fibrosis, cannot be aspirated. (How-ever, it should be noted that not all cases of acutemyelofibrosis are examples of M7 AML.)

The blood film and bone marrow aspirate featuresare of prime importance in the diagnosis of AML.The bone marrow biopsy is of secondary impor-tance except in those cases in which an adequateaspirate cannot be obtained. Assignment to a FABcategory is more readily done on the basis of theblood and bone marrow aspirate findings and is notalways straightforward from tissue sections. It mayalso be impossible, in tissue sections, to distinguishM1 and M0 AML from acute lymphoblastic leukaemia(ALL) unless immunohistochemistry is employed.Antibodies reactive with CD68, CD117, lysozyme,neutrophil elastase and myeloperoxidase (MPO)are useful in distinguishing AML from ALL intrephine biopsy sections [6–8]. The antigen (calpro-tectin) detected by the MAC387 antibody is alsomyeloid-associated, being expressed by cells of themonocyte lineage and by mature cells of the granu-locyte lineage. To distinguish AML with erythroid ormegakaryocytic differentiation from ALL, it is nec-essary to use antibodies reactive with antigens such as glycophorin and spectrin (erythroid) or CD42b,CD61 and von Willebrand’s factor (megakary-ocytic). Some cases of AML, particularly thosebelonging to the FAB M0 category, give negativereactions with some of the antibodies commonlyused to detect myeloid differentiation (see Table 2.6).This and other problems in diagnosis will be dis-cussed under ‘Problems and pitfalls’ (see page 166).

Table 4.2 The WHO classification of the acute myeloidleukaemias [5].

AML with recurrent cytogenetic translocationsAML with t(8;21)(q22;q22), AML1(CBFα)/ETOAcute promyelocytic leukaemia (AML with

t(15;17)(q22;q11–12) and variants, PML/RARα)AML with abnormal bone marrow eosinophils

(inv(16)(p13q22) or t(16;16)(p13;q22),CBFβ/MYH11)AML with 11q23 (MLL) abnormalities

AML with multilineage myelodysplasia*With prior myelodysplastic syndromeWithout prior myelodysplastic syndrome

AML and myelodysplastic syndromes, therapy-relatedAlkylating agent-relatedEpipodophyllotoxin-relatedOther types

AML not otherwise categorizedAML minimally differentiatedAML without maturationAML with maturationAcute myelomonocytic leukaemiaAcute monocytic leukaemiaAcute erythroid leukaemiaAcute megakaryocytic leukaemiaAcute basophilic leukaemiaAcute panmyelosis with myelofibrosis

* Defined as dysplastic features in two or more cell lines.

Acute myeloblastic leukaemia (M1 and M2AML)

The term acute myeloblastic leukaemia indicatesleukaemia in which lineage commitment (differen-tiation) is to one of the granulocyte lineages, usuallythe neutrophil lineage. Cases can be dividedbetween the FAB categories M1 and M2, dependingon whether leukaemic cells are predominantlymyeloblasts or whether, alternatively, further mat-uration to promyelocytes and later cells is occur-ring (see Table 4.1). Cases of AML assigned to FAB M1 and M2 categories would, in the WHOclassification, fall into various categories including‘t(8;21)-associated AML’, ‘AML with and withoutmaturation’, ‘AML with multilineage dysplasia’ and‘acute basophilic leukaemia’.

Peripheral blood

Anaemia, thrombocytopenia and a high white cellcount are usual. Neutropenia is common in M1AML but cases of M2 AML may have a normal orhigh neutrophil count. The majority of cases haveconsiderable numbers of circulating myeloblasts.These are large cells, usually about twice the diame-ter of an erythrocyte. They have a high nucleocyto-plasmic ratio. The nucleus has a diffuse chromatinpattern and one or more nucleoli. The cytoplasm isweakly or moderately basophilic and may containscanty azurophilic granules. In some cases, particu-

larly but not exclusively M2 AML, the blasts containAuer rods; these are cytoplasmic crystals formed byfusion of primary granules with which they sharestaining characteristics. In M2 AML the peripheralblood may also contain morphologically abnormalpromyelocytes and other maturing cells. Occa-sional patients with M2 AML have eosinophilia orbasophilia. In AML associated with t(8;21)(q22;q22)the peripheral blood may show large blasts withbasophilic cytoplasm and often a single long thin Auer rod, together with morphologicallyabnormal maturing cells. In AML associated witht(6;9)(p23;q34) there may be peripheral bloodbasophilia.

Bone marrow cytology

The bone marrow is markedly hypercellular. Num-bers of megakaryocytes and developing erythroidcells are usually reduced. In M1 AML the bone mar-row is almost totally replaced by myeloblasts, someof which may contain scanty granules or Auer rods(Fig. 4.1). In M2 AML myeloblasts are relatively lessnumerous and there are considerable numbers ofmaturing cells (Fig. 4.2). Many of these are mor-phologically abnormal and often difficult to cat-egorize; abnormalities include: (i) hypogranularity;(ii) bizarre nuclear shapes; and (iii) the presence ofAuer rods, not only in blasts but sometimes also in promyelocytes, myelocytes and neutrophils. Insome cases of M2 AML the maturing granulocytic

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Fig. 4.1 BM aspirate, M1 AML.Note that some of the blast cellsresemble lymphoblasts in that theyare small and round with a highnucleocytoplasmic ratio and nogranules. The presence of anagranular neutrophil andoccasional blasts with granulessuggests the correct diagnosis. MGG ×940.


component includes, or is composed of, basophils or eosinophils. In both M1 and M2 AML the ery-throcytes and megakaryocytes may show dysplastic features.

In M2 AML associated with t(8;21) the bone mar-row, in addition to maturing cells of neutrophil lin-eage, commonly shows increased eosinophils; theseare usually cytologically normal. Erythroid cells andmegakaryocytes do not show dysplastic features. InM2 AML associated with t(6;9), the bone marrowcommonly shows not only blasts of basophil lineagebut also increased numbers of mature basophils;there is often associated myelodysplasia and Auerrods are commonly present. In M2 AML associatedwith 12p-, blasts are also of basophil lineage butthere is very little maturation so that they mayappear undifferentiated by light microscopy.

Cytochemistry

The myeloid nature of M2 AML is evident from the features of differentiation observable in May–Grünwald–Giemsa (MGG)-stained films but in M1AML cytochemical staining [9] is often necessary toconfirm the diagnosis. Either MPO or Sudan black B(SBB) staining must be positive in at least 3% ofblasts to satisfy the FAB criteria of AML [1]; usuallyboth stains are positive. These stains are also veryuseful in identifying Auer rods and reveal theirpresence in some cases in which they are not

identifiable in the MGG stain. A naphthol AS-Dchloro-acetate esterase (chloro-acetate esterase)stain is also positive in myeloblasts in the majorityof cases of M1 and M2 AML. Metachromatic stain-ing with toluidine blue is useful in the diagnosis ofcases with basophil differentiation but little matura-tion, for example cases of M2Baso AML associatedwith 12p-.

Bone marrow histology

The marrow is markedly hypercellular (greater than95% cells) in most cases. Often the cellularity seenin biopsy sections is greater than that estimatedfrom aspirated fragments in films [10]. The mor-phology of the neoplastic cells in tissue sections is different from that seen in films of aspirates. Intissue sections, myeloblasts have large round to ovalnuclei, delicate chromatin, one or more small well-defined nucleoli and scant basophilic cytoplasm(Fig. 4.3). In a half to two thirds of all cases there is a dense homogeneous infiltrate of blasts whereas,in the remainder, there is a mixture of blasts, moremature haemopoietic cells and inflammatory cellssuch as plasma cells, lymphocytes and mast cells[10,11]. Evidence of maturation is often apparent inM2 AML (Fig. 4.4). Leder’s chloro-acetate esterasestain is usually positive in M2 AML (depending on the method of processingasee page 59) but isoften negative in M1 AML. Dysplastic changes are

Fig. 4.2 BM aspirate, M2 AML,showing two myeloblasts, one ofwhich contains a long slender Auerrod, and abnormal maturing cells.MGG ×940.

commonly detected in megakaryocytes and erythroidprecursors (see page 172). In M2 AML associatedwith t(8;21) there is often a prominent infiltrate ofeosinophils scattered among the blasts. Reticulinfibrosis is present in up to a third of cases [12] butcollagen fibrosis is rare. Areas of bone marrownecrosis are sometimes present. Following chemo-therapy, the marrow is hypoplastic and often showsnecrosis, stromal oedema and gelatinous change.Residual leukaemic cells are often more apparent intissue sections than in aspirate films. However, theycan be difficult to distinguish from foci of immatureregenerating granulocytic or erythroid precursors.

In M2 AML there are usually sufficient maturinggranulocytic cells for a diagnosis of AML to be madefrom H&E-stained sections. However, a provisionaldiagnosis of M1 AML may require immunohisto-chemistry for confirmation if the diagnosis rests ona trephine biopsy specimen alone.

Cytogenetics and molecular genetics

Various subtypes of AML, characterized by specificchromosomal abnormalities, are included within theFAB M1 and M2 categories [8]. Of these, the com-monest is M2 AML associated with t(8;21)(q22;q22)

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Fig. 4.3 Section of BM trephinebiopsy specimen, M1 AML. There is a relatively uniform populationof small blast cells with a highnucleocytoplasmic ratio and prominent nucleoli. Plastic-embedded, H&E ×970.

Fig. 4.4 Section of BM trephinebiopsy specimen, M2 AML. There is a mixture of blast cells andmaturing cells of the granulocyticseries. Paraffin-embedded, H&E ×960.


and formation of an AML1-ETO fusion gene, whichis recognized as a separate entity in the MIC, MIC-M and WHO classifications. A much less commonsubtype is that associated with t(6;9)(p23;q34) andformation of a DEK-CAN fusion gene.

Acute (hypergranular) promyelocyticleukaemia

The majority of cases of acute promyelocyticleukaemia have hypergranular promyelocytes andare therefore designated acute hypergranular pro-myelocytic leukaemia, or M3 AML. A minority ofcases have abnormal promyelocytes which are eithermicrogranular or hypogranular when examined bylight microscopy; such cases are variously referredto as the variant form of acute promyelocyticleukaemia, M3 variant AML, acute hypogranularpromyelocytic leukaemia or acute microgranularpromyelocytic leukaemia. Acute promyelocyticleukaemia is recognized as a specific entity in allleukaemia classifications. Rapid, correct diagnosisof M3 and M3 variant AML is of critical importanceif early death from haemorrhage is to be avoided.

Peripheral blood

In M3 AML, the peripheral blood white cell count is not usually greatly elevated and the number ofcirculating leukaemic cells tends to be low. There isusually anaemia. The platelet count may be dispro-portionately low as a consequence of complicating

disseminated intravascular coagulation. The ab-normal promyelocytes are large cells, usually two to three times the diameter of an erythrocyte. Theircytoplasm is packed with granules which stainbright pink or reddish-purple. Some cells containbundles of Auer rods (‘faggot cells’) or giant gran-ules. No Golgi zone is apparent. The nucleus is usually round or oval but cytoplasmic granulation is so marked that the nuclear outline is difficult to discern.

In M3 variant AML, the white cell count is usu-ally elevated. Again, there is usually anaemia andmarked thrombocytopenia. Abnormal promyelocytesare characteristically more frequent in the periph-eral blood in M3 variant AML than in typical,hypergranular M3 AML. The promyelocytes mayappear completely agranular or may have fine,dust-like reddish granules (Fig. 4.5). Some cellscontain bundles of Auer rods or other crystallineinclusions. The nucleus is usually deeply lobed, oftenwith two large lobes joined by a narrow bridge. The cytoplasm is usually weakly or moderatelybasophilic but some cases have promyelocytes withmore marked basophilia and cytoplasmic protru-sions or blebs. A careful search in cases of M3 vari-ant AML often discloses a minor population of moretypical, hypergranular promyelocytes, occasionallywith multiple Auer rods.


Bone marrow aspiration is often difficult since the

Fig. 4.5 PB film, M3 variant AML, showing hypogranularpromyelocytes with characteristicdeeply lobulated nuclei. Severalcells have very fine granules. MGG ×940.

hypercoagulable state leads to clotting of the speci-men, even during aspiration. However, the bonemarrow aspirate is important in diagnosis since, inM3 AML, there may be only infrequent leukaemiccells in the peripheral blood and, in M3 variantAML, the bone marrow often contains a higher pro-portion of typical hypergranular cells than does theperipheral blood.

The bone marrow aspirate is usually intenselyhypercellular. The number of blasts is relatively lowsince the predominant cell is an abnormal promye-locyte (Fig. 4.6); in the majority of cases there arefewer than 30% of blasts in the marrow. In M3AML the predominant cell is a hypergranular pro-myelocyte while, in M3 variant, the predominantcell is a hypogranular promyelocyte with a variableadmixture of hypergranular forms. There is a markedreduction in the number of normal maturing gran-ulocytes. Erythroid cells and megakaryocytes arealso considerably reduced in number but are cyto-logically normal.

Cytochemistry

Cytochemical stains are unnecessary in typical hyper-granular M3 AML but are important in confirming a diagnosis of M3 variant AML. Typically, there are positive reactions with MPO, SBB and chloro-acetate esterase and negative reactions with non-specific esterase.


There is usually marked hypercellularity with ahomogeneous infiltrate of abnormal promyelocytes(Fig. 4.7). These cells have a characteristic appear-ance; they have prominent large granules that fillthe cytoplasm and often obscure the nucleus. Thenucleus may be oval or bilobed and has a singleprominent nucleolus. Faggot cells may be detect-able. In M3 variant AML the granules are muchsmaller and may be inconspicuous; the nuclei areoften bilobed. Occasional cases of M3 AML show little increase in cellularity at presentation, an un-usual feature in de novo AML.

Because of their hypergranularity, the leukaemiccells of M3 AML can be readily recognized in H&E-stained sections of trephine biopsy specimens. Aproportion of cases of M3 variant AML can also berecognized from cytological features. Other cases ofM3 variant AML, with very infrequent hypergranu-lar cells, require histochemistry (Leder’s stain) orimmunohistochemistry (e.g. demonstration of neu-trophil elastase) for confirmation if the diagnosisrests on the trephine biopsy specimen alone.

Collagen fibrosis may be observed at presentationin M3 AML. This is greatly increased by treatmentwith all-trans retinoic acid (ATRA) [13]. ATRA therapy leads to maturation of the leukaemic clonewith the bone marrow remaining hypercellular incontrast to the hypocellular marrow that is seen

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Fig. 4.6 BM aspirate, M3 AML,showing heavily granulatedpromyelocytes, one of whichcontains a giant granule. MGG ×940.


when chemotherapy is administered. Extensive bonemarrow necrosis has been reported in associationwith hyperleucocytosis caused by ATRA [14].


M3 and M3 variant AML are uniformly associatedwith t(15;17)(q22;q21), a PML-RARα fusion gene orboth.

Acute myelomonocytic leukaemia

Acute myelomonocytic leukaemia (M4 AML)shows significant evidence of both granulocytic andmonocytic differentiation. The granulocytic dif-ferentiation is usually neutrophilic but, in somevariants, it is eosinophilic (M4Eo) or basophilic(M4Baso). Cases of AML assigned to FAB M4 cat-egory would, in the WHO classification, fall into various categories including ‘AML with abnormalbone marrow eosinophils’, ‘AML associated with11q23 rearrangements’ and ‘acute myelomonocyticleukaemia’.

Peripheral blood

There is usually anaemia and thrombocytopeniawith an elevated white cell count and circulatingleukaemic cells of both granulocytic and monocyticlineages. Myeloblasts and monoblasts show the usualcytological features of these lineages (see below).

Maturation of leukaemic cells is usual so that theleukaemic cell population commonly includes bothmonocytes and neutrophils. However, some casesof M4 AML have no evidence in the peripheralblood of the significant monocytic component whichis present in the bone marrow. In AML associatedwith inv(16)(p13q22) or t(16;16)(p13;q22), thereare usually only occasional eosinophils in the periph-eral blood and these are not morphologically veryabnormal. In AML associated with t(6;9)(p23;q34),there may be basophilia with some dysplasticbasophils.


The bone marrow is hypercellular and shows a vari-able mixture of cells of granulocytic and monocyticlineages (Fig. 4.8). In the majority of cases, matura-tion of leukaemic cells is occurring and maturemonocytes as well as maturing cells of granulocyticlineage can be readily recognized. However, thereare a minority of cases in which, despite a peripheralblood monocytosis, the bone marrow cannot be dis-tinguished morphologically from that of M2 AML.In such cases the significant monocytic compo-nent can be confirmed either by cytochemistry (seebelow) or by assays of serum or urinary lysozyme.The explanation of such cases is probably twofold.Firstly, leukaemic monocytes may be infrequent in the bone marrow because of early migration tothe peripheral blood. Secondly, promonocytes can

Fig. 4.7 Section of BM trephinebiopsy specimen, M3 AML,showing abnormal promyelocyteswith irregular, often bilobulated,nuclei containing prominentnucleoli and prominentcytoplasmic granules. Plastic-embedded, H&E ×970.

be difficult to distinguish morphologically frompromyelocytes.

Megakaryocytes and erythroid precursors areusually reduced. Dysplastic features are sometimespresent in these lineages.

In AML associated with inv(16)(p13q22) ort(16;16)(p13;q22), the bone marrow aspirate oftenshows a mixture of cells of monocyte and eosino-phil lineages but cells of neutrophil lineage are rela-tively infrequent (Fig. 4.9). Some basophils may alsobe present. The cells of eosinophil lineage are a mix-ture of myelocytes and mature eosinophils. In thegreat majority of cases the eosinophil myelocytes

are morphologically abnormal with prominent pro-eosinophilic granules, which are basophilic intheir staining characteristics, mixed with typicaleosinophilic granules. The mature eosinophils mayshow cytological abnormalities such as nuclearhyper- or hypolobulation or the presence of occa-sional pro-eosinophilic granules. Some cells ofgranulocyte lineage, mainly immature cells, showAuer rods. There is usually maturation in themonocyte lineage so that mature monocytes arepresent. Megakaryocytes and erythroid cells do notshow dysplastic features. It should, however, benoted that not all cases of AML associated with

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Fig. 4.8 BM aspirate, M4 AML,showing four myeloblasts, onemonoblast and a monocyte. MGG ×940.

Fig. 4.9 BM aspirate, M4 AML with eosinophilia (M4Eo), showing characteristic eosinophilmyelocytes, eosinophils, amonocyte and blast cells. MGG ×940.


these chromosomal rearrangements have the cyto-logical features of M4Eo; some cases fall into eitherthe M4 or M2 category of AML.

In AML associated with t(6;9)(p23;q34), the bonemarrow commonly shows, in addition to an increasein monocytes and their precursors, an increase ofmature basophils and sometimes an increase ofneutrophils or eosinophils. There are associatedmyelodysplastic features.

Cytochemistry

A double esterase stain, combining chloro-acetateesterase and non-specific esterase, is important inconfirming a diagnosis of M4 AML. In AML associ-ated with inv(16), the eosinophils may show aber-rant chloro-acetate esterase positivity. In addition,SBB and MPO stains may show Auer rods in occa-sional eosinophils and their precursors as well as in neutrophil precursors. A toluidine blue stain isuseful for confirming basophil differentiation inM4Baso AML.


The marrow is markedly hypercellular and there is an infiltrate composed of variable numbers ofmyeloblasts, monoblasts and maturing cells of bothlineages. Maturing granulocytes may be neu-trophils, eosinophils or both. Monoblasts have large

irregular nuclei with delicate chromatin and pro-minent nucleoli; cytoplasm is abundant and may bevacuolated. The distribution of monoblasts is notuniform: they are often seen in small clusters, par-ticularly in the paratrabecular areas [10]. Dysplasticchanges are often seen in other haemopoietic lin-eages and reticulin fibrosis may be present.

Trephine biopsy sections in AML associated withinv(16) often show increased eosinophils and pre-cursors (Fig. 4.10). However, M4Baso, associatedwith t(6;9), cannot be distinguished from othercases of M4 AML since basophil granules are dissolved during processing. M4 and M4Eo AMLcan usually be readily recognized in H&E-stainedsections so that histochemistry and immunohisto-chemistry are not needed in most cases.


M4 AML is preferentially associated with transloca-tions having an 11q23 breakpoint and rearrangementof the MLL gene. In addition, there are several othersubtypes within the M4 AML category defined by associated cytogenetic and molecular geneticabnormalities. These are M4 and M4Eo AML asso-ciated with inv(16)(p13q22) or t(16;16)(p13;q22)and formation of a CBFβ-MYH11 fusion gene andM4Baso associated with t(6;9)(p23;q34) and a CAN-DEK fusion gene. The former is a WHO category butthe latter is not.

Fig. 4.10 Section of BM trephinebiopsy specimen, M4Eo AML,showing monoblasts, myeloblasts,eosinophil myelocytes andeosinophils. Plastic-embedded,H&E ×970.

Acute monocytic/monoblastic leukaemia

AML showing predominantly or entirely monocyticdifferentiation is categorized as acute monocytic oracute monoblastic leukaemiaaM5 AMLathe for-mer showing maturation of leukaemic cells tomature monocytes and the latter showing littlematuration. The FAB group have assigned to thesecategories (designated M5) those cases of AML inwhich there is monocytic differentiation and inwhich less than 20% of bone marrow non-erythroidcells are granulocytes or their precursors. Cases arefurther divided into acute monoblastic leukaemia(M5a) and acute monocytic leukaemia (M5b) onthe basis of whether or not the leukaemic clone isshowing maturation (see Table 4.1). Cases of AMLassigned to the FAB M5 category would, in the WHOclassification, fall into various categories including‘AML associated with 11q23 rearrangements’ and‘acute monocytic leukaemia’.

Peripheral blood

The peripheral blood usually shows anaemia, throm-bocytopenia and leucocytosis with circulatingleukaemic cells which are variously monoblasts,promonocytes or monocytes. Monoblasts are largerthan myeloblasts, usually with a diameter aboutthree times that of an erythrocyte. Their shape variesfrom round to oval or irregular. The cytoplasm isvoluminous and varies from weakly to strongly

basophilic; it may contain very infrequent granules.The nucleus ranges from round to lobulated and isusually nucleolated; nucleoli vary from being large,single and prominent to being smaller and multiple.Promonocytes are large cells with an oval or lobu-lated nucleus and moderately basophilic cytoplasmcontaining fairly numerous azurophilic granules.Monocytes in M5 AML resemble normal monocytesin having lobulated nuclei and weakly basophilic,sometimes vacuolated, cytoplasm. They may showcytological abnormalities such as nuclei of bizarreshapes. In M5b AML, the peripheral blood containsmonocytes and a variable number of promonocytesand monoblasts. In some cases of M5a AML the periph-eral blood contains large numbers of monocytes andpromonocytes, even though the predominant cell inthe bone marrow is a monoblast. In other cases theperipheral blood leukaemic cells, like those in themarrow, are almost exclusively monoblasts.


The bone marrow is hypercellular and numbers of megakaryocytes and erythroid precursors arereduced. In M5a AML (Fig. 4.11) the great majorityof cells are monoblasts. All cases in which at least80% of bone marrow non-erythroid cells are mono-blasts are classified as M5a, regardless of whetherthere are maturing cells in the peripheral blood. InM5b AML the marrow contains a mixture of mono-blasts, promonocytes and monocytes (Fig. 4.12).

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Fig. 4.11 BM aspirate, M5a AML,showing large blast cells withmoderately abundant cytoplasm.MGG ×940.


Cytochemistry

Cytochemical stains [9] are useful in confirming thenature of M5 AML, particularly in those cases ofM5a with negligible maturation. Monoblasts areoften negative for MPO and SBB, although positiveresults are obtained with promonocytes. The mostuseful stains are those for ‘non-specific’ esterasessuch as α-naphthyl acetate esterase or α-naphthylbutyrate esterase. An alternative is the demon-stration of strong, fluoride-sensitive, positivity fornaphthol-AS-acetate esterase, another ‘non-specific’esterase. Monocyte differentiation can also bedemonstrated by using a suspension of the bac-terium Micrococcus lysodeikticus to show lysozymeactivity. However, this test is now little used sincethe availability of monoclonal antibodies providesanother means of confirming monocyte differentia-tion. Positive reactions with monoclonal antibodies,such as those of the CD11b and CD14 clusters, pro-vide useful confirmation of the diagnosis of M5 AMLin cases with negative reactions for non-specificesterases.


The marrow is intensely hypercellular. In M5a AML(Fig. 4.13) there is a homogeneous infiltrate ofmonoblasts whereas in M5b AML (Fig. 4.14) thereis a variable proportion of more mature cells of

monocyte lineage. The monoblasts are similar tothose seen in M4 AML. The more mature monocy-toid cells are smaller than monoblasts and haveirregular nuclei that are often convoluted or lobu-lated, with delicate chromatin but without nucleoli.

Cytochemical stains to confirm the monocyte lin-eage are not generally applicable to tissue sections.Chloro-acetate esterase activity is usually absent.Immunohistochemistry is useful, particularly inM5a AML in which the diagnosis is less readilymade from H&E-stained sections. Monoclonal anti-bodies that can be employed include antilysozyme,CD64, CD68, CD11b, CD14, CD15 and MAC387. Of CD68 monoclonal antibodies, the PG-M1 cloneshows good specificity for M4 and M5 AML, whilethe KP1 clone is a sensitive marker of myeloid differentiation but does not discriminate betweenmonocytic and granulocytic differentiation [15].


Chromosomal rearrangements with an 11q23 break-point and with rearrangement of the MLL gene arestrongly associated with M5 AML, particularly M5aAML. Other subtypes of M5 AML defined by theassociated cytogenetic abnormality include M5aassociated with t(8;16)(p11;p13), which is distinctivedue to the frequent occurrence of haemophagocytosisby leukaemic cells (and, clinically, due to a rela-tively high incidence of coagulation abnormalities).

Fig. 4.12 BM aspirate, M5b AML, showing monoblasts,promonocytes and a monocyte.MGG ×940.

Acute erythroleukaemia

Acute erythroleukaemia (M6 AML) describes anAML in which erythroid cells represent a major partof the leukaemic population. The FAB group hasrecommended that cases be assigned to this cate-gory (M6) when at least 50% of bone marrow cellsare erythroid and at least 30% of the remainingnon-erythroid cells are blasts (see Table 4.1). Othershave considered that a case can also reasonably be classified as erythroleukaemia if erythroid cellsshow prominent cytological abnormalities and constitute between 30 and 50% of bone marrow

nucleated cells [16]. There is a problem in assigning to a FAB category cases with almost exclusively erythroid cells including many very primitive ery-throid cells [17]. In such cases, fewer than 30% ofnon-erythroid cells may be blasts and consequentlythe FAB criteria for M6 AML may not be fulfilled;nevertheless this seems to be the category wherethey fit most naturally. We suggest a designation of M6 variant AML with criteria for diagnosis be-ing that more than 50% of bone marrow cells areerythroid and more than 30% are immature eryth-roid cells resembling proerythroblasts or basophilicerythroblasts.

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Fig. 4.13 BM trephine biopsyspecimen, M5a AML, showingmonoblasts which are large cellswith plentiful cytoplasm and aprominent nucleolus. One blast(upper left) has phagocytosed redcells. H&E ×940. (By courtesy of Dr FM Wood, Vancouver.)

Fig. 4.14 BM trephine biopsyspecimen, M5b AML, showingreplacement of haemopoieticmarrow and fat spaces by a mixtureof monoblasts, promonocytes andmonocytes. Plastic-embedded, H&E ×390.


It appears likely that a very high proportion of cases of M6 AML represent transformation ofunderlying MDS.

Cases of AML assigned to FAB M6 category would,in the WHO classification, fall into various cat-egories. Most would probably be designated ‘AMLwith multilineage dysplasia’ and others ‘acute erythroid leukaemia’.

Peripheral blood

The peripheral blood almost always shows anaemia,neutropenia and thrombocytopenia, usually withsome circulating blasts. There may also be circulat-ing erythroid precursors.


The bone marrow is hypercellular and shows botherythroid hyperplasia and, except in M6 variant, asignificant population of blasts (Fig. 4.15). The mor-phology of erythroid cells varies between cases. Insome patients, erythroid cells show striking cytolog-ical abnormalities which may include nuclear lobu-lation, karyorrhexis, multinuclearity, gigantism ormegaloblastic or sideroblastic erythropoiesis. In otherpatients, cytological abnormalities are quite minor.The associated blasts are usually myeloblasts, whichmay contain Auer rods, or a mixed population in-cluding monoblasts or megakaryoblasts. Maturing

granulocytes often show dysplastic features such as hypogranularity and nuclear hypolobulation.Megakaryocytes are also commonly dysplastic withfeatures such as nuclear hypolobulation or the pres-ence of micromegakaryocytes.

Cytochemistry

Cytochemical stains may show various abnor-malities. SBB, MPO and chloro-acetate esterase methods can be employed to confirm the nature ofmyeloblasts; the first two stains will also demon-strate Auer rods. An α-naphthyl acetate esterasestain can be used to identify monoblasts. Erythro-blasts may be positive with a periodic acid–Schiff(PAS) stain; such positivity is not shown by normalerythroid precursors but it is not confined to neo-plastic erythroblasts. Erythroblasts in M6 AML mayalso show focal staining for α-naphthyl acetateesterase and acid phosphatase. An iron stain mayshow the presence of ring sideroblasts.


There is marked hypercellularity of the marrowwith intense erythroid hyperplasia (Figs 4.16 and4.17). The erythroid precursors are usually mar-kedly abnormal and may have bizarre appearances.They often show nuclear lobulation or fragmenta-tion, marked variation in size or megaloblastic

Fig. 4.15 BM aspirate, M6 AML,showing two myeloblasts andnumerous erythroblasts. MGG ×940.

change. They are arranged in sheets without theformation of normal erythroblastic islands. Mega-karyocytic dysplasia is often seen. Non-erythroidblasts (myeloblasts or monoblasts) may be relativelyinconspicuous although, by definition, they mustmake up more than 30% of the non-erythroid cellsin the marrow except in the condition that we des-ignate M6 variant AML.

The diagnosis of M6 AML can usually be readilymade from H&E-stained sections. In cases with predominantly primitive erythroid cells, immuno-histochemistry may be needed. Useful reagents are

antibodies reactive with glycophorin, spectrin orhaemoglobin A.


M6 AML is often associated with abnormalities ofchromosomes 5 and 7 and with complex chromo-somal abnormalities.

Acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (M7 AML) has

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Fig. 4.16 Section of BM trephinebiopsy specimen, M6 AML,showing a disorganized marrowcontaining large numbers ofdysplastic erythroblasts andnumerous myeloblasts. Paraffin-embedded, H&E ×390.

Fig. 4.17 Section of BM trephinebiopsy specimen, M6 AML,showing ill-organizederythroblasts. Some havecharacteristic elongated nucleoliabutting on the nuclear membrane.Paraffin-embedded, H&E ×940.


blasts constituting at least 30% of bone marrowcells, with megakaryoblasts being the predominantform. There may be admixture with cells of otherlineages, for example, myeloblasts. The diagnosis isoften difficult, particularly in adults, because of thepaucity of leukaemic cells in the peripheral bloodand the difficulty in obtaining a bone marrow aspi-rate. A trephine biopsy is then of critical importancein making the diagnosis. In children, circulatingblasts are often present and the bone marrow canmore often be aspirated easily.

Cases of AML assigned to FAB M7 categorywould, in the WHO classification, fall into various cat-egories including ‘acute megakaryocytic leukaemia’and ‘acute panmyelosis with myelofibrosis’.

Peripheral blood

Commonly, the peripheral blood shows only pan-cytopenia with very infrequent or no circulatingleukaemic blasts. Such patients usually lack organ-omegaly and have a fibrotic marrow; this condi-tion has been described as ‘acute myelofibrosis.’Other patients have features more typical of acuteleukaemia, with hepatomegaly, splenomegaly andsignificant numbers of circulating blasts. Mega-karyoblasts are similar in size to myeloblasts. Theyhave a high nucleocytoplasmic ratio and agranular,moderately basophilic cytoplasm. In some casesthere are distinctive features that suggest their

nature, such as formation of peripheral cytoplasmicblebs or an association with circulating micro-megakaryocytes, but in other cases there are no features to suggest lineage.


Aspiration may be impossible or a poor aspirate con-taining scanty blasts may be obtained (Fig. 4.18). Inaddition to megakaryoblasts, the aspirate may con-tain some micromegakaryocytes or other markedlydysplastic megakaryocytes. There may be admixturewith myeloblasts. Erythroid precursors sometimesshow dysplastic features.

Cytochemistry

Cytochemistry is often not very useful. SBB, MPOand chloro-acetate esterase stains are negativeexcept in any associated myeloblasts. PAS, acidphosphatase and α-naphthyl acetate esterase stainsmay be positive in cells showing cytoplasmic matu-ration but not in more immature cells of megakary-ocyte lineage. The PAS stain sometimes shows adistinctive pattern with positivity being confined tocytoplasmic blebs. The differential staining patternwith α-naphthyl acetate esterase (positive) and α-naphthyl butyrate esterase (negative) can be useful in distinguishing megakaryoblasts frommonoblasts, since the latter cells give positive

Fig. 4.18 BM aspirate of a patientwith Down’s syndrome and M7 AML, showing twomegakaryoblasts and two giant platelets. MGG ×940.

reactions for both of these non-specific esterases.Immunocytochemistry with monoclonal antibodiesdirected at platelet glycoproteins is now the mostpracticable way to confirm the diagnosis.

Bone marrow histology [18,19]

The marrow histology is very variable. In thosecases that present clinically as acute myelofibrosis,the marrow is largely replaced by fibrous tissue containing blasts and dysplastic megakaryocytes(Fig. 4.19). In other cases the marrow is very hyper-cellular with an infiltrate of blasts; in some cases the blasts are relatively small and monomorphicwhile, in others, they are large and pleomorphic.Cases with a hypercellular marrow usually showincreased reticulin and scattered collagen fibres.Dyserythropoiesis is common.

Immunohistochemical staining for von Wille-brand antigen or for platelet GpIIIa (CD61) or GpIb(CD42b) is useful in the identification of mega-karyoblasts. Immunohistochemistry is necessary for diagnosis in those cases with predominantlymegakaryoblasts with few megakaryocytes.


M7 AML in infants is strongly associated either with constitutional trisomy 21 or with acquiredt(1;22)(p13;q13).

Acute myeloid leukaemia with minimalevidence of myeloid differentiation

The availability of ultrastructural cytochemistry andof monoclonal antibodies detecting antigens specificfor myeloid cells has revealed cases of acute leukae-mia which are negative for markers of lymphoidlineages but have insufficient evidence of myeloiddifferentiation to meet the original FAB criteria forAML. Such cases have fewer than 3% SBB- or MPO-positive blasts; blasts are also negative for α-naphthylacetate esterase and lysozyme activity. When suchcases can be demonstrated to have peroxidase activ-ity at ultrastructural level, or to be positive for myeloidantigens, such as CD13, CD14, CD33, CD117 orMPO, they should be classified as AML. The FABgroup has suggested the designation AML M0 [20].Cases of AML assigned to FAB M0 category would, inthe WHO classification, fall into various categoriesincluding ‘AML minimally differentiated’.

Peripheral blood

The peripheral blood usually shows anaemia, neu-tropenia, thrombocytopenia and the presence ofcirculating blasts (Fig. 4.20). The blasts are agranu-lar and have cytological features similar to those ofthe blasts of AML M1, ALL L2 or, more rarely, ALLL1 (see page 236). They tend to be large with pro-minent nucleoli and abundant, often basophilic,

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Fig. 4.19 Section of BM trephinebiopsy specimen, M7 AML. There iscollagen fibrosis and an infiltrate of dysplastic megakaryocytes andsmall blast cells. Plastic-embedded,H&E ×390.


cytoplasm but there are no constant features per-mitting the diagnosis from cytomorphology alone.The diagnosis may be suspected when apparentlyundifferentiated blasts are associated with dysplasticneutrophils or marked poikilocytosis.


The bone marrow features do not differ from thoseof M1 AML except that the blasts have scanty, ifany, granules and have no Auer rods. Other lin-eages may show dysplastic features, suggesting thatALL is not the correct diagnosis.

Cytochemistry

Cytochemistry with all the stains that are usuallyused to identify cells of various myeloid lineages iseither negative or is positive in fewer than 3% ofblasts. Immunocytochemistry for the above anti-gens is therefore used to confirm the diagnosis.


The trephine biopsy appearances do not differ fromthose of M1 AML. It is also often not possible to dis-tinguish M0 AML from ALL, although the presenceof dysplastic features suggests a diagnosis of AML.The diagnosis of AML cannot be confirmed by Leder’sstain, which is uniformly negative in M0 AML.

Immunological markers, however, can permit thedistinction. Lymphoblasts usually express CD45 andmay express CD79a or CD3, whereas myeloblastsdo not express CD79a or CD3 and are often CD45-negative or only weakly positive. Monoclonal orpolyclonal antibodies reactive with myeloid anti-gens, including anti-MPO and CD15, may be posi-tive in M0 AML.


M0 AML is preferentially associated with abnor-malities of chromosomes 5 and 7, trisomy 13 andtranslocations and deletions involving 12p [21],suggesting a relationship to M6 AML and to therapy-related and myelodysplasia-related AML.

Acute panmyelosis with myelofibrosis

The WHO expert group [5] has drawn attention to arare entity in which there is trilineage differentia-tion associated with bone marrow reticulin fibrosis.This entity has some features in common with thosecases of M7 AML that are associated with the clini-copathological features of ‘acute myelofibrosis’.

Peripheral blood

The blood film shows no specific features. Theremay be circulating blast cells.

Fig. 4.20 PB film from a patientwith M0 AML superimposed on MDS. Note the agranularneutrophil, anisocytosis andpoikilocytosis. MGG ×940.

Bone marrow aspirate

The bone marrow aspirate is often inadequate fordiagnosis because of the associated reticulin fibrosis.Increased blast cells and multilineage dysplasia maybe apparent.


Bone marrow histology is critical in making thediagnosis, with immunohistochemistry beingimportant in showing trilineage differentiation.Immature cells are increased and dysplasia may beprominent. Reticulin is increased.

Acute eosinophilic leukaemia

The term ‘eosinophilic leukaemia’ covers a hetero-geneous group of disorders in which eosinophils are increased in the peripheral blood. Acuteeosinophilic leukaemia falls into the FAB categoriesof M2 or M4 AML and can be designated morespecifically as M2Eo and M4Eo. There is no precisepercentage of eosinophils and precursors whichleads to a case being categorized as M2Eo or M4EoAML but cases of M4 AML with cytologically atyp-ical eosinophils are usually categorized as M4Eo,even if the eosinophil percentage is relatively low.This latter group would not, however, usually bereferred to as ‘eosinophilic leukaemia’. Eosinophilicleukaemia, which is not synonymous with ‘AML

with abnormal bone marrow eosinophils’, is notrecognized specifically in the WHO classification.Cases without inv(16) would fall mainly into theWHO category ‘AML with maturation’. For a case tobe classified as acute eosinophilic leukaemia, blastcells must constitute at least 20% of bone marrowcells. Cases of chronic eosinophilic leukaemia haveperipheral blood and bone marrow eosinophiliaand fewer than 20% bone marrow blasts. They areclassified with the myeloproliferative disorders.

Peripheral blood

The peripheral blood shows mature eosinophils and eosinophil precursors. Eosinophils may be cytologically atypical but this is not diagnostically useful since atypical eosinophils are also seen inreactive eosinophilia. Eosinophil precursors oftenshow nucleocytoplasmic asynchrony and immaturegranules with basophilic staining characteristics(Fig. 4.21).


The bone marrow shows an increase, often very mar-ked, in eosinophils and their precursors (Fig. 4.22).Occasionally, Charcot–Leyden crystals are present,these being formed by crystallization of eosinophilgranule contents. Other myeloid lineages (e.g. neutrophils or monocytes) may also be part of theleukaemic clone.

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Fig. 4.21 PB film from a patientwith acute eosinophilic leukaemia(M2Eo AML), showing eosinophilprecursors with a mixture ofprimary and secondary granules.MGG ×960. (By courtesy of Dr A Smith, Southampton.)


Cytochemistry

Eosinophils and precursors show strong peroxidaseactivity.


Bone marrow trephine biopsy sections show an in-crease of eosinophils and their precursors (Figs 4.23and 4.24).


There is no specific cytogenetic association with

acute eosinophilic leukaemia. Rare cases have hadt(8;21)(q22;q22).

Acute basophilic leukaemia

Acute basophilic leukaemia is a rare form of AMLwhich is not identified specifically in the FABclassification. Cases usually fall into the M2 or M4 categories and can be designated M2Baso andM4Baso. Some cases with little or no maturationfall into the M1 or M0 categories. Blasts in thesecases can be identified as being of basophil lineage,either because they have basophil-type granulesidentifiable on light microscopy or by ultrastructural

Fig. 4.23 Section of BM trephinebiopsy specimen from a patientwith acute eosinophilic leukaemia(same case as in Fig. 4.21), showingtwo Charcot–Leyden crystals in an area of necrotic bone marrow.H&E ×384.

Fig. 4.22 BM aspirate from apatient with acute eosinophilicleukaemia (same case as in Fig. 4.21), showing eosinophils and their precursors and twoCharcot–Leyden crystals. MGG×384. (By courtesy of Dr A Smith,Southampton.)

examination. The WHO classification has a specificcategory for acute basophilic leukaemia.

Peripheral blood

The blood film usually shows both blast cells andmaturing basophils. The latter may be hypogranular.


The bone marrow aspirate shows more than 20%blast cells, usually with maturation to dysplasticbasophils.


Trephine biopsy sections show increased blast cellsbut are of no use currently in the detection ofbasophilic differentiation since basophil granulesare dissolved during processing.

Acute mast cell leukaemia

Acute mast cell leukaemia is a rare condition whichcan occur de novo or as a transformation of systemicmastocytosis. It is not included in the FAB classi-fication. The WHO classification assigns it to a sepa-rate category of ‘mast cell diseases’ but, since themast cell is derived from the haemopoietic stem cell,

it seems to us more appropriate that it is classified asa form of AML.

Peripheral blood

The blood film shows immature mast cells whichhave round, oval or lobulated nuclei and a variablenumber of granules of mast cell type (Fig. 4.25).


The bone marrow aspirate shows immature cells ofmast cell lineage. Immunocytochemistry for mastcell tryptase can be used to demonstrate mast celldifferentiation if this is not certain from cytologicalfeatures.

Cytochemistry

Mast cells stain metachromatically with toluidineblue. They are chloro-acetate esterase-positive.


Trephine biopsy sections show effacement of thebone marrow by immature mast cells. These havemore voluminous cytoplasm than most other blastcells so that, in an H&E-stained section, the round,oval or lobulated nuclei appear spaced apart

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Fig. 4.24 Section of BM trephinebiopsy specimen from a patientwith acute eosinophilic leukaemia(same case as in Fig. 4.21), showingeosinophils and their precursorsand two Charcot–Leyden crystals.H&E ×960.


(Fig. 4.26). With Giemsa staining, granules may be apparent. Immunohistochemistry for mast celltryptase confirms the lineage.

Hypoplastic AML

A minority of cases of AML have a bone marrowwhich is hypocellular rather than hypercellular.Hypoplastic AML is defined variously, for example,as AML with bone marrow cellularity less than 50%[22] or less than 40% [23]. Blasts constitute at least20% of nucleated cells. Cases of hypoplastic AML

may belong to various FAB categories but not M3.They may arise apparently de novo or be preceded bya hypoplastic variant of myelodysplasia. Clinicaland haematological features differ from those oftypical cases of AML with a hypercellular bone mar-row. Hepatomegaly and splenomegaly are not com-monly present. The median age is higher than thatof AML in general [22]. The prognostic significanceof hypoplastic AML is not yet clear. In one series,patients who were treated had a low remission rateand treated patients, overall, did not survive longerthan those who received no specific treatment [22].

Fig. 4.25 PB film in acute mast cellleukaemia, showing circulatingmast cell precursors. MGG ×940.

Fig. 4.26 BM trephine biopsysection in acute mast cellleukaemia, showing replacement ofmarrow by mast cells. H&E ×376.

However, in another series, treated patients had ahigh remission rate and improved survival [23].

Hypoplastic AML is not identified specifically inthe WHO classification. Some cases fall into the categories designated ‘AML with multilineage dys-plasia’ and ‘AML, therapy-related’.

Peripheral blood

In contrast to cases of typical AML, pancytopenia isusual and peripheral blood blasts are often absent or infrequent. Blasts may contain Auer rods.


The bone marrow aspirate is often hypocellular andmay therefore not be optimal for diagnosis. Blastsare increased and commonly show granulocyticrather than monocytic differentiation. There maybe associated dysplastic features.

Cytochemistry

The blasts in hypoplastic AML have no specific cyto-chemical features.


Because a poor aspirate is often obtained, trephinebiopsy is usually important in diagnosis. The mar-

row shows irregular hypoplasia (Fig. 4.27) withsmall foci of blasts separated by fat cells (Fig. 4.28).Blasts make up more than 20% of bone marrownucleated cells. There are often dysplastic changesin the other haemopoietic lineages. Reticulin maybe increased.


Cytogenetic abnormalities are non-specific but mayinclude abnormalities of chromosomes 5 and 7.

Cytochemistry in AML

Cytochemistry permits the confirmation of a diag-nosis of AML in all categories except M0 and M7. Itis most important in M1 and M5a AML, which canbe confused with ALL and high grade lymphomarespectively. Recommended cytochemical stains areeither MPO or SBB (for identification of granulocyticdifferentiation) and a non-specific esterase stain suchas α-naphthyl acetate esterase (for identification ofmonocytic differentiation).

Immunocytochemistry and flow cytometry in AML

Immunophenotyping [8] (Table 4.3) is of consider-able importance in confirming the diagnosis of M0and M7 AML, which may otherwise be confused

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Fig. 4.27 Section of BM trephinebiopsy specimen, hypoplastic AML.The marrow is hypocellular withpreservation of fat cells. However,normal haemopoietic cells are notseen. Plastic-embedded, H&E ×97.


with ALL. It is also useful in the recognition of M6 variant AML since the primitive cells may be so immature that they are not readily recognizablefrom cytological features. Immunophenotyping isan alternative to cytochemistry for confirming themyeloid lineage of M1 and M5 AML. Other FABcategories are usually readily recognizable withoutrecourse to immunophenotyping. Immunopheno-typing is now generally performed by flow cytome-try rather than by immunocytochemistry.

Immunohistochemistry in AML

Monoclonal and polyclonal antibodies that are use-ful in confirming a diagnosis of AML are shown inTable 4.4. Their use is most important in M0, M1,M5a and M7 categories. In M2, M3, M4 and M5bsubtypes it is usually possible to make a diagnosis ofAML from H&E-stained sections. It is our practice to perform immunohistochemical stains wheneverthere is diagnostic difficulty, for example, when bone

Fig. 4.28 Section of BM trephinebiopsy specimen, hypoplastic AML(same case as in Fig. 4.27). Highpower examination reveals most ofthe cells present to be myeloblasts.Plastic-embedded, H&E ×970.

Table 4.3 Monoclonal and polyclonal antibodies useful for flow cytometry immunophenotyping in the diagnosis of AML.

Specificity Antibodies

Panmyeloid CD13, CD33, CD65, CD117, anti-myeloperoxidaseMarkers of maturation CD15, CD11bMarkers of monocytic differentiation CD14, CD11b, CD64Erythroid markers Anti-glycophorinMegakaryocytic markers CD41, CD42a, CD42b, CD61Markers of immaturity CD34, anti-terminal nucleotidyl transferase

Table 4.4 Monoclonal and polyclonal antibodies useful in immunohistochemistry for the diagnosis of AML.

Specificity Antibodies

Granulocytic and/or monocytic markers Anti-myeloperoxidase, CD64, CD68, anti-neutrophil elastaseMegakaryocyte/platelet markers CD42b, CD61, anti-von Willebrand’s factorErythroid markers Anti-glycophorin, anti-spectrinMast cell marker Mast cell tryptaseMarkers of immaturity CD34, anti-terminal deoxynucleotidyl transferase

marrow aspiration has failed and there are only lownumbers of circulating blast cells.

Cytogenetics and molecular genetics in AML

It is the somatic mutation that occurred in the stemcell giving rise to the leukaemic clone that deter-mines the clinical and pathological features of AML.The molecular genetic abnormality is therefore oneof the most fundamental characteristics of any caseof AML and it is appropriate that it be incorporatedinto classification of this disease. The MIC classi-fication sought to do this, when cytogenetic analysiswas added to morphology and immunophenotyp-ing in classifying acute leukaemia [3]. With theadvent of molecular genetic analysis, it became pos-sible to identify genes involved in the oncogenicevent and a MIC-M classification, incorporating theresults of molecular genetic analysis [4], becamepossible. The MIC-M categorization is open-ended,new categories being added when the oncogenicevents have been defined (Table 4.5).

Some cases of AML following topoisomerase-II-interactive drugs have the same clinical featuresand cytogenetic and molecular genetic abnormal-ities as are seen in de novo AML. They can bedescribed by the term ‘de novo-type’ AML. The WHOclassification incorporates the four numericallymost important cytogenetic categories of de novoAML, associated with simple chromosomal rear-rangements and a single major genetic event, but categorizes separately cases with the same cytogenetic abnormality that follow therapy withtopoisomerase-II-interactive drugs. The MIC-Mclassification categorizes primarily by molecularevent. It is applicable to cases of de novo and de novo-type AML in which there is one major oncogenicevent. It is not applicable to many cases of AML in the elderly, myelodysplasia-related AML andtherapy-related AML following alkylating agentsand related drugs, all of which are consequent on an accumulation of multiple genetic events. Thistype of AML shows a different range of cytogeneticabnormalities. The most characteristic are mono-somy 5, del(5)(q), monosomy 7, del(7)(q), trisomy8, trisomy 9, del(11)(q), del(12)(p), monosomy 18,monosomy 19, del(20)(q), trisomy 21 and complexchromosomal rearrangements.

Problems and pitfalls in the diagnosis of AML

It is sometimes difficult to distinguish cases of M0and M1 AML from ALL on the basis of examinationof H&E-stained sections. Some histological featuresare useful. ALL is usually associated with efface-ment of the bone marrow whereas, in AML, theremay be residual myeloid cells showing dysplasticfeatures. ALL blasts tend to have more scanty cytoplasm and more chromatin condensation. Ifdifficulty is experienced in making the distinctionand if diagnosis depends on the trephine biopsy sections, immunohistochemistry should be used.However, it should be noted that immunohisto-chemistry for MPO and other myeloid antigens isless sensitive for the detection of myeloid differenti-ation than cytochemistry and immunophenotypingby flow cytometry. For this reason, there are somecases of M0 and M1 AML that cannot be distin-guished from ALL histologically, even with the helpof immunohistochemistry.

Other diagnostic problems relate mainly to M6AML, M7 AML and hypoplastic AML. M6 AML andmegaloblastic anaemia can be confused with eachother in trephine biopsy sections or, less often, in abone marrow aspirate. This is a very serious errorwhich must be avoided. It arises mainly because thediagnosis of AML is not considered or because a caseof M6 has megaloblastic erythropoiesis. Diagnosticerror can be avoided by careful attention to cytolog-ical details (see page 371) supplemented, when necessary, by assays of vitamin B12 and folic acid. Atherapeutic trial of haematinic agents is sometimesuseful.

M7 AML and acute panmyelosis with myelo-fibrosis, when accompanied by dense fibrosis, canbe confused with chronic idiopathic myelofibrosis(see page 204). The clinical and haematological fea-tures of these three conditions differ. Marked spleno-megaly is usual in idiopathic myelofibrosis but notin M7 AML or acute panmyelosis with fibrosis. Aleuco-erythroblastic anaemia with marked poikilo-cytosis is likewise usual in idiopathic myelofibrosisbut not in M7 AML or acute panmyelosis. Bonemarrow aspiration usually fails or yields inadequatematerial for diagnosis in all these conditions. How-ever, histology shows increased blast cells in M7AML and acute panmyelosis and not in idiopathicmyelofibrosis. AML with myelofibrosis can also be

166 CHAPTER FOUR


confused with bone marrow infiltration by a non-haemopoietic tumour with secondary myelofibrosis.Dysplastic megakaryocytes and metastatic tumourcells can be distinguished by immunohistochemistry.

Hypoplastic AML must be distinguished fromaplastic anaemia and from hypoplastic MDS. In-creased reticulin deposition and dysplastic mega-karyocytes suggest a diagnosis of AML or MDS

Table 4.5 A morphological, immunophenotypic, cytogenetic, molecular genetic (MIC-M) classification of acute myeloidleukaemia (modified from [4,24,25]).

Cytogenetic abnormality Molecular genetic abnormality FAB category

t(1;11)(p32;q23)* MLL-AF1p fusion M5t(1;11)(q21;q23)* MLL-AF1q fusion M4t(1;11)(q23;p15) NUP98-PMX1 M2t(2;11)(q35;p15) NUP98-HOXD13 Therapy-relatedinv(3)(q21q26) or EVI1 dysregulation Various

t(3;3)(q21;q26)t(3;5)(q25.1;q34) NPM-MLF1 fusion Varioust(3;12)(q26;p13) Fusion of various genes at 3q26 with Various

TEL at 12p13t(3;21)(q26;q22) Heterogeneous, mainly AML1-EAP, Variable

AML1-EVI1 and AML1-MDS1t(4;11)(q21;q23)* MLL-AF4 fusion M5t(4;12)(q11–12;p13) BTV-TEL (BTV-ETV6) M0t(5;17)(q32;q21) NPM-RARα fusion M3-liket(6;9)(p23;q34) DEK-CAN fusion M2Basot(6;11)(q27;q23)* MLL-AF6 fusion M4 or M5t(7;11)(p15;p15) NUP98-HOXA9 fusion M2inv(8)(p11q13) MOZ-TIF2 fusion M7*t(8;16)(p11;p13) MOZ-CBP fusion M4 or M5t(8;21)(q22;q22)† AML1-ETO fusion M2t(9;11)(p21–22;q23)* MLL-AF9 fusion M5t(9;22)(q34;q11) BCR-ABL fusion M0, M1 or M2t(10;11)(p12;q23)* MLL-AF10 fusion M5t(10;11)(p11.2;q23)* MLL-ABI1 fusion AMLt(10;11)(p13;q14) CALM-AF10 fusion M4ins(11;9)(q23;p22p23)* MLL-AF9 fusion M5inv(11)(p15q22) NUP98-DDX10 fusion Varioust(11;16)(q23;p13)* MLL-CBFβ fusion Variablet(11;17)(q23;q21)* MLL-AF17 fusion M5t(11;17)(q23;q21) PLZF-RARα fusion M3-liket(11;19)(q23;p13.1)* MLL-ELL fusion M4 or M5t(11;19)(q23;p13.3)* MLL-ENL fusion M4 or M5t(11;20)(p15;q11) NUP98-TOP1 fusion t-MDS or AMLt(11;22)(q23;q13)* MLL-p300 fusion AML+11, 11q+ or normal* Partial tandem duplication of MLL AMLt(12;13)(p13;q12) ETV6(TEL)-CDX2 fusion AMLt(12;22)(p13;q11) MN1-TEL fusion Variablet(15;17)(q22;q21)† PML-RARα fusion M3inv(16)(p13q22)† or CBFβ-MYH11 fusion M4Eo

t(16;16)(p13;q22)†t(16;21)(p11;q22) FUS-ERG fusion Variablet(X;11)(q13;q23)* MLL-AFX fusion M4 or M5

* Grouped together in the WHO classification as ‘AML with 11q23 (MLL) abnormalities.† Specific WHO categories of AML.

rather than aplastic anaemia. In addition, inhypoplastic AML and hypoplastic MDS there maybe some circulating blasts and the bone marrowaspirate, although inadequate for diagnostic pur-poses, may also show blast cells. There is no increasein blast cells in aplastic anaemia. Accurate differ-ential diagnosis of these three conditions requiresgood quality sections of trephine biopsy specimens,which need to be examined carefully with highpower magnification so that the proportion of blast cells can be estimated. If there are more than20% blast cells, the diagnosis is AML and, if blastsare increased but less than 20%, the diagnosis isMDS.

AML of M5a subtype can sometimes be confusedwith large cell non-Hodgkin’s lymphoma. If mono-cytes are very immature, they may lack peroxidaseand non-specific esterase activity. Flow cytometryimmunophenotyping or immunohistochemistry isthen very important in making the correct diagnosis.

The myelodysplastic syndromes

As discussed at the beginning of this chapter, theMDS are diseases consequent on a clonal haemo-poietic disorder characterized by dysplastic, ineffec-tive haemopoiesis. There is thus often a discrepancybetween a hypercellular bone marrow and peripheralcytopenia. In any one patient, different haemopoi-etic lineages are not necessarily affected to the samedegree and there may be defective production ofcells of one lineage while in another lineage normalnumbers of cells are produced. There may even be increased production of cells of one or more lineagesafor example, neutrophils, monocytes orplateletsadespite other features typical of MDS (seepage 215).

The MDS are predominantly diseases of theelderly with an incidence of the order of 70cases/100 000/year.

Clinical features of MDS result from the variouscytopenias; there may be haemorrhage, susceptibil-ity to infections and symptoms of anaemia. Somepatients have hepatomegaly and splenomegaly. MDSshow a tendency to evolve into more severe formsof MDS and into acute leukaemia. Most cases ofMDS are apparently primary but a minority are sec-ondary to exposure of the bone marrow to knownmutagens, such as alkylating agents. There are

some differences in laboratory and clinical featuresbetween primary and secondary MDS.

Diagnosis of MDS requires consideration of clinical,peripheral blood, bone marrow and cytogenetic fea-tures. Peripheral blood and bone marrow aspiratefindings are most important and, in a straightfor-ward case, may be all that is required for diagnosis.Bone marrow trephine biopsy in general offers onlysupplementary information; however, sometimes itis necessary for confirmation of the diagnosis, forexample, when an excess of blasts or an abnormallocalization of blasts is detected in a patient who hasother features suggestive but not diagnostic of MDS.Bone marrow trephine biopsy is particularly impor-tant in patients with secondary MDS, in whom ahypocellular bone marrow with increased fibrosisoften leads to a non-diagnostic aspirate. In somepatients, a firm diagnosis cannot be made on cyto-logical and histological features alone, but diagnosisis possible when these are supplemented by cyto-genetic analysis or other tests giving informationabout clonality. An iron stain should be performedin all patients with suspected myelodysplasia; thisdemonstrates any ring sideroblasts as well as permit-ting an assessment of iron stores. An MPO or SBBstain should be performed at least in all patientswith any increase in blasts; this will facilitate thedetection of Auer rods, which are of importanceboth in diagnosis and in classification.

The MDS are a heterogeneous group of disorderswith very variable prognoses. They can be dividedinto various disease categories which have moreuniform clinicopathological characteristics. Untilnow the most widely used categorization has beenthat proposed by the FAB group [1,2,26]. The FABclassification is based on the presence or absence ofsignificant sideroblastic erythropoiesis, on the num-bers of monocytes in the peripheral blood and onthe number of blasts in the peripheral blood andbone marrow (Table 4.6). The FAB categories are:1 refractory anaemia (RA) or refractory cytopenia;2 refractory anaemia with ring sideroblasts (RARS);3 refractory anaemia with excess of blasts (RAEB);4 chronic myelomonocytic leukaemia (CMML); and5 refractory anaemia with excess of blasts in trans-formation (RAEB-T).

More recently, a WHO classification of MDS hasbeen proposed (Table 4.7) [5]. The most importantdifference from the FAB classification is that the

168 CHAPTER FOUR


RAEB-T category has been abolished. Cases withmore than 20% of bone marrow blasts are classifiedas AML. In addition, CMML has been classified as amixed myelodysplastic/myeloproliferative diseaserather than as one of the MDS. The WHO group hasalso made a distinction between cases of RA andRARS with and without dysplasia of other lineages,since this is of prognostic significance.

Some morphological abnormalities are char-acteristic of MDS, without being specific for them,while others show sufficient specificity to be usefulin confirming the diagnosis. Although the MDS areheterogeneous they also have many features in

common. We will therefore describe these syn-dromes as a group before discussing specific cat-egories of disease. CMML will be discussed mainlyin the next chapter (see page 215). The other FABcategories of MDS will be discussed in this chapter,retaining the FAB definitions since the WHOclassification has been published only in outline.

Peripheral blood

Anaemia is seen in the great majority of patients.Red cells are usually normochromic and either normocytic or macrocytic. In patients with sidero-blastic erythropoiesis there is commonly a dimorphicblood film with a mixture of a minority population ofhypochromic microcytes and a majority populationof normochromic cells which are either normocyticor, more commonly, macrocytic; Pappenheimerbodies, the nature of which can be confirmed withan iron stain, may be present. Microcytosis is seenin certain rare variants including acquired haemo-globin H disease. Some patients have occasional cir-culating erythroblasts which may include dysplasticforms such as megaloblasts and, in patients withsideroblastic erythropoiesis, ring sideroblasts.

Neutropenia is common, particularly in RAEB andRAEB-T. Neutrophils often show dysplastic features

Table 4.6 The FAB classification of the myelodysplastic syndromes [1,2,8,26].

Category Peripheral blood Bone marrow

Refractory anaemia (RA) Anaemia* Blasts <5%, ringed or refractory cytopenia* Blasts ≤1% and sideroblasts ≤15%

Monocytes ≤1 × 109/l of erythroblastsRefractory anaemia with Anaemia Blasts <5%, ringedringed sideroblasts (RARS) Blasts ≤1% and sideroblasts >15%

Monocytes ≤1 × 109/l of erythroblastsRefractory anaemia with Anaemiaexcess of blasts (RAEB) Blasts >1% or Blasts ≥5%

Blasts <5% and Blasts ≤20%Monocytes ≤1 × 109/l

Chronic myelomonocytic Blasts <5% and Blasts up to 20%leukaemia (CMML) Monocytes >1 × 109/l promonocytes

Granulocytes often often increasedincreased

Refractory anaemia with excess of Blasts ≥5% or Auer rods in blasts in or Blastsblasts in transformation (RAEB-T) blood or marrow >20%

but <30%

* Or in the case of refractory cytopenia, either neutropenia or thrombocytopenia.

Table 4.7 The WHO classification of the myelodysplasticsyndromes [5].

Refractory anaemiaWith ringed sideroblastsWithout ringed sideroblasts

Refractory cytopenia with multilineage dysplasia5q- syndromeRefractory anaemia with excess blastsMyelodysplastic syndrome, unclassifiableMyelodysplastic syndromes, therapy-related

Alkylating agent-relatedEpipodophyllotoxin-related

including reduced granulation and the acquired orpseudo-Pelger–Huët anomaly. Hypogranular andagranular neutrophils (Fig. 4.29) are consequent ondefective formation of secondary granules; agranularneutrophils are highly specific for the MDS [27].The acquired Pelger–Huët anomaly refers to hypo-lobulation of nuclei associated with dense chromatinclumping (Fig. 4.29); nuclei of mature neutrophilsmay be completely non-lobed, dumb-bell or peanut-shaped, or bilobed with the shape resembling a pair of spectacles. This abnormality resembles theinherited Pelger–Huët anomaly, hence its name.The acquired anomaly is highly characteristic of the

MDS and almost pathognomonic [27]. Eosinophiland basophil counts are commonly reduced but, ina small minority of patients, are increased; dysplas-tic forms with abnormalities of either nuclear shapeor cytoplasmic granulation can occur. Monocytosisis sometimes present and monocytes may showcytological abnormalities such as increased cyto-plasmic basophilia or nuclei of unusual shape. Blastcells may be present in the peripheral blood in allcategories of MDS but particularly in RAEB andRAEB-T. They usually have the cytological featuresof myeloblasts with scanty cytoplasm and few granules. Auer rods are sometimes present. Other

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Fig. 4.29 PB film, MDS, showinganisocytosis, poikilocytosis and twopseudo-Pelger–Huët neutrophils,one of which is also hypogranular.MGG ×940.

Fig. 4.30 BM aspirate, RA,showing a binucleatemicromegakaryocyte which isbudding platelets. MGG ×940.


granulocyte precursors are quite uncommon in theperipheral blood.

The platelet count is usually either normal orreduced. In a minority of patients it is increased.Dysplastic features which may be noted in plateletsinclude hypogranular and agranular forms (‘grey’platelets) and giant platelets.

Patients with MDS have an increased incidence of auto-immune thrombocytopenia [28].


The bone marrow is hypercellular in the majority of patients but is sometimes normocellular and inabout 10% of patients is hypocellular. Hypercel-lularity may be due to hyperplasia of erythroid orgranulocytic series or both.

Erythropoiesis may be normoblastic, macronor-moblastic or megaloblastic. In patients with sidero-blastic erythropoiesis there are some erythroblastswith poorly haemoglobinized or vacuolated cyto-plasm. Other dysplastic features may include: (i) binuclearity and multinuclearity; (ii) internuclearbridges; (iii) nuclear lobulation; (iv) irregularity orfragmentation of nuclei; (v) gigantism; (vi) increasedpyknosis; and (vii) basophilic stippling. The bonemarrow erythroid component (calculated from cellularity and the percentage of erythroblasts) hasbeen found to be predictive of response to therapywith erythropoietin plus GM-CSF [29].

Granulopoiesis is usually hyperplastic. Defects ofgranulation may be apparent from the promyelo-cyte stage onwards and defects of nuclear lobulationmay also be present.

Megakaryocyte numbers are usually normal orincreased but sometimes decreased. One of the fea-tures most specific for the MDS is the presence ofmicromegakaryocytes [27], cells of about the size ofa myeloblast with one or two small round nuclei(Fig. 4.30). Megakaryocytes may also be of nor-mal size but have a large non-lobulated nucleus(Fig. 4.31); this abnormality is less specific for MDSbut is characteristic of cases with 5q- as an acquiredchromosomal abnormality [30]. Other megakary-ocyte abnormalities include bizarre nuclear shapesand the presence of multiple separate nuclei. Poorgranulation of megakaryocyte cytoplasm is also com-mon in the MDS and has been found to be highlyspecific [31].

The bone marrow aspirate may show non-specific abnormalities such as increased numbers of macrophages, sea-blue histiocytes, lymphocytes,plasma cells or mast cells.

Cytochemistry

The cytochemical stain of most value is an iron stain.This should be performed in all cases of suspectedMDS, in order to quantify iron stores and to detectand enumerate ring sideroblasts and other abnormal

Fig. 4.31 BM aspirate, RA, 5q- syndrome, showing amegakaryocyte of normal size with a hypolobulated nucleus.MGG ×940.

sideroblasts. Ring sideroblasts have iron-positivegranules in a circle close to the nuclear membrane(Fig. 4.32). Other abnormal sideroblasts have scat-tered iron-positive granules which are both largerand more numerous than those of normal sidero-cytes. Ring sideroblasts are highly suggestive ofMDS if the other known causes of sideroblastic erythropoiesis (see page 36) can be eliminated.Abnormal sideroblasts, other than ring sideroblasts,are common both in MDS and in other disorders oferythropoiesis so are not useful in the differentialdiagnosis of suspected MDS. Other cytochemicalstains are of use in identifying abnormal cells of

megakaryocyte lineages, in characterizing blastsand in detecting Auer rods. MPO and SBB stainswill identify myeloblasts and may also show cells ofthe neutrophil lineage to have defective primarygranules. Non-specific esterase stains are useful foridentifying monoblasts; non-specific esterase andPAS stains are valuable for identifying abnormalmegakaryocytes.


In the majority of cases the marrow is hypercellular(Fig. 4.33), but a significant minority have a hypo-

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Fig. 4.32 BM aspirate, RARS,showing numerous ringsideroblasts, several of which can be seen to have defectivelyhaemoglobinized cytoplasm. Perls’ stain ×940.

Fig. 4.33 BM trephine biopsysection, RA, showing markedhypercellularity withdisorganization of haemopoiesisand marked dyserythropoiesis.Note the apoptotic erythroblastswith peripheral condensation of their nuclear chromatin. Plastic-embedded, H&E ×390.


cellular marrow [32,33]. There may be considerablevariation of the cellularity between adjacent inter-trabecular spaces [34]. In addition to cytologicalevidence of dysplasia, there is derangement of nor-mal architecture. In histological sections, dysplasiais most obvious in the erythroid precursors andmegakaryocytes; however, the acquired Pelger–Huët anomaly can be detected in good quality sec-tions of paraffin-embedded material and, in goodplastic-embedded material, Auer rods may also be identified. Disturbance of normal architectureresults in groups of granulocytic precursors beingfound in the central parts of intertrabecular spaces

(Fig. 4.34) and erythroid precursors and megakary-ocytes in the paratrabecular regions. Erythroblasticislands may be poorly formed or very large and erythroid precursors may be multinucleated or shownuclear budding or fragmentation, megaloblasticchange or cytoplasmic vacuolation (Fig. 4.35). Mega-karyocytic dysplasia is present in the vast majorityof cases and is usually more apparent in histologicalsections than in marrow films. They are usuallyincreased in number and clustering is often seen(Fig. 4.36). Typically, they have hypolobulatednuclei which are often hyperchromatic; the smalldysplastic megakaryocytes are usually referred to as

Fig. 4.34 BM trephine biopsysection, RAEB-T, showingincreased numbers of blastsforming a small cluster (centre) (an abnormal localization ofimmature precursors or ALIP).Plastic-embedded, H&E ×970.

Fig. 4.35 BM trephine biopsysection in MDS, showing numerousimmature erythroid cells includinga binucleate form; the erythroblastsare not grouped into a compacterythroid island. Paraffin-embedded, Giemsa ×970.

micromegakaryocytes [33,35]. Emperipolesis isincreased [36]. Immunohistochemical staining withanti-GpIIIa (CD61) and anti-GpIb (CD42b) may beused to accentuate the abnormal megakaryocytes(Fig. 4.37) [37]. Increased numbers of apoptoticerythroid and granulocytic precursors are com-monly seen in MDS consequent on ineffectivehaemopoiesis (see Fig. 4.33) [38]. In a minority ofcases, haemopoietic cells, particularly megakary-ocytes, are present within sinusoids [36]. Reticulinfibrosis has been reported in a fifth [39] to almost ahalf [33] of cases of MDS, as defined by the FAB

group. It is more common in chronic myelomono-cytic leukaemia (now designated a myelodysplastic/myeloproliferative disorder) than in other subtypes[33,39]. The presence of reticulin fibrosis correlateswith megakaryocyte numbers and atypia [39].Severe collagen fibrosis is rare in all subtypes[33,39]. It is seen most often in secondary MDS.Reticulin fibrosis correlates with unfavourable cyto-genetic abnormalities and is indicative of a worseprognosis. Other non-specific reactions are com-monly seen including oedema, ectasia of sinusoids,increased numbers of plasma cells and increased

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Fig. 4.36 BM trephine biopsysection, RA, showing an aggregateof dysplastic megakaryocytes.Paraffin-embedded, H&E ×192.

Fig. 4.37 BM trephine biopsysection, therapy-related MDS,showing dysplastic smallmegakaryocytes. Paraffin-embedded, immunoperoxidasewith a CD61 monoclonal antibody ×970.


numbers of lymphoid follicles. Haemosiderin-ladenmacrophages are a frequent finding, particularly inpatients who have received transfusions.

One feature that has been the subject of muchdebate is the significance of small groups of imma-ture granulocytic precursors (promyelocytes andmyeloblasts) in a central position within intertra-becular spaces (see Fig. 4.34). This has been termedabnormal localization of immature precursors (ALIP).Some studies have found this phenomenon to be an independent predictor of prognosis and to beassociated with an increased incidence of leukae-mic transformation [40]. Although ALIP is morefrequent in the subtypes of myelodysplasia withincreased numbers of blasts in the marrow, severalrecent studies have failed to confirm any inde-pendent influence on prognosis [33,34,41]. Others,however, have confirmed prognostic significanceindependent of the blast percentage in an aspirate[36] or in histological sections [42]. It should benoted that it can be difficult, particularly in paraffin-embedded sections, to distinguish between smallgroups of immature erythroid precursors and theclusters of immature cells of granulocytic lineagethat are seen in ALIP. Aggregates of immature erythroid cells, which can mistakenly be taken asevidence of ALIP, can be distinguished by their positive reactions with antibodies to glycophorin orspectrin, whereas the immature cells in ALIP mayreact positively with CD68, antimyeloperoxidase andantineutrophil elastase antibodies. Other histological

features found to be of poor prognostic significance,in one study using multivariate analysis, were anelevated blast percentage, increased haemosiderin,megakaryocyte atypia and reduced erythropoiesis,while increased mast cells were of good prognosticsignificance [42].

Immunocytochemistry and flow cytometry in MDS

Immunophenotyping can be used to confirm thelineage of any blast cells present but, in general,immunophenotyping is of little value in MDS.

Immunohistochemistry

The value of immunohistochemistry in MDS can besummarized as follows:1 abnormal topography can be detected, e.g. thepresence of ALIP or the presence of erythroid cellsor megakaryocytes in a paratrabecular position;2 immature cells in ALIP can be distinguished fromclusters of immature erythroid cells (Fig. 4.38);3 abnormally large erythroid islands can beidentified;4 micromegakaryocytes can be identified and thepresence of megakaryocyte clustering or dysplasiahighlighted (see Fig. 4.37);5 prognostic information can be gained by the use of CD34 antibodies (Fig. 4.39)athe presence ofmore than 1% of CD34-positive cells is indicative ofa worse prognosis in MDS as a whole and within the

Fig. 4.38 Trephine biopsy sectionfrom a patient with RARS, showinga clump of early erythroid cells thatcan be distinguished from an ALIPby their weak but definite reactionwith antiglycophorin. Paraffin-embedded, immunoperoxidase,antiglycophorin ×940.

RAEB category [43]; clusters of CD34-positive cellsare predictive of leukaemic transformation; and6 making a distinction between hypocellular MDSand aplastic anaemia can be facilitated (see below).


The cytogenetic abnormalities associated with MDSare heterogeneous. The most characteristic abnor-malities are monosomies, deletions and unbalancedtranslocations. Abnormalities often observed includemonosomy 5, monosomy 7, trisomy 8, del(5)(q),del(7)(q), del(9)(q) and del(20)(q). In MDS second-ary to alkylating agents, monosomies and deletionsof chromosomes 5 and 7 are common, whereas MDSsecondary to topoisomerase-II-interactive drugs ischaracterized by balanced translocations with 3q26,11q23 and 21q22 breakpoints.

Clonal cytogenetic abnormalities can confirm thediagnosis of MDS. The type of abnormality found isalso of prognostic significance.

MDS is typically associated with multiple onco-genic events, formation of fusion genes, mutations ofoncogenes and both mutation and loss of cancer sup-pressing genes. Genes that may be mutated include N-RAS, p53, IRF1, BCL2, p15INK4b, EVI1 and MLL.

Problems and pitfalls

Diagnostic errors can result from a failure to assessclinical features, peripheral blood and bone marrow

cytology, bone marrow histology and the results of cytogenetic analysis in all cases. Cytology andhistology are complementary in the investigation ofMDS since sometimes one will give informationthat could not be gained from the other. For ex-ample, ring sideroblasts and neutrophil dysplasia arebest detected in an aspirate, whereas ALIP is detectedonly by means of trephine biopsy. Similarly, it issometimes cytogenetic analysis that permits anunequivocal diagnosis when other features havebeen suggestive of MDS but not pathognomonic.

Some cases of MDS have pathological featuresthat are strongly suggestive of the diagnosis. Inother patients the diagnosis of MDS is a presump-tive one, based on the presence of features that arecharacteristic but not diagnostic. In the latter groupthe exclusion of other diagnoses is particularlyimportant. A careful clinical assessment is essential,in order to exclude relevant systemic illness andexposure to drugs, alcohol, heavy metals and growthfactors. Some of the non-neoplastic causes of bonemarrow dysplasia are discussed on page 392. Im-portant pitfalls are relevant drug exposure that hasnot been disclosed to the pathologist and unex-pected HIV positivity.

If dysplastic features are confined to the erythroidlineages, it is important to consider alternative causesof dyserythropoiesis such as the congenital dysery-thropoietic anaemias and various thalassaemic con-ditions. Unstable haemoglobins are also sometimesassociated with quite marked dyserythropoiesis.

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Fig. 4.39 Trephine biopsy sectionfrom a patient with RAEB showingnumerous CD34-positive immaturecells. Paraffin-embedded,immunoperoxidase anti-CD34×388.


It can sometimes be difficult to distinguish mega-loblastic erythropoiesis as a consequence of MDSfrom that attributable either to a deficiency of vita-min B12 or folic acid or to the administration ofdrugs that interfere with DNA synthesis (Fig. 4.40).A useful feature is the lack of associated white cell changesagiant metamyelocytes and hyperseg-mented neutrophilsawhen megaloblastosis is a fea-ture of MDS. However, it should be noted that whitecell abnormalities may be lacking in megaloblasticerythropoiesis caused by drug exposure. Occasion-ally cohesive clumps or sheets of erythroid cells, all

at a similar stage of maturation, can be confusedwith infiltration by carcinoma cells (Fig. 4.41).

The differential diagnosis of RARS includes sider-oblastic erythropoiesis secondary to drugs or heavymetals (see page 397), copper deficiency (see page397), the mitochondrial cytopathies (see page 407)and thiamine-responsive anaemia with diabetesmellitus and sensorineural deafness. The latter con-dition may show granulocytic and megakaryocyticdysplasia in addition to ring sideroblasts [44].

The differential diagnosis of hypoplastic AML andhypoplastic MDS (Fig. 4.42) has been discussed on

Fig. 4.40 BM trephine biopsysection from an HIV-positivepatient taking a high dose ofzidovudine. The patient hadmegaloblastic erythropoiesis and acluster of early megaloblasts wasconfused with ALIP; the linearnucleoli of the megaloblasts are a clue to their true nature. Paraffin-embedded, H&E ×940.

Fig. 4.41 BM trephine biopsysection from a patient with RARSshowing large sheets of dysplasticerythroid cells separated by dilatedsinusoids; the growth patternappears so cohesive that theappearance could be confused withinfiltration by carcinoma cells.Paraffin-embedded, H&E ×94.

Immunohistochemistry has been foundto be of some value in making a distinction betweenhypoplastic MDS and aplastic anaemia [45,46].Cases of hypocellular MDS have been found to have higher numbers of CD34-positive cells andcells expressing proliferating cell nuclear antigen[45].

Lymphoid aggregates are sometimes present inMDS, giving rise to a differential diagnosis withnon-Hodgkin’s lymphoma, particularly T-cell lym-phoma, with associated dysplastic features. It shouldalso be remembered that some patients with lym-phomatous infiltration of the bone marrow have

secondary dysplastic changes resembling those ofMDS (Fig. 4.43).

In the rare patients with MDS who present withisolated thrombocytopenia it is sometimes difficultto make a distinction from auto-immune throm-bocytopenic purpura. Dysplastic features may bevery minor.

It should be noted that, even with a carefulassessment of clinical features and use of all avail-able diagnostic methods, it may still not be possibleto make a firm diagnosis of MDS. In such patientsfollow-up with regular review of the diagnosis isnecessary.

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Fig. 4.42 BM trephine biopsysection from a patient withhypoplastic MDS (RAEB) showing:(a) a disorganized marrow of lowcellularity; (b) at higher power it is apparent that blast cells areincreased. Paraffin-embedded ×388 and ×970.

(a)

(b)


The FAB categories and other identifiedsubtypes of MDS

Refractory anaemia

RA is characterized by ineffective erythropoiesis,with or without ineffective granulopoiesis andthrombopoiesis but, as defined by the FAB group,there are insufficient monocytes, blast cells or ringsideroblasts for the case to qualify for inclusion inother categories of MDS (see Table 4.6). RA is usu-ally either an incidental diagnosis in the elderly or isdiagnosed because of symptoms of anaemia. Manycases belonging to the FAB category of RA fall intothe WHO category of ‘refractory anaemia withoutringed sideroblasts’ but others are designated ‘5q-syndrome’ (see below) or ‘refractory cytopenia withmultilineage dysplasia’.

Peripheral blood

Often morphological and numerical abnormalitiesare confined to the erythroid series but somepatients, particularly those with secondary MDS,manifest anomalies of other lineages. A minority ofpatients have thrombocytosis.


The bone marrow is usually hypercellular as a resultof erythroid hyperplasia (Fig. 4.44). A minority of

patients show marked erythroid hypoplasia, some-times with an apparent arrest of erythropoiesis at the proerythroblast stage. Erythropoiesis usuallyshows dysplastic features but in some patients erythropoiesis is ineffective although dysplastic features are quite minor. Ring sideroblasts may be present but constitute no more than 15% of erythroblasts.

In RA the granulocyte series and megakaryocytesmay be apparently normal or may be hyperplasticor dysplastic (see Figs 4.30 and 4.31).


There are often no histological features of diagnosticimportance in trephine biopsies of patients with RA. The marrow is usually hypercellular but hypo-cellular forms do occur. Erythroid hyperplasia anddyserythropoiesis are usually present and are easilyseen in tissue sections (see Fig. 4.33). The granulo-cytic series may appear relatively normal. Dysplasticmegakaryocytes are found in most cases; however,they are not universally present and, in theirabsence, the diagnosis of myelodysplasia may beeasily overlooked if the clinical and cytological features are not taken into account.

Refractory cytopenia

A small proportion of patients with MDS are not anaemic but have refractory neutropenia or

Fig. 4.43 BM trephine biopsysection from a patient with lowgrade T-cell lymphoma withsecondary myelodysplasia; smallhypolobulated megakaryocytes are apparent. Paraffin-embedded,H&E ×376.

thrombocytopenia. The FAB group has recommendedthat, if such cases lack the features of the other cate-gories of MDS, they should be grouped with refrac-tory anaemia and be designated refractory cytopenia.

Patients with refractory neutropenia show abnor-malities predominantly of the neutrophil lineage,while patients with refractory thrombocytopeniahave increased, dysplastic megakaryocytes and oftendysplastic platelets.

Refractory anaemia with ring sideroblasts

RARS is also referred to as primary acquired sider-oblastic anaemia. The FAB group criteria for thisdiagnosis are the presence of more than 15% of ring sideroblasts among bone marrow erythroblasts,with monocytes and blast cells being insufficientlyincreased to permit assignment to other MDS cate-gories (see Table 4.6). In the WHO classification themajority of cases fall into the category ‘refractoryanaemia with ring sideroblasts’ and a minority intothe category ‘refractory cytopenia with multilineagedysplasia’. Sideroblastic anaemia is usually either an incidental diagnosis in the elderly or is diagnosedbecause of symptoms of anaemia.

Peripheral blood

There is anaemia which is sometimes normocyticbut more often macrocytic. The film is dimorphic,consequent on the presence of a minor population

of hypochromic and microcytic red cells. Occasionalcells contain Pappenheimer bodies. There may be asmall number of circulating erythroblasts, amongwhich may be some ring sideroblasts. Abnormal-ities of neutrophils and platelets can occur but areuncommon. A significant minority of patients havethrombocytosis.


The bone marrow is usually hypercellular andshows erythroid hyperplasia. Erythropoiesis is usu-ally normoblastic or macronormoblastic. A propor-tion of erythroblasts, which correspond to the ringsideroblasts, are micronormoblasts or show defec-tive haemoglobinization or cytoplasmic vacuola-tion (Fig. 4.45). Other dysplastic features in red cellsare uncommon. Abnormalities may occur in otherlineages but they are uncommon except when theMDS is secondary.

By definition, in RARS an iron stain shows thatmore than 15% of erythroblasts are ring sidero-blasts (see Fig. 4.32). They may be as frequent as 70or 80% of erythroblasts and may be associated withother abnormal sideroblasts. Iron stores are com-monly increased.


Trephine biopsy is not usually very useful in thediagnosis of RARS. Bone marrow histology may be

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Fig. 4.44 BM aspirate, RA, showing erythroid hyperplasia andhypogranular neutrophils; note adysplastic binucleated erythroblast.MGG ×940.


relatively normal with the only abnormality beingerythroid hyperplasia with large, poorly formederythroid islands. There is often an increase in stain-able iron within macrophages. Ring sideroblasts canbe seen in plastic-embedded sections of trephinebiopsies and occasionally in paraffin-embedded marrow clot sections (Fig. 4.46); they are not visiblein sections of paraffin-embedded or other decal-cified trephine biopsy specimens. The granulocyticseries is usually normal. Dysplastic megakaryocytesare present in a minority of cases.

Refractory anaemia with excess of blasts

RAEB as defined by the FAB group (see Table 4.6)has:• either an increase of peripheral blood blasts tomore than 1% but less than 5%;• or an increase of bone marrow blasts to at least5% but not more than 20%;• and no Auer rods;• and a monocyte count of less than 1 × 109/l (or thecase falls into the CMML category) (see page 215).

Fig. 4.45 BM aspirate, RARS,showing five erythroblasts, two of which show defectivelyhaemoglobinized, heavilygranulated cytoplasm. MGG ×940.

Fig. 4.46 BM clot section, RARS,showing two ring sideroblasts withblue iron-containing granulesarranged around the nucleus.Paraffin-embedded, Perls’ stain ×970.

Diagnosis of RAEB usually follows the develop-ment of symptoms of anaemia or the occurrence of bruising, bleeding or infection. This FAB cat-egory corresponds to the WHO category ‘refractoryanaemia with excess blasts’.

Peripheral blood

The peripheral blood shows normocytic or macrocyticanaemia and may also show some hypochromicmicrocytic cells. In addition, there may be neu-tropenia, mild monocytosis or thrombocytopenia.Dysplastic features in neutrophils and platelets arecommonly present. There may be some circulat-ing blasts which are usually, but not necessarily,myeloblasts.


The bone marrow is usually hypercellular. Any or all lineages may be hyperplastic and trilineagedysplasia is common. The percentage of blasts isusually increased, although a case may qualify to be categorized as RAEB on the basis of increasedperipheral blood blasts alone. Erythropoiesis maybe sideroblastic but, because of the excess of blasts,the case is categorized as RAEB rather than asRARS.

An iron stain may show ring sideroblasts, otherabnormal sideroblasts and increased iron stores.Either an MPO or a SBB stain should be performed

routinely both to confirm the lineage of the blastsand to exclude the presence of Auer rods, whichwould lead to the case being categorized as RAEB-T(see below).


Bone marrow biopsy is not usually essential for diag-nosis but can give useful supplementary informa-tion. The majority of cases have increased or normalcellularity with only a small number of cases beinghypocellular. Dyserythropoiesis and megakaryocyticdysplasia are seen in almost all cases (Fig. 4.47).Blasts are increased in number but it is not uncom-mon for the percentage of blasts seen in the biopsysections to be less than that observed in marrowaspirates taken at the same time [33]. ALIP is seenin most cases.

Refractory anaemia with excess of blasts intransformation

As defined by the FAB group, the diagnosis ofRAEB-T requires that:• either peripheral blood blasts are at least 5%;• or bone marrow blasts are greater than 20% butless than 30%;• or there are Auer rods in blasts, either in the bone marrow or in the peripheral blood (see Table4.6).

In practice, most cases have more than 20% blasts

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Fig. 4.47 BM trephine biopsysection, RAEB, showing dysplasticmegakaryocytes includingmicromegakaryocytes.Plastic-embedded, H&E ×390.


in the bone marrow whether or not they show theother features. However, occasional cases are cat-egorized as RAEB-T on the basis of one of the othercriteria alone. In the WHO classification, cases withmore than 20% bone marrow blasts would be cat-egorized as AML rather than as MDS.

Patients with RAEB-T are almost always symp-tomatic at diagnosis with bruising, bleeding, infec-tion or symptoms of anaemia. Pallor, bruising,hepatomegaly and splenomegaly are common.

Peripheral blood

Most patients have anaemia, neutropenia and throm-bocytopenia and show morphological abnormalitiesof all lineages. Some circulating blasts are usuallypresent and they may contain Auer rods. There maybe monocytosis, particularly in those patients inwhom the disease represents a transformation ofCMML rather than RAEB.


The bone marrow is usually hypercellular with tri-lineage myelodysplasia, an increase of blasts andsometimes Auer rods in the blasts. Sideroblastic erythropoiesis is not inconsistent with a diagnosis of RAEB-T.


Trephine biopsy is not usually essential for diagnosisbut can give useful supplementary information. Inaddition to trilineage dysplasia there is an increasein numbers of blasts, often with ALIP. Reticulinfibrosis has been reported to be less common thanin the other subtypes [33].

The 5q- syndrome

Among patients with MDS a group of patients can be delineated who have what is designated the5q- syndrome. This entity is a specific category in the WHO classification. Patients tend to be middle-aged or elderly women with a relatively good prognosis. Haemopoietic cells show an interstitial deletion of the long arm of chromosome 5 as a single acquired chromosomal anomaly. Such patientsmost often have a refractory anaemia, often macro-

cytic, with or without ring sideroblasts and alsohave characteristic megakaryocytes; these are morethan 30 µm in diameter but have non-lobulatednuclei (see Fig. 4.31). The platelet count is usuallynormal or even increased. The disease usually fallsinto the RA category of the FAB classification butsometimes into the RARS category.

Refractory macrocytosis

Occasional patients with myelodysplasia have refrac-tory macrocytosis but are not anaemic and lack features which would lead to their being assigned toany of the FAB categories of MDS. When erythro-poiesis is clonal, they should be recognized as havingMDS and can reasonably be grouped with RA. Withlong-term follow-up, anaemia and other features ofovert MDS develop.

Refractory sideroblastic erythropoiesis

Occasional patients are seen with primary acquiredsideroblastic erythropoiesis who lack anaemia andother features which would allow them to beassigned to one of the FAB categories of MDS [47].Nevertheless, erythropoiesis is clonal and suchpatients should be recognized as having MDS. Theycan reasonably be grouped with RARS. With dis-ease progression, anaemia occurs.

Secondary myelodysplasia

Myelodysplasia may be secondary to deliberate oraccidental exposure to mutagenic agents such ascytotoxic chemotherapy, benzene and irradiation.Such cases can be categorized according to the FABrecommendations but, because they have distinctivefeatures, it is useful to consider them separately.Secondary MDS usually occurs at a younger agethan primary MDS and has a much worse prognosis.Bone marrow failure and evolution to acute leukae-mia occur much earlier. Many cases of secondaryMDS fall into the WHO category of ‘AML andmyelodysplastic syndromes, therapy-related’.

Peripheral blood

Abnormalities in the peripheral blood are usuallymore marked than in primary MDS. Even cases

which fall into the RA and RARS categories commonly show neutropenia, thrombocytopenia,monocytosis and evidence of trilineage dysplasia.


The bone marrow may be hypercellular but is often hypocellular. A poor aspirate may be obtainedbecause of associated bone marrow fibrosis. Obvi-ous trilineage myelodysplasia is common, even incases which meet the criteria for RA and RARS.There are usually at least some ring sideroblasts.Hypogranular neutrophils, pseudo-Pelger–Huët neu-trophils and micromegakaryocytes are also common.


Because of the frequent difficulty experienced inobtaining a good aspirate, trephine biopsy is oftenimportant in diagnosis. The cellularity is more vari-able than in primary myelodysplasia and stromalinjury may be prominent, with disruption of fatcells and extravasation of non-nucleated red cellsinto the interstitium. There is often a marked increasein reticulin and, in contrast to primary myelodys-plasia, there may be collagen fibrosis. There is oftena severe degree of dysplasia affecting all three lin-eages with numerous micromegakaryocytes.

Malignant histiocytosis

Malignant histiocytosis is a disease caused by theproliferation in tissues of a neoplastic clone of cellsof monocyte/macrophage lineage; the abnormalcells show variable phagocytic activity. This diseasemay be regarded as the tissue counterpart of acutemonocytic leukaemia. It differs from monocytic sarcoma in that the cells of the neoplastic clone are widely distributed in peripheral tissues ratherthan forming localized tumours. This disease is notspecifically recognized by the WHO classificationand would fall into the category ‘histiocytic neo-plasms, unclassifiable’.

It appears likely that, in the past, a significant proportion of diagnoses of malignant histiocytosis[48,49] or of histiocytic medullary reticulosis [50](usually regarded as a form of the same disease)were actually misdiagnoses [51–55]. The majorityof cases misinterpreted as malignant histiocytosis

were either reactive histiocytosis consequent onviral or other infections or reactive histiocytosisoccurring as a response to large cell anaplastic lymphoma and other T-lineage lymphomas. A lesscommon cause of confusion is a T-cell lymphoma inwhich the lymphoma cells are themselves phago-cytic [56]. It is important that the term malignanthistiocytosis be restricted to cases in which neoplasticcells are of monocyte lineage. The term histiocyticmedullary reticulosis is probably best abandonedsince the recent availability of immunophenotypingand other techniques has led to the recognitionthat, in the great majority of cases, the histiocyticproliferation and florid haemophagocytosis weresecondary to a T-cell lymphoma [54,55] or a viralinfection [53]. The reactive haemophagocytic syn-dromes are discussed further on pages 119–123.

The diagnosis of malignant histiocytosis rests onclinical, histological, cytochemical and immuno-phenotypic grounds. Neoplastic cells are primitiveand, although phagocytosis occurs, it is not promi-nent [51]. Neoplastic cells can be demonstrated, bycytochemical staining or immunophenotyping, tobelong to the monocyte lineage and not to the T-lymphocyte lineage whereas, in haemophagocyticsyndromes consequent on a T-cell lymphoma, thereis an admixture of reactive mature phagocytic histi-ocytes and immature neoplastic cells of lymphoidlineage [54].

Common clinical features of malignant histiocy-tosis are hepatomegaly, splenomegaly, lymphadeno-pathy, skin infiltration and systemic symptoms suchas malaise, fever and weight loss.

Peripheral blood

Pancytopenia is common. Small numbers of imma-ture cells of monocyte/macrophage lineage may bepresent in the blood (Fig. 4.48).


At the onset of disease, the bone marrow may showminimal or no infiltration by neoplastic cells. Withmore advanced disease there may be heavy infiltra-tion (Fig. 4.49). The majority of neoplastic cells havethe morphological features of monoblasts or, inolder terminology, ‘reticulum cells’. Cells are largeand usually have a round nucleus with nucleoli and

184 CHAPTER FOUR


a diffuse chromatin pattern. Cytoplasm is plentifuland moderately basophilic. A variable number ofmaturing cells with kidney-shaped nuclei and moreabundant cytoplasm are also present [51]. Somecells are phagocytic and are seen to have ingestedgranulocytes and their precursors, erythroblasts andplatelets; however, phagocytosis is much less markedthan in reactive haemophagocytosis.


Bone marrow infiltration has been reported to bemore commonly detected by trephine biopsy than

by bone marrow aspiration [57]. The bone marrowmay appear normal at the time of diagnosis or mayshow a mild focal infiltrate of neoplastic cells. Theremay be intrasinusoidal neoplastic cells [58]. In thelater stages of the disease diffuse replacement ofhaemopoietic tissue commonly occurs (Fig. 4.50)[49,59,60]. The infiltrate is largely composed ofimmature cells with large pleomorphic nuclei whichmay be lobulated and contain prominent nucleoli;there are moderate amounts of basophilic cytoplasm.Mitoses are usually numerous. A variable compo-nent of more mature cells of monocytic lineage maybe present.

Fig. 4.48 PB, malignanthistiocytosis, showing anaemia,thrombocytopenia and threeabnormal cells of monocytelineage, one of which has aphagocytic vacuole. MGG ×940.

Fig. 4.49 BM aspirate, malignanthistiocytosis associated witht(9;11)(p22;q23). MGG ×376.(By courtesy of Dr R Brunning,Minneapolis.)

Genetics and cytogenetics

Some cases of malignant histiocytosis have beenassociated with translocations that are also associ-ated with AML with monocytic differentiation,such as t(9;11)(p22;q23) [60] and t(8;16)(p11;p13).


The diagnosis of malignant histiocytosis is fraughtwith pitfalls and should be made with great circum-spection. A minor degree of haemophagocytosismay be seen but marked haemophagocytosis sug-gests an alternative diagnosis. In children, familialor sporadic lymphohistiocytosis is likely and investi-gation for herpesvirus infection is indicated. Inadults, reactive haemophagocytosis is often causedby mycobacterial or herpesvirus infection or by a T-cell lymphoma. Molecular analysis to demonstrateT-cell receptor gene rearrangement can be useful.Cytogenetic analysis is indicated if malignant his-tiocytosis appears a likely diagnosis, since it mayconfirm the diagnosis.

True histiocytic lymphoma

True histiocytic lymphoma initially presenting atextramedullary sites may subsequently infiltrate thebone marrow. The patterns of infiltration reportedare interstitial, patchy focal and diffuse [61]. In the

WHO classification the designation ‘histiocytic sar-coma’ is used.

Langerhans’ cell histiocytosis

Langerhans’ cell histiocytosis, previously known ashistiocytosis X, is a heterogeneous disease or groupof diseases characterized by proliferation of Langer-hans’ cells [62]. Recent evidence suggests that theproliferation is clonal and neoplastic [63]. Localizedand disseminated forms occur. Haematologicalinvolvement occurs in the disseminated forms ofthe disease, which in the past have been referred toby the eponymous terms Letterer–Siwe disease ofinfants and Hand–Schüller–Christian disease. Bonemarrow infiltration is seen mainly in infants andchildren.

Peripheral blood

The peripheral blood may be normal or there maybe pancytopenia as a consequence either of hyper-splenism or of bone marrow infiltration. Rarely, aleukaemia of Langerhans’ cells occurs.


The bone marrow aspirate may show Langerhans’cells together with a mixed population includingeosinophils, monocytes, lipid-laden macrophages,

186 CHAPTER FOUR

Fig. 4.50 BM trephine biopsysection, malignant histiocytosis(same case as in Fig. 4.49). Paraffin-embedded, H&E ×188. (By courtesyof Dr R Brunning, Minneapolis.)


lymphocytes and plasma cells [64,65]. Haemo-phagocytosis may occur. Langerhans’ cells are largeand slightly irregular in shape. The nucleus is somewhat irregular and sometimes grooved withdelicately clumped chromatin and inconspicuousnucleoli (Fig. 4.51). The cytoplasm is weaklybasophilic with occasional azurophilic granules.Ultrastructural examination demonstrates Birbeckgranules.

Cytochemistry

Langerhans’ cells show tartrate-resistant acid phos-phatase activity [66] and are negative for non-specific esterase.

Immunophenotyping

Langerhans’ cells are positive for CD1a and HLA-DR.


In those cases with marrow involvement, the bonemarrow contains clusters or sheets of Langerhans’cells together with eosinophils, neutrophils, lym-phocytes, plasma cells, monocytes, phagocyticmacrophages, lipid-laden macrophages and giantcells (Fig. 4.52). Xanthomatous transformation andfibrosis may occur [64,65]. Langerhans’ cells have a

characteristic appearance; the nuclei are usuallyconvoluted or twisted and longitudinal groovesmay be present.

Immunohistochemistry

Immunohistochemical staining shows Langerhans’cells to express S100 protein, although only a smallproportion of cells may stain positively in somecases. Staining with the lectin, peanut agglutinin,gives a characteristic staining pattern of a cytoplas-mic halo with a paranuclear dot. Langerhans’ cellsare positive for vimentin and CD1a. Positivity forCD1a is currently the most reliable immunopheno-typic marker of Langerhans’ cells; the value of newanti-TRAP monoclonal antibodies remains to beestablished.

Genetics and cytogenetics

A familial tendency to the development of Langer-hans’ cell histiocytosis has been described [67]. Aclonal cytogenetic abnormality, t(7;12)(q11.2;p13),has been reported in a single case of eosinophilicgranuloma [68].


It should be noted that Langerhans’ cell histiocytosisoften causes focal lesions, even when widespread

Fig. 4.51 BM aspirate inLangerhans’ cell histiocytosis. MGG ×940. (By courtesy of Dr R Brunning, Minneapolis.)

throughout the skeleton, and a targeted biopsy of aradiologically suspicious lesion may be more infor-mative than a standard iliac crest trephine biopsy.

Langerhans’ cell histiocytosis has been confusedwith systemic mastocytosis, hairy cell leukaemiaand malignant melanoma. Careful assessment ofcytological features, supplemented by immunohis-tochemistry, will resolve difficulties.

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