J Clin Pathol 1992;45:855-859

Bone marrow histology 3: Value of bone marrow

core biopsy in acute leukaemia, myelodysplasticsyndromes, and chronic myeloid leukaemia

D A Winfield, S V Polacarz

IntroductionThe standard approach to the diagnosis ofacute leukaemia and myelodysplasia has beenbased on the morphology and percentage ofmalignant haemopoietic cells in peripheralblood and in an aspirated sample of bonemarrow.The FAB group reports on acute leukaemia

have emphasised the value of additional infor-mation gained from cytochemistry,' andimmunological studies and cytogenetics areincreasingly being used to provide an accuratediagnosis in acute leukaemia2 and myelodys-plasia.

Aspirated bone marrow only provides asmall sample which is diluted with sinusoidalblood and provides no information on thearchitectural changes occurring within themarrow cavity as a result of the leukaemicprocess. An adequate core biopsy samplepermits a more accurate analysis of the degreeof involvement of the marrow by the leukaemicprocess and may provide additional informa-tion on the response of normal marrow ele-ments to the malignant infiltrate. Despite theseadvantages marrow core biopsy is not widelyaccepted as an essential requirement in theinvestigation of haemopoietic malignancies.One reason for this may be the view thatdecalcification and paraffin-wax embedding ofsuch biopsy specimens produces distortionand shrinkage, resulting in difficulty in inter-preting cellular morphology. This problem canbe overcome by using plastic embedding ofbiopsy specimens,3 but satisfactory morpho-logical details may also be obtained withimproved methods of processing paraffin-waxembedded material,4" with the additionaladvantage that such processing also allows theuse of immunocytochemical techniques.

This review will assess the value of marrowcore biopsy as a routine diagnostic procedurein the investigation of acute leukaemia, myelo-dysplasia, and chronic myeloid leukaemia.

HaematologyDepartment, RoyalHallamshire Hospital,Sheffield S10 2JFD A WinfieldDepartment ofPathology, Universityof Sheffield MedicalSchoolS V PolacarzCorrespondence to:Dr D A WinfieldAccepted for publication20 March 1992

Acute myeloblastic leukaemia (AML)Aspirated bone marrow permits accurate diag-nosis and FAB classification in most cases ofAML. The value of a core biopsy specimen isthat it provides a more accurate assessment oftotal marrow cellularity and blast cell numbersboth of which are usually numerically greaterin biopsy samples than in aspirated marrow.

Biopsy material also allows for accurate quanti-tation of normal marrow components such as

plasma cells, lymphocytes, mast cells, eosino-phils and macrophages which are sometimes

found in increased numbers in acute leukae-mia.6 Whether this additional information is ofany prognostic value is at present uncertain.The newer techniques of processing core

biopsy specimens make recognition of cyto-morphological features easier, but the appear-ances of cells are often different from thoseseen in Romanowsky stained marrow smears.Despite these differences it is usually possibleon marrow section to distinguish accuratelybetween acute myeloblastic and lymphoblasticleukaemia, but morphological classificationinto the AML FAB subtypes can be difficultand a comparison of biopsy specimens andaspirates resulted in agreement in only 57% of

7cases.Enzyme histochemistry is routinely used for

diagnosis on marrow aspirates but is limited onparaffin-wax embedded sections to the detec-tion of chloroacetate esterase by the Lederprocedure.8 The use of plastic embeddingprocedures allows for a wide range of his-tochemical reactions, including chloroacetateesterase in myeloid cells (fig 1), a-naphthylbutyrate esterase in monocyte/macrophagecells, alkaline phosphatase in fibroblast-likereticulum cells and acid phosphatase in mega-karyocytes, monocytes, and T lymphocytes.9The usefulness of the information obtainedfrom marrow core enzyme histochemistryrequires further evaluation, although the con-firmed value of chloroacetate esterase in deter-mining cells of the myeloid series indicates thatthe Leder procedure should be routinely usedon marrow biopsy specimens.Monoclonal antibodies detecting myeloid

antigens are now available for use on paraffinwax sections of marrow biopsy specimens andhave been evaluated in the immunophenotyp-ing ofAML. '"1 Positive reactivity is obtainedwith a range of antibodies but because AMLsubtypes exhibit a heterogenic phenotype asingle antibody rarely produces diagnosticallyuseful information with the exceptions of anti-glycophorin C in erythroleukaemia and bothanti-platelet glycoprotein III and anti-factorVIII related antigen in megakaryoblastic leu-kaemia. Immunophenotyping of marrow sec-tions remains at the research stage, but it islikely that this technique will be of increasingvalue in routine diagnostic work.

HYPOPLASTIC AML

In the hypoplastic variant ofAML core biopsyof marrow is essential for an accurate diag-nosis. The incidence of this condition is about5% of all cases of AML. The diagnosticfeatures are a hypocellular marrow (less than

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50% cellularity) with more than 30% primitivemyeloid cells (fig 2). It is essential that thecellular morphology is carefully assessed in allhypoplastic core biopsy specimens to avoidmistakes in diagnosis, particularly betweenhypoplastic AML and hypoplastic anaemia.Antecedent myelodysplastic features are foundin one third of cases and there is often a historyof previous exposure to leukaemogenicagents.12 13The pathogenesis of the hypocellu-larity is uncertain, but serial biopsy specimensusually have a constant hypocellular pattern,indicating that this condition is a true variantofAML.

Megakaryoblastic leukaemia (FAB M7) andacute myelofibrosisAre these two terms describing separate enti-ties or are they the same disease? Both arecharacterised by absence of clinical splenome-galy, pancytopenia, and a rapidly fatal course.Blast cells may be present in the peripheralblood and are characterised as megakaryo-blasts by a positive platelet peroxidase reactionat ultrastructural level. An adequate marrowaspirate cannot usually be obtained and diag-nosis requires core biopsy with features ofincreased numbers of blast cells, increased

megakaryocytes of atypical morphology, and adense increase in reticulin (figs 3 and 4).

In a comparative study of FAB M7 andacute myelofibrosis there were similar morpho-logical findings with the exception of moreprominent lymphocytic aggregates in acutemyelofibrosis. These diseases can be separatedinto different entities by immunohistochemicalreactivity to monoclonal anti-factor VIIIrelated antigen. In FAB M7 the blast cells inthe core biopsy specimen are positive; in acutemyelofibrosis they are negative.'4 This finding,together with a previous report of heterogene-ity of the blast cells in acute myelofibrosis,'5suggests that acute myelofibrosis results from aproliferation of more primitive stem cells.

Increased marrow reticulin fibres may alsobe associated with cellular dysplastic changes,but this can be considered as a separate entityand will be discussed in the section dealingwith myelodysplastic syndromes.

Acute lymphoblastic leukaemia (ALL)In most cases ofALL core biopsy provides onlylimited additional information compared withstandard diagnostic techniques used on aspir-ated marrow or peripheral blood. Marrow

Figure 1 Bone marrowcore biopsy specimen froma case of acutemyelomonoblasticleukaemia (FAB M4).Positive staining withnaphtholAS-Dchloroacetate esterasereaction in myeloblasts(Leder's stain).

Figure 2 Bone marrowcore biopsy specimen froma case of hypoplastic AMLshowing fatty hypocellularmarrow with foci of blastcells (haematoxylin andeosin).

Figure 3 Bone marrowcore biopsy specimen froma case of megakaryoblasticleukaemia (FAB M7)with prominent atypicalmegakaryocytes and blastcells (haematoxylin andeosin).

Figure 4 Pronouncedfibrosis in a case ofmegakaryoblasticleukaemia (FAB M7)(Gomori reticulin stain).

Figure 5 Bone marrowcore biopsy specimen froma case ofMDS showingALIP and occasionaldyserythropoietic features(1) (haematoxylin andeosin).

Figure 6 Bone marrowcore biopsy specimen froma case ofCML showingparatrabecular blast cellsand promyelocytes withmaturing myeloid cells inthe intertrabecular marrowspace (haematoxylin andeosin).

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Value of bone marrow biopsy in haematological malignancy and MDS

cellularity and blast cell numbers may be moreaccurately assessed on core biopsy but FABclassification is very difficult.7 Increasedamounts of reticulin are found in about 70% ofcases of ALL. There is a strong associationbetween an increase in reticulin and lympho-blasts expressing B cell lineage markers, butwhether marrow fibrosis is of any prognosticimportance is at present uncertain. 6 There arealso a few reported cases ofALL which on corebiopsy fulfil the diagnostic criteria for hypo-plastic acute leukaemia.'3 These must be dif-ferentiated from the more commonly reportedcases of transient marrow aplasia which mayprecede the development of common ALL.'7Bone marrow necrosis can sometimes be sus-pected in the marrow aspirate of ALL atpresentation or relapse because cellular fea-tures are lost and background staining isamorphous. In such situations a trephinebiopsy specimen is required for confirma-tion. 8

Currently, the newer immunohistologicaltechniques are limited in the additional infor-mation they can provide and their use isrestricted to the detection ofT cell ALL withpolyclonal anti-CD3.V0

Myelodysplastic syndromes (MDS)The MDS comprise a group of diseasescharacterised by morphological abnormalitiesin myeloid, erythroid, and megakaryocytic celllines. The FAB cooperative group'9 havedefined, by cytological criteria, five subtypes:refractory anaemia (RA), refractory anaemiawith ring sideroblasts (RARS); refractoryanaemia with excess of blasts (RAEB); refrac-tory anaemia with excess of blasts in trans-formation (RAEB-t); and chronic myelo-monocytic leukaemia (CMML). Recognitionof these subtypes may be difficult in a corebiopsy specimen,20 although improved accu-racy of diagnosis can be achieved if peripheralblood blast cell and monocyte counts are alsoknown.2" A core biopsy specimen does allow adisturbed marrow cellular spatial orientation tobe recognised, resulting in the presence ofmyeloid precursor cells away from their normalposition along endosteal surfaces. This featureis termed abnormal localisation of immatureprecursors (ALIP) and is defined as the pres-ence of three or more myeloblasts or promyelo-cytes clustering centrally in the marrow (fig 5).There must be more than three ALIPs persection before they can be considered to be ofdiagnostic importance,20 and it is important torecognise that aggregates of immature eryth-roid and megakaryocytic cells, as demon-strated by immunochemistry, may also mimicthe appearance of an ALIP (pseudo-ALIP).2223 AlthoUgh ALIP is typically asso-ciated with MDS, it is not unique to this groupof diseases and can sometimes be seen inmarrow treated with chemotherapy in AMLand chronic myeloid leukaemia, and also fol-lowing bone marrow transplantation.22 Doesthe finding of ALIP have prognostic as well asdiagnostic importance? There seems to be an

association between ALIP and shorter patientsurvival, not only in RAEB, RAEB-t, andCMML where ALIP is most common, but alsoin RA and RARS where, by definition, there isno excess of myeloblasts in the marrow aspir-ate. 22-24

Dysmegakaryopoiesis is found in up to 80%of cases of MDS25 and abnormal mega-karyocytic morphology may be easier to detectin a core biopsy specimen than in aspiratedmarrow. As well as having diagnostic impor-tance the severity of dysmegakaryopoiesis mayalso indicate a poorer prognosis.2" Character-istic features are hypo- and hyperlobulatedmegakaryocytes and megakaryoblasts with ahigh nuclear: cytoplasmic ratio, delicatenuclear chromatin, and one or more nucleoli.20Micromegakaryocytes are frequently presentand although they can be recognised in ahaematoxylin and eosin preparation, they aremore easily seen after staining with periodicacid schiff (PAS) or by immunochemistryusing monoclonal antibody anti-CD61 direc-ted against glycoprotein IIIa.25 The relativelyrare 5q-syndrome is suspected from the find-ing of a uniform population of monolobulatedmicromegakaryocytes.20

Dyserythropoiesis is present in most cases ofMDS,20 normally manifest as megaloblasticmaturation or as defective maturation in whichthe erythron is composed of cells at the samestage of development. A predominance ofproerythroblasts can also be seen.20 26

Accurate quantitation of marrow blast cellsis required for a correct subtyping ofMDS andfor differentiation from acute leukaemia.Although quantitation is more difficult in acore biopsy specimen than in aspirated mar-row, a good concordance in blast cell estima-tion between core and aspirate samples hasbeen reported.2'A core biopsy specimen is of special value in

the detection of increased concentrations ofmarrow reticulin, present in about 50% ofsamples of MDS.20 21 26 This is usually ofmoderate degree, although several recentreports have described cases of myelodysplasticsyndrome associated with a pronounceddegree of fibrosis.27-29 The prognostic impor-tance of this finding is at present unclearbecause it is suggested that this condition maybe associated with an increased27 or adecreased28 29 survival. A biopsy specimen isalso important for accurate diagnosis of therare MDS hypoplastic variant, characterisedby pancytopenia, marrow hypoplasia, and alow incidence of progression to acute leukae-mia.'0The assessment of a biopsy sample in MDS

requires not only consideration of the dyshae-mopoietic features but should also includequantitation of eosinophils and plasma cells.Eosinophilia, defined as greater than eighteosinophils per microscopic field (x 400magnification), has been recorded in a quarterof cases and may be associated with improvedsurvival. Plasmacytosis of more than twoplasma cells per microscopic field (x 400magnification) is seen in 18% of all MDS

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cases, and in as many as one half of theCMML subtype, although no prognostic valuehas been attached to this finding.2" 26

Chronic myeloid leukaemia (CML)The diagnosis of CML is based on typical

morphological features of excess myeloidactivity in blood and bone marrow aspirate, a

low leucocyte alkaline phosphatase score, thepresence of the Philadelphia chromosome[t(9;22(q34;ql 1)] and the molecular conse-

quences of the Ph translocation with formationof the BCR-ABL fusion gene on chromosome22. A core biopsy specimen provides onlylimited additional diagnostic informationalthough it has been suggested that two sub-types can be identified. One has proliferationof predominantly granulocytic precursor cells,the other a mixed proliferation of granulocyticand megakaryocytic lines (chronic mega-karyocytic-granulocytic myelosis) . The sug-gestion was made that these morphologicalsubtypes show differences in clinicalbehaviour, but more recent work has shown no

clinical, evolutionary,or prognostic differencesbetween the two groups.32The characteristic morphological pattern in

chronic myeloid leukaemia is of blast cells andpromyelocytes located in the paratrabecularand perivascular region with increasing pro-portions of more mature myeloid cells in thecentral intertrabecular spaces (fig 6). Accuratedetermination of blast cells and promyelocyticnumbers may provide independent prognosticinformation. Patients with pronounced infiltra-tion (four to eight layers of blasts and promye-locytes) compared with minimal infiltration(one to three layers) have an increased proba-bility of early blast cell transformation.33An advantage of marrow biopsy in CML is

that it allows marrow fibrosis to be assessed. Innewly diagnosed patients up to half haveincreased amounts of reticulin in more than50% of the cellular areas of the biopsy speci-men. Increased fibrosis is strongly associatedwith poor prognostic features such as spleno-megaly, anaemia, increased peripheral bloodand marrow blasts, and additional karyotypicabnormalities, and it has therefore been sug-gested that fibrosis can be a useful prognosticfactor at diagnosis. " In sequential biopsyspecimens taken during the course of thedisease many patients show a progressiveincrease in marrow fibrosis which correlateswith the number of megakaryocytes, and thistoo is associated with a worsening prog-nosis.35

ConclusionA core biopsy specimen complements theperipheral blood and marrow aspirate findingsin providing additional information for the

diagnosis and assessment of prognosis in acuteleukaemias, myelodysplastic syndromes, andchronic myeloid leukaemia. In recommendingthe routine use of core biopsy it is essential thatadequate, well processed samples are availablefor morphological interpretation and for the

assessment of newer cytochemical and immu-nohistological techniques. Also important is agreater awareness of the benefits of closecooperation between histopathologists andhaematologists in ensuring that maximal infor-mation is gained from bone marrow samples inhaematological malignancies.

SV Polacarz is supported by the Yorkshire Cancer ResearchCampaign. We are grateful to Mrs J Peel and Mrs M Raymondfor their help in preparing the manuscripts.

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