metastatic tumours marrow... · increasingly, bone marrow aspiration and trephine biopsy are being...

when skeletal radiology and isotopic bone scans[8,11] are normal.

Considering the small volume of tissue sampled,both bone marrow aspiration and trephine biopsyare relatively sensitive techniques for detectingbone marrow infiltration by metastatic tumours. Intwo autopsy studies which simulated biopsy pro-cedures, it was estimated that, when osseous metas-tases were present, a bone marrow aspirate wouldgive positive results in 28% of cases [12] and a sin-gle trephine biopsy in 35–45% [13]. Trephinebiopsy is more sensitive than bone marrow aspira-tion and sensitivity is increased by performing bilat-eral biopsies or by obtaining a single large biopsy.The sensitivity of aspiration is increased if largenumbers of films are examined and if a clot sectionis also examined. It is common for tumour cells tobe detectable in trephine biopsy sections whennone are demonstrable in films of an aspirate[8,14]. Overall, about three quarters of metastasesdetected by a trephine biopsy are also detected bysimultaneous bone marrow aspirate. Discrepancybetween biopsy and aspirate findings usually resultsfrom a desmoplastic stromal reaction to the tumourwhich renders neoplastic cells more difficult to aspirate than residual haemopoietic cells. It is also,to some degree, a consequence of the different vol-umes of tissue sampled. Because of its greater sensi-tivity, trephine biopsy should always be performedwhen metastatic malignancy is suspected. How-ever, tumour cells are seen occasionally in aspiratefilms when trephine biopsy sections appear normal[2,8,14] and the two procedures should thereforebe regarded as complementary.

Increasingly, bone marrow aspiration andtrephine biopsy are being performed as staging pro-cedures at the time of diagnosis in a number of solidtumours, principally neuroblastoma in children and

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METASTATIC TUMOURS

The bone marrow is one of the more commonorgans to be involved by tumours that metastasizevia the bloodstream. In adults the tumours mostoften seen are carcinomas of the prostate gland,breast and lung, although any tumour that givesrise to blood-borne metastases may infiltrate themarrow [1,2]. In children neuroblastoma, rhab-domyosarcoma, Ewing’s sarcoma, other primitiveneuro-ectodermal tumours (PNET) and retinoblas-toma account for the majority of metastases [3,4].Bone marrow metastases from squamous cell carci-noma, other than that of the lung, and from soft tissue tumours of adults are uncommon [1]. Intra-cranial tumours rarely metastasize outside the cranial vault. Of those cases reported with bonemarrow involvement, glioblastoma multiforme hasbeen the most frequent [5]; examples of metastaticmedulloblastoma [6] and oligodendroglioma [7]have also been recorded.

Infiltration of the marrow may be suspected onthe basis of: (i) bone pain; (ii) pathological fractures,lytic lesions or sclerotic lesions demonstrated radio-logically; (iii) unexplained ‘hot spots’ on isotopicbone scans; (iv) hypercalcaemia or elevated serumalkaline phosphatase activity; or (v) unexplainedhaematological abnormalities. The haematologicalabnormality most suggestive of marrow infiltration,though not specific for it, is leuco-erythroblasticanaemia (see below). Metastases are also demon-strated occasionally when bone marrow examina-tion is carried out for staging purposes in theabsence of any features suggestive of bone marrowinfiltration. Overall, the presence of leuco-ery-throblastic anaemia is a relatively insensitive indica-tion of infiltration since it is observed in less thanhalf of patients in whom bone marrow metastasescan be demonstrated by biopsy [8–10]. Aspiratesand trephine biopsies are occasionally positive even

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METASTATIC TUMOURS 431

carcinomas of the breast and lung in adults. Suchinvestigations are indicated when there is a sig-nificant probability of bone marrow metastases andwhen knowledge of their presence would affect thechoice of primary treatment. Biopsy may be indi-cated, for example, when radical surgery or radio-therapy with curative intent is to be undertaken orwhen intensive chemotherapy with autologousbone marrow transplantation is being considered.

It can be important to suggest the likely primarysite of metastatic lesions detected in the bone mar-row. This is particularly so in the case of adenocar-cinoma since, although many such tumours are relatively resistant to therapy, those originating inthe breast and prostate gland may respond to hor-monal therapy. Identification of metastatic thyroidcarcinoma is likewise important although, in prac-tice, this tumour is rarely found unexpectedly inbone marrow biopsy samples since malignant thy-roid tumours generally manifest themselves clearlyat their primary site. Investigation for an unknownprimary tumour is therefore rarely necessary in thiscontext and radio-isotope imaging is the preferredstaging technique for known thyroid malignancies.

The main areas of difficulty in the diagnosis ofmetastatic tumour in bone marrow are: 1 distinguishing metastatic tumour cells fromtumours of haemopoietic cellsafor example, mar-row involvement by high grade non-Hodgkin’s lym-phoma (NHL) or M7 AML (WHO categories of acutemegakaryocytic leukaemia and acute panmyelosiswith myelofibrosis); 2 determining the site of origin of metastatictumour when the primary is unknown; 3 detecting small foci of metastatic tumour in biop-sies performed as part of tumour staging; and4 identifying scanty metastatic malignant cells inseverely sclerotic deposits.

Immunohistochemistry is very useful in theidentification of metastatic tumours in the bonemarrow (Table 10.1).

Peripheral blood

Normocytic normochromic anaemia is commonlypresent when there is infiltration of the bone marrow by malignant cells; other cytopenias areless common. In a third to a half of patients withbone marrow infiltration there are nucleated red

cells and neutrophil precursors in the bloodadesignated leuco-erythroblastic anaemia when the patient is also anaemic. The presence of a leuco-erythroblastic anaemia correlates with the degree of reactive bone marrow fibrosis rather than with the extent of malignant infiltration [9]; it is most commonly seen in association with carcinoma ofthe breast, stomach, prostate gland and lung.Sometimes bone marrow infiltration is identified inthe absence of anaemia or any other abnormality inthe peripheral blood.

Significant numbers of circulating malignant cellsare rare but may occur in the small cell tumours of childhood, particularly neuroblastoma, rhab-domyosarcoma and medulloblastoma. Circulatingneoplastic cells may also be seen in adult patientswith carcinoma but this is a very rare occurrence.

Patients with metastatic malignant cells in thebone marrow may show peripheral blood abnor-malities which are caused by the underlying malignant disease but are not directly due to bone marrow infiltration. Such abnormalities can include: (i) iron deficiency anaemia; (ii) theanaemia of chronic disease; (iii) micro-angiopathichaemolytic anaemia; (iv) neutrophilia; (v) eosino-philia; (vi) thrombocytopenia; (vii) thrombocytosis;and (viii) increased rouleaux formation.

Bone marrow cytology

When bone marrow infiltration has led to reactivemyelofibrosis, attempts at aspiration may result in a‘dry tap’ or a ‘blood tap’, or a small amount of mar-row containing haemopoietic cells, tumour cells orboth may be aspirated with difficulty. When there isan associated increase in bone turnover, the aspiratemay contain a mixture of tumour cells, osteoblastsand osteoclasts (Fig. 10.1). Sometimes the aspirateis wholly or partly necrotic and this observationshould lead to the suspicion of malignant infiltra-tion. When a satisfactory aspirate is obtained, it maycontain large numbers of tumour cells mixed with avariable number of residual haemopoietic cells, ortumour cells may be scanty and found only after aprolonged search. Examination of the tail and edgesof the film and examination of many films are important if scanty tumour cells are to be detected.The detection of scattered neoplastic cells in films of bone marrow aspirates is enhanced by the use of

Table 10.1 Antigens expressed by non-haemopoietic cells, useful for demonstration of metastatic tumours byimmunohistochemistry in fixed, decalcified bone marrow trephine biopsy specimens.

Comments

Cells in occasional cases ofanaplastic large cell lymphomaare also positive

Cells in occasional cases ofanaplastic large cell lymphomaare also positive

Anaplastic large cell lymphomasand non-neoplastic plasma cellsare also positive

Metamyelocytes and matureneutrophils are also positive

Of limited diagnostic value inmetastases from an unknownprimary source but may assist inprognostication in breastcarcinoma



Perineurial cells, a subset ofmacrophages, cells of Langerhanscell histiocytosis and 20% ofbreast cancers are positive

Varying proportions of cells arenegative in many tumours

Newly produced reticulin,chondrocytes and lategranulocyte precursors may bepositive

Extensive background nuclearstaining occurs with some fixationprotocols

Endothelial cells and some adultmalignant epithelial tumours mayalso be positive

Specificity

Epithelial cells (cytoplasmicexpression)

Epithelial cells (cytoplasmicexpression)

Epithelial cells (membraneexpression)

Epithelial cells (membraneexpression, often apical orperiluminal distribution)

Prostatic epithelial cells(cytoplasmic expression)

Prostatic epithelial cells(preferential apical/periluminaldistribution)

Thyroid epithelial cells(cytoplasmic expression)

Thyroid medullary C-cells(cytoplasmic expression)

Oestrogen-sensitive cells,including those of breast, ovary and endometrium (nuclear expression)

Oestrogen-sensitive cells,including those of breast, ovary and endometrium (nuclear expression)

Tumours of diverse origins withexcessive wild-type P53 ormutant P53 (nuclearexpression)

Malignant melanoma (nuclearand cytoplasmic expression)

Malignant melanoma(cytoplasmic expression)

Malignant melanoma(cytoplasmic expression)

Neuro-ectodermal tumours(especially neuroblastoma) andsmall cell carcinomas from lungand other sources (nuclear andcytoplasmic staining)

Primitive neuro-ectodermaltumours, especially neuro-blastoma (cytoplasmicexpression)

Primitive neuro-ectodermaltumours, especially neuro-blastoma (cytoplasmicexpression)

Antibody

CAM5.2, AE1,MNF116

AE3

E29

Polyclonal antiseraand 85A12

ER-PR8, PSA 28/A4

PASE/4LJ

DAK-Tg6 1D4

Polyclonal antiseraand CAL-3-F5

1D5

1A6

D07

Polyclonal antisera

HMB-45

Melan A

Polyclonal antisera

NB84

NeuN

Antigen

Low molecular weightcytokeratins

High molecular weightcytokeratins

Epithelial membraneantigen

Carcino-embryonicantigen (epitopesinclude CD66)

Prostate-specificantigen

Prostatic acidphosphatase

Thyroglobulin

Calcitonin

Oestrogen receptor

Progesterone receptor

P53

S100 protein

Melanosome matrixprotein gp100-cl

Melanoma-associatedMART-1 geneproduct

Protein gene product9.5 (PGP9.5)aubiquitin related

Uncharacterized

Uncharacterized

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Table 10.1 (cont’d)

Antigen Antibody Specificity Comments

Some normal T lymphocytes andcells in many cases of ALL are alsopositive

Cytoplasmic staining is found inmost neuroblastomas andoccasional cases of Ewing’ssarcoma/PNET. Consistent goodperformance with the currentlyavailable McAb is difficult toachieve in fixed tissue

Megakaryocytes are also positive

Some non-endothelial spindle celltumours, normal and neoplasticearly haemopoietic cells are alsopositive

Macrophages, monocytes,megakaryocytes and plasma cellsare also positive

Megakaryocytes, erythroid cells andsome epithelial cell types are alsopositive

Primitive neuro-ectodermaltumours, especially Ewing’ssarcoma (membraneexpression)

Rhabdomyosarcoma (cytoplasmicexpression)

Rhabdomyosarcoma (nuclearexpression)

Rhabdomyosarcoma (nuclearexpression)

Rhabdomyosarcoma (cytoplasmicexpression)

Endothelium and tumours ofendothelial origin (cytoplasmicexpression)




12E7, HO36-1.1

D33

5.8A

F5D

MG-1 andpolyclonalantisera

F8/86 andpolyclonalantisera

QBEnd10

JC70a

Ulex europaeusagglutinin-1(the lectin is usedas an antibodyequivalent)

CD99 (MIC2)

Desmin

MyoD1

Myogenin

Myoglobin

von Willebrand factor(previously known asFactor VIII-relatedantigen)

CD34

CD31

a-L-fucosyl residues,including thoseexpressed on bloodgroup H coreglycoprotein

Fig. 10.1 BM aspirate, carcinomaof prostate, showing carcinomacells and an osteoclast. MGG ×377.


appropriate monoclonal antibodies such as thosereactive with cytokeratins, carcino-embryonic anti-gen, human milk-fat globulin and epithelial mem-brane antigen (EMA) [15,16]. Positive reactionswith such antibodies allow single neoplastic cells tobe identified with more confidence.

Malignant cells are usually considerably largerthan any haemopoietic cells other than megakary-ocytes. However, in the small cell tumours of child-hood, malignant cells may be similar in size to blastcells and acute leukaemia then enters into the dif-ferential diagnosis. Malignant cells are commonlycohesive and therefore occur as tight clumps with orwithout dispersed cells. Sometimes only irregularlydistributed, dispersed cells are present. Neoplasticcells are usually pleomorphic with regard to size,shape and nuclear characteristics. Cell outlines maybe indistinct or cells may appear smudged. Somecells may be multinucleated. The nuclei are oftenhyperchromatic and may contain nucleoli. Mitoticfigures may be numerous. Carcinoma cells usuallyhave moderately abundant cytoplasm which showsa variable degree of basophilia and may contain vacuoles; they are sometimes phagocytic. In thesmall cell tumours of childhood, cytoplasm may be scanty, thus increasing the resemblance toleukaemic blast cells, and sometimes, because oftheir marked fragility, the cells are represented onlyby single or clustered bare nuclei. It should also be noted that neuroblastoma cells are positive for α-naphthyl acetate esterase activity [17]. However,

they do not resemble cells of the monocyte lineagecytologically and they lack α-naphthyl butyrateesterase activity.

It is not usually possible to predict the tissue oforigin from the cytological features of neoplasticcells in films of bone marrow aspirates. In view ofthis, it is important also to examine histological sec-tions of marrow particles, particularly if a trephinebiopsy has not been performed. Sections may showfeatures, such as gland formation, which are helpfulin suggesting the tissue of origin. In a small percent-age of cases, cytological features in aspirate filmsmay suggest the tissue of origin. Melanoma cellsmay be recognized by the presence of pigment (Fig. 10.2), the nature of which can be confirmed byspecific stains (see below). Such stains may be posi-tive even when no pigment is detected in routinelystained films but otherwise the cells of amelanoticmelanoma cannot be distinguished from other neoplastic cells. Melanin may also be present inmacrophages. Clear cell carcinomas are distinctiveand suggest a renal primary; the cells have a rel-atively small nucleus and abundant, very weaklybasophilic cytoplasm (Fig. 10.3). Cells of metastaticcarcinoid tumour also have a relatively small nucleusand moderately abundant cytoplasm (Fig. 10.4). Inchildren, neuroblastoma (Fig. 10.5) may sometimesbe identified by the presence of extracellular blue–grey fibrillar material or by the presence of cellswith irregular ‘tails’; rosettes of tumour cells are distinctive and are found in up to two thirds of

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Fig. 10.2 BM aspirate, malignantmelanoma, showing melanomacells containing melanin. MGG×940. (By courtesy of Dr J Luckit,London.)

Fig. 10.3 BM aspirate, carcinomaof kidney, showing ‘clear cells’ with voluminous pale cytoplasm.MGG ×377. (By courtesy of Dr D Gill, Brisbane.)

Fig. 10.4 BM aspirate, carcinoidtumour, showing cells withrelatively small nuclei and avariable amount of cytoplasm.MGG ×377.

Fig. 10.5 BM aspirate,neuroblastoma, showing neoplasticcells which are relatively small andhave a high nucleocytoplasmicratio and a diffuse chromatinpattern. Neurofibrillary bundles areapparent. MGG ×376.


patients [18]. Rosettes are uncommon in othersmall cell tumours of childhood but small numbersmay be seen in Ewing’s sarcoma [18] and otherPNET. In metastatic rhabdomyosarcoma there maybe multinucleated giant cells or spindle-shaped binucleated rhabdomyoblasts [4]. The cytoplasm is often vacuolated and large vacuoles may coalesceto form lakes [19] (Fig. 10.6). Such cells are periodicacid–Schiff (PAS)-positive. In some cases, some of the tumour cells are phagocytic. Less specificchanges, such as foamy or vacuolated cytoplasm ordisplacement of nuclei by cytoplasmic mucin, may

be noted in metastatic adenocarcinoma originatingfrom various primary sites (Fig. 10.7). In squamouscell carcinoma, metastatic tumour cells have some-times been noted, with Romanowsky stains, to havea reddish cytoplasmic margin; the cytoplasm adja-cent to the nucleus is more basophilic [20]. In smallcell carcinoma of the lung, the neoplastic cells areusually smaller than those of most carcinomas butare nevertheless still larger than haemopoieticblasts. They have scanty, weakly basophilic cyto-plasm and nuclei with coarse chromatin and incon-spicuous nucleoli. The nuclei may appear to be bare

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Fig. 10.6 BM aspirate,rhabdomyosarcoma, showingcoalescing vacuoles. MGG ×940.

Fig. 10.7 BM aspirate, carcinomaof breast, showing adenocarcinomacells with secretory globules. MGG ×377.


and ‘moulded’ by the nuclei of adjacent tumourcells (Fig. 10.8).

Non-haemopoietic neoplastic cells in a bone mar-row aspirate must be distinguished from lymphomacells, blast cells of acute leukaemia and the neoplas-tic cells of Langerhans cell histiocytosis or systemicmastocytosis. Other cells which are sometimes con-fused with malignant cells include: (i) osteoblasts;(ii) osteoclasts; (iii) stromal fibroblasts; (iv) endo-thelial cells; (v) atypical megakaryocytes; and (vi)crushed erythroblasts.

When the bone marrow is infiltrated by malig-nant cells there may be associated reactive changesincluding increased plasma cells or mast cells, granu-locytic or megakaryocytic hyperplasia, increasedmacrophages and increased storage iron. Gelatinousdegeneration is rare but may be seen in severelycachectic patients.

Immunocytochemistry

Immunocytochemistry can be useful both toconfirm the presence of carcinoma cells in a bonemarrow aspirate and to detect infrequent cells (Fig. 10.9). A number of studies have been under-taken to assess the value and reliability of immuno-cytochemistry for epithelial antigens as a means ofassessing the extent of bone marrow involvementby metastatic carcinoma (see page 455).

Cytogenetics and molecular genetics

Cytogenetic analysis may be useful in suggestingthe non-haemopoietic nature of malignant cellsinfiltrating bone marrow and in confirming thespecific diagnosis in small cell tumours of child-hood. For example, t(2;13)(q35;q14) can be demon-strated in many cases of rhabdomyosarcoma [21]and the variant translocation, t(1;13)(p36;q14)[22], in a minority. In neuroblastoma, +7 and 17q+ are typical, the latter often resulting from anunbalanced translocation with chromosome 1 inwhich 1p is lost [23]. Ewing’s sarcoma and otherPNET may be associated with t(11;22)(q24;q12);examples of Ewing’s sarcoma have also been foundwith t(7;22)(p22;q12) and t(21;22)(q22;q12). Re-current cytogenetic abnormalities have also beenreported in several types of adult sarcomas but wehave not yet seen examples of these involving bonemarrow.

Molecular genetic analysis in Ewing’s sarcomashows rearrangement of the EWS gene at 22q12. Inalveolar rhabdomyosarcoma, the PAX3 and PAX7genes at 2q25 and 1p36, respectively, are rear-ranged resulting in formation of PAX3-FKHR andPAX7-FKHR fusion genes [22]. Neuroblastoma maybe associated with N-MYC oncogene amplification;when present, such amplification is associated withan adverse prognosis.

Fig. 10.8 BM aspirate, small cellcarcinoma of lung, showingcarcinoma cells with scantycytoplasm and ‘moulding’ of cellsby adjacent cells. MGG ×940.

Bone marrow histology

Marrow infiltration by metastatic tumour may befocal or diffuse. Reticulin and collagen fibrosis arecommonly present. Marked fibrosis is most fre-quent in carcinomas of the breast and prostategland but is also found relatively commonly inmetastases from cancers of the stomach and lung[9,24,25]. The proportion of tumour cells to stromais variable and, in cases with severe myelofibrosis,there may be associated osteosclerosis (Figs 10.10and 10.11). Failure to recognize tumour cells withinthe fibrous stroma can result in a mistaken diag-nosis of idiopathic myelofibrosis. The degree of

differentiation of metastatic tumour is very variable and it is often impossible to be certain of the site of the primary tumour on purely morphologicalgrounds. Frequently, metastases are undifferentiatedand the differential diagnosis includes poorly differ-entiated carcinoma, high grade NHL and malignantmelanoma (Figs 10.12–10.14); immunohistochem-istry is invaluable (see below) [26]. In undifferenti-ated or poorly differentiated carcinomas it is notusually possible to determine the site of origin of thetumour. In tumours showing differentiation it maybe possible to determine the type of carcinoma and suggest the likely site of originafor example, inmetastatic squamous carcinoma, lung is the most

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Fig. 10.9 BM aspirate, metastaticcarcinoma of breast. (a) MGG ×940.(b) Immunoperoxidase with anti-cytokeratin antibody ×940.

(a)

(b)

Fig. 10.10 BM trephine biopsysection, carcinoma of the breast,showing osteosclerosis andreplacement of the marrow bydense fibrous tissue containingtumour cells. Paraffin-embedded,H&E ×39.

Fig. 10.11 BM trephine biopsysection, carcinoma of the breast(same case as in Fig. 10.10),showing a group of tumour cellswith hyperchromatic nuclei andvacuolated cytoplasm. Paraffin-embedded, H&E ×390.

Fig. 10.12 BM trephine biopsysection, poorly differentiatedprostatic carcinoma, showingosteosclerosis and replacement ofthe marrow by dense fibrous tissuecontaining tumour cells. Paraffin-embedded, H&E ×97.


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Fig. 10.13 BM trephine biopsysection, poorly differentiatedprostatic carcinoma, showingexpression of cytokeratin bytumour cells (same case as in Fig. 10.12). Paraffin-embedded,peroxidase–antiperoxidase, anti-cytokeratin monoclonal antibody×97.

Fig. 10.14 BM trephine biopsysection (as for Fig. 10.13), ×390.

likely primary site. Squamous differentiation is recognized by the formation of keratin and the pres-ence of intercellular bridges. A mixed pattern of dif-ferentiation, with squamous cell carcinoma, smallcell carcinoma and adenocarcinoma in any combi-nation, is highly suggestive of origin from the lung.

Metastatic adenocarcinoma (Fig. 10.15) can bediagnosed on the basis of the formation of glands,the presence of signet ring cells and/or the presenceof mucin (best detected using a combined diastase-treated PAS–alcian blue stain). A mucin stain facil-itates the detection of small numbers of carcinoma

cells which may be difficult to detect when they are present as an interstitial infiltrate (Fig. 10.16).Some adenocarcinomas produce large amounts ofextracellular mucin, also detectable with a mucinstain (Fig. 10.17). It should be noted that, veryrarely, signet ring cells occur in lymphomas [27].Metastatic adenocarcinoma may arise from primarytumours in the gastro-intestinal tract, breast,prostate gland, ovary, endometrium, pancreas andmany other sites. The two primary sites whoseidentification is most important because of theirsensitivity to hormonal therapy are breast and

Fig. 10.15 BM trephine biopsysection, well-differentiatedprostatic carcinoma, showing atumour composed of small, well-defined glandular structures.Plastic-embedded, H&E ×97.

Fig. 10.16 BM trephine biopsysections, adenocarcinoma, showinginterstitial infiltrate. (a) H&E ×370.(b) Alcian blue stain ×370. (Bycourtesy of Dr S Wright, London.)

(a)

(b)


Fig. 10.17 BM trephine biopsysections, adenocarcinoma, showingintracellular mucin and abundantextracellular mucin. (a) H&E ×370.(b) PAS stain ×370.

(a)

(b)

Fig. 10.18 BM trephine biopsysection, prostatic adenocarcinoma,showing microglandular patternwith extensive new boneformation. Paraffin-embedded,H&E ×188.

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prostate gland. Prostatic origin is suggested by acribriform, microglandular pattern associated withfibrosis and new bone formation (Fig. 10.18).Occasionally, a macroglandular pattern is present(Fig. 10.19), also usually accompanied by fibrosisand neo-osteogenesis. Identification of breast carci-noma is usually based on its morphological resem-blance to primary breast cancer, including ductformation and, particularly in lobular carcinoma,the presence of cell columns forming ‘Indian file’patterns (Fig. 10.20). Intracytoplasmic lumina aresometimes visible within individual breast carci-noma cells and these can be highlighted by com-

bined alcian blue–PAS staining (Fig. 10.21). Most,but not all, bone marrow metastases from breastcancer are associated with fibrosis and new boneformation. Immunohistochemical staining is notusually helpful in confirming the primary site ofsuspected metastatic breast carcinoma. Becausemetastatic lobular carcinoma of the breast can pro-duce an interstitial infiltrate with little cellular reac-tion, its detection can be difficult. Routine use ofimmunohistochemistry when a biopsy is carried outfor staging purposes has therefore been advised [28].Adenocarcinoma composed predominantly of rib-bons or villous formations of columnar epithelium

Fig. 10.19 BM trephine biopsysection, macroglandular pattern ofprostatic adenocarcinoma. Paraffin-embedded, H&E ×188.

Fig. 10.20 BM trephine biopsysection, carcinoma of the breast,showing tumour cells withhyperchromatic nuclei arranged incords and strands in a dense fibrous(desmoplastic) stroma. Paraffin-embedded, H&E ×390.

with intracellular mucin (goblet cells) is usually oflarge bowel origin (Fig. 10.22). Clear cell carcino-mas have large amounts of pale cytoplasm due tothe presence of abundant glycogen or lipid; mucinstains are negative. Likely primary sites of meta-static clear cell carcinoma include kidney, ovary andlung. In rare cases when metastatic follicular carcinoma of the thyroid gland is present, it may besuspected on morphological grounds if follicles con-taining colloid are seen.

Metastatic small cell carcinoma of the lung com-monly involves the bone marrow (see below). The

cells are usually small with intensely hyperchro-matic, round or oval nuclei and scant cytoplasm(Fig. 10.23). Necrosis is common and there is oftensmearing of nuclei which can render interpreta-tion difficult. Morphological variants of small cellcarcinoma also occur, in which the cells are slightly larger and have either a fusiform or poly-gonal shape. The principal differential diagnosis of metastatic small cell carcinoma is that of NHL.Metastases from other tumours with neuro-endocrine differentiation, such as malignant carci-noid tumours, may also occasionally spread to bone

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Fig. 10.21 BM trephine biopsysection, lobular carcinoma ofbreast, showing intracytoplasmiclumen formation. Alcian blue-positive material is present at theperiphery of what appears to be a cytoplasmic vacuole and PAS-positive material is present withinthe centre. Electron microscopy has shown that these structures inlobular breast carcinoma representtrue lumina, formed withinindividual cells, rather than simplesecretory vacuoles. Paraffin-embedded, combined alcian blue–PAS stain ×960.

Fig. 10.22 BM trephine biopsysection, metastatic adenocarcinomafrom a colonic primary tumour,showing a glandular and villouspattern. Paraffin-embedded, H&E ×376.


marrow; these may have distinctive morphology,with nests and ribbons of monomorphic cells hav-ing nuclei with dense, evenly distributed chromatin(Fig. 10.24).

Malignant melanoma is found in the bone marrow in approximately 5% of patients with disseminated disease [29]. If melanin is present inthe tumour cells (Fig. 10.25) or associated macro-phages, the diagnosis is relatively easy although, if the patient is not already known to havemelanoma, the nature of any pigment presentshould be confirmed by either a Masson–Fontana or

a Schmorl stain for melanin. However, not infre-quently, metastatic malignant melanoma is ame-lanotic (Fig. 10.26) and immunohistochemistryshould then be considered for confirmation.Malignant melanoma should be suspected ifmetastatic tumour is composed of polygonal orspindle cells with prominent nucleoli.

The differential diagnosis of metastatic spindlecell tumour within the marrow includes carcinomashowing spindle cell differentiation, malignantmelanoma and various sarcomas. Sarcomas rarelymetastasize to the marrow and, when they do, the

Fig. 10.23 BM trephine biopsysection, small cell carcinoma of thebronchus, showing a sheet of smallcells with hyperchromatic ovoidnuclei and scanty cytoplasm; inplaces there is nuclear ‘moulding’.Plastic-embedded, H&E ×390.

Fig. 10.24 BM trephine biopsysection, carcinoid tumour(unknown primary site), showingrelatively uniform cells with ovoidhyperchromatic nuclei andeosinophilic cytoplasm; in someareas the cells are arranged intrabeculae. Plastic-embedded, H&E ×390.

primary tumour is usually readily apparent.Occasionally, Kaposi’s sarcoma and other angiosar-comas may present in the bone marrow or may besampled when bone marrow is investigated for rea-sons other than staging in patients (e.g. individualswith AIDS) who have known primary tumourselsewhere. Bone marrow involvement by Kaposi’ssarcoma is rare but has been observed both in AIDS[30] and in occasional HIV-negative patients [31].Genomic sequences of the human herpesvirus 8(HHV8) have been demonstrated in many cases ofKaposi’s sarcoma and this virus is believed to be

involved in the pathogenesis of the neoplasm [32]. The bone marrow may be extensively replacedby abnormal tissue composed of slit-like vascularchannels lined by spindle-shaped endothelial cells(Fig. 10.27). Large, plump nuclei of neoplasticendothelial cells protrude into the abnormal vascu-lar channels, some of which are engorged with erythrocytes. Haemosiderin-laden macrophages areincreased. Angiosarcomas, including Kaposi’s sar-coma, show widely varying degrees of vascular dif-ferentiation and may form reticular or solid spindlecell areas (Fig. 10.28). Immunohistochemistry to

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Fig. 10.25 BM trephine biopsysection, malignant melanoma,showing a focal tumour infiltratecomposed of cells containing largeamounts of melanin; the tumourcells have hyperchromatic nuclei,and some have prominent nucleoli.(It should be noted that it isunusual to see large amounts ofmelanin in metastatic malignantmelanoma.) Paraffin-embedded,H&E ×390.

Fig. 10.26 BM trephine biopsysection, amelanotic melanoma,composed of fairly bland epithelioidcells. Immunohistochemistry wasrequired to confirm the true natureof this tumour; cells expressed S100protein and the antigen recognizedby HMB-45. Paraffin-embedded,H&E ×376.


demonstrate expression of endothelial antigensmay be helpful in cases lacking obvious vessel for-mation. However, it should be noted that one caseof Kaposi’s sarcoma has been reported which lackedexpression of von Willebrand factor although therewas positive staining with Ulex europaeus lectin [31].

Many of the malignant tumours that occur inchildhood are composed of small cells with rela-tively uniform round nuclei. The differential diag-nosis of bone marrow infiltration by such cells in a child includes NHL (usually lymphoblastic orBurkitt’s lymphoma), metastatic neuroblastoma,rhabdomyosarcoma, Ewing’s sarcoma, other PNET

and retinoblastoma. In order to make a specificdiagnosis, the clinical features, morphologicalfindings and histochemical and immunohistochem-ical staining characteristics all need to be consid-ered. The marrow findings in lymphoblastic andBurkitt’s lymphoma are described on pages 239 and282; both express CD45 and B-cell immunopheno-typic markers. Neuroblastoma is the most commonmalignant solid tumour in children and oftenmetastasizes to the bone marrow. The majority ofcases occur in children under 4 years of age. Theneoplastic cells are slightly larger than small lym-phocytes with regular, round hyperchromatic nuclei

Fig. 10.27 BM trephine biopsysections, showing Kaposi’s sarcomain a patient who did not have AIDS.Paraffin-embedded, H&E. (a) ×240.(b) ×960. (By courtesy of Dr RMConran and Dr VB Reddy, Aurora,Colorado.)

(a)

(b)

Fig. 10.28 BM trephine biopsysections, metastatic Thorotrast-induced angiosarcoma, showingrefractile Thorotrast. Paraffin-embedded, H&E: (a) ×97; (b) ×390;(c) ×960.

(a)

(b)

(c)

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and little cytoplasm [33] (Fig. 10.29). Rosettes arepresent in a minority of cases, consisting of tumourcells arranged around central fibrillary materialwhich is pink in H&E-stained sections (Fig. 10.30).The cells of neuroblastoma may show focal PASpositivity but this is usually less marked than thatseen in rhabdomyosarcoma and Ewing’s sarcoma.In all of these neoplasms, PAS positivity is difficultto detect in fixed tissue sections; it should be soughtby staining of aspirate films, as described above.

In one series, rhabdomyosarcoma was found to have metastasized to the bone marrow in 16%

of cases [1]. Several histological variants are recognized:1 embryonal, which may be further subdividedinto myxoid, spindle cell or round cell patterns [34],with the round cell subtype being represented mostcommonly in bone marrow metastases; 2 alveolar, characterized by a pattern of irregularspaces lined by tumour cells [35]; and 3 pleomorphic, which is very rare and not usuallyseen in children.

In any of these subtypes there may be a few mul-tinucleated rhabdoid cells with peripheral nuclei. A

Fig. 10.29 BM trephine biopsysection, neuroblastoma, showingextensive, diffuse infiltration bysmall cells with scanty cytoplasm.Paraffin-embedded, H&E ×376.

Fig. 10.30 BM trephine biopsysection, neuroblastoma, withrosette formation. Note thecollections of pink material aroundwhich neuroblastoma cells areclustered. Paraffin-embedded, H&E×376.

pseudo-alveolar pattern may be produced by cellsadhering to the margins of vascular channels [19].Erythrophagocytosis may be seen [36]. Diagnosisdepends on the recognition of skeletal muscle dif-ferentiation. Rhabdomyoblasts may be oval, spin-dle, tadpole or strap-shaped. They have abundantpink granular cytoplasm which may show cross-striations (Fig. 10.31). The number of rhabdomy-oblasts present is highly variable; in many patientsthe majority of cells are undifferentiated round orspindle cells. Ewing’s sarcoma and related PNET aremalignant tumours that may arise in bone or softtissue. Most patients are in the second decade of life

and approximately 35% of cases develop bone mar-row metastases [1]. The tumour cells are approxi-mately twice the size of small lymphocytes and haveround to oval vesicular nuclei (Fig. 10.32). Theymay show PAS-positive cytoplasmic staining forglycogen, either finely dispersed or forming largeblocks of positively stained material (Fig. 10.33).Haemophagocytosis by malignant cells has beenreported in rhabdomyosarcoma but is not a specificfeature; it also occurs rarely in other neoplasms,including small cell carcinoma, medulloblastoma,breast carcinoma and haemangio-endothelioma[37,38].

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Fig. 10.31 BM trephine biopsysection, rhabdomyosarcoma,showing elongated cells withplentiful eosinophilic cytoplasm(rhabdomyoblasts). Plastic-embedded, H&E ×390.

Fig. 10.32 BM trephine biopsysection, Ewing’s sarcoma, showingirregular groups of cells in a fibrousstroma; the cells have ovoid nucleiwith indistinct nucleoli and scantycytoplasm. Plastic-embedded, H&E ×390.


Immunohistochemistry

In bone marrow trephine biopsies containingmetastatic poorly differentiated tumours, immuno-histochemistry with a small panel of antibodies isuseful to demonstrate lymphoid antigens (CD45,CD20 and CD3), epithelial markers (cytokeratinsand EMA) and melanoma-associated antigens(S100 protein and the antigens recognized by anti-bodies HMB-45 and Melan A) [15,26,39,40].

The prostatic origin of a metastatic adenocarci-noma may be confirmed by immunohistochemicalstaining with antibodies that react with prostate-specific antigen [41]. Since these antibodies mayalso react with some colonic tumours [42], the useof parallel immunostaining for prostate-specific acidphosphatase is recommended. Use of both antibod-ies considerably improves sensitivity and specificityin confirming the prostatic origin of metastatic can-cer. Unfortunately, there are no antigens withequivalent tissue specificity for breast carcinoma. Of the markers available, α-lactalbumin (which isexpressed in most primary breast cancers) has beenclaimed to be relatively specific [43] but is not widelyused. Nuclear expression of oestrogen and proges-terone receptors may be demonstrated immunohis-tochemically (Fig. 10.34) but these antigens are alsoexpressed in a variety of other adenocarcinomas,particularly those arising in the uterus or ovary. Itshould be noted that human milk-fat globulin,

despite its name, is another antigen that is expressedby many adenocarcinomas and has no specificity for those of breast origin. Immunohistochemicalstaining for thyroglobulin is useful in confirmingthe thyroid origin of metastatic tumour cells.

Small cell carcinoma reacts positively with anti-bodies directed against protein gene product 9.5(PGP9.5), an antigen expressed by neuro-ectodermal cells, but it should be noted that poly-clonal anti-PGP9.5 antisera cross-react with somelymphomas [44]. Small cell carcinomas also oftenexpress various sizes of cytokeratin filaments thatcan be detected by their distinctive intracytoplasmicdot-like or perinuclear ring patterns of immunohis-tochemical staining. Other tumours showing neuro-endocrine differentiation, such as carcinoid tumoursand medullary carcinoma of the thyroid gland, maymetastasize to the bone marrow; immunohisto-chemical staining for chromogranin or for PGP9.5identifies neuro-endocrine differentiation in thesetumours [45,46]. In addition, medullary carcino-mas of the thyroid may express calcitonin.

In cases of metastatic melanoma, immuno-histochemical staining for S100 protein is usuallypositive [26]. Newer antibodies such as HMB-45and Melan A also react well with metastatic malig-nant melanoma in bone marrow trephine biopsysections. Spindle cell carcinomas and melanomasexpress cytokeratins and S100 protein (or othermelanoma-associated antigens), respectively. Use

Fig. 10.33 BM trephine biopsysection, Ewing’s sarcoma, showinglarge granules of glycogen in thecytoplasm. Plastic-embedded, PAS ×390.

of vimentin to distinguish such spindle cell tumoursfrom true sarcomas is unreliable, since many non-sarcomatous tumours with spindle cell morphologyexpress vimentin; apart from variants of angiosar-coma, however, metastatic sarcomas are exception-ally rarely seen in bone marrow biopsies. In difficultcases of angiosarcoma or Kaposi’s sarcoma, theendothelial origin of malignant cells can be demon-strated by immunohistochemical staining for vonWillebrand factor, CD31 and CD34. The Ulex euro-paeus lectin can also be used to stain endothelium.

Neuroblastoma cells usually express neurone-specific enolase and PGP9.5; less consistently, chro-mogranin (Fig. 10.35) and the antigens detected by

antibodies NB84 and NeuN are expressed. In rhab-domyosarcomas, immunohistochemical stainingfor desmin, myogenin and MyoD is usually posi-tive [47] although staining for myoglobin, which issaid to be more specific, is variable [34]. Ewing’ssarcomas with t(11;22)(q24;q12) express the MIC2gene product, CD99 [48,49], but so do some ALL.

The role of bone marrow examination in thestaging of solid tumours

Examination of the bone marrow by aspiration andtrephine biopsy is an established part of the stagingof neuroblastoma in children in most large centres.

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Fig. 10.34 BM trephine biopsysections, interstitial infiltration by oestrogen receptor-positivebreast carcinoma. (a) H&E; (b) immunostained to shownuclear expression of oestrogenreceptors. Paraffin embedded,×390.

(a)

(b)


The bone marrow biopsy is positive at the time ofinitial diagnosis in approximately half of all pati-ents, most of whom have evidence of metastaticspread at other sites [50]. Discordance betweenmarrow aspirate and trephine biopsy findings iscommon. In one reported series, the trephinebiopsy alone was positive in 20% of cases whereasneoplastic cells were seen in the aspirate films when trephine biopsy sections appeared normal in7%. Taking bilateral bone marrow aspirates andtrephine biopsies from the iliac crests increases thesensitivity of the staging procedure by approxi-mately 10%. Particularly careful examination of aspi-rate films and trephine biopsy sections is necessarybecause infiltration is often extremely focal [51,52].In cases in which the marrow appearances are suspicious but not diagnostic of infiltration, im-munocytochemical staining of aspirate films usingantibodies reactive with neuro-ectodermal anti-gens (UJ13A and UJ127.11) may be of value in confirming marrow involvement [53,54]. Immu-nohistochemical staining of paraffin-embedded sections of trephine biopsies for neurone-specificenolase has not been found to increase the sensi-tivity of marrow biopsy as a means of detectingtumour cells [46]. Detection of PGP9.5 expression is considerably more reliable and should be per-formed in all cases; its use permits demonstration oftiny clusters and even single cells in patients withminimal bone marrow involvement (Fig. 10.36).

The additional utility of immunohistochemicalstaining with newer antibodies, such as NB84[55,56] and NeuN [57], has yet to be established.Assessment of marrow infiltration by neuroblas-toma is more difficult in patients who have beentreated with chemotherapy; it is essential thattrephine biopsies are of adequate size and quality[58–60]. It has been suggested that the appearancesin post-treatment marrow trephine biopsy sectionsshould be divided into four grades: 1 grade 1, normal (or hypocellular) marrow;2 grade 2, marrow with reticulin fibrosis as the onlyabnormality;3 grade 3, distorted architecture with collagenfibrosis; and4 grade 4, marrow with obvious tumour cells, withor without other abnormalities [33].

Grades 2–4 were all considered by Reid andHamilton [33] to be compatible with continuedbone marrow involvement by tumour, even whenindividual neoplastic cells could not be identifiedwith certainty. It remains unclear whether this isreally true for grades 2 and 3 [61] and whether thedifferent grades correlate with differences in clinicaloutcome. An important additional feature to recog-nize in follow-up bone marrow samples obtainedduring treatment of neuroblastoma is differentia-tion of the primitive small cells to produce large,ganglion-like cells and areas of pink, fibrillary tissueresembling neuropil (Fig. 10.37). Ganglion-like

Fig. 10.35 Bone marrow trephinebiopsy section, neuroblastoma.Immunoperoxidase forchromogranin ×390. (By courtesyof Professor D Evans, London.)

cells may show superficial resemblance to mega-karyocytes and the neuropil-like tissue requires dif-ferentiation from fibrous tissue. Immunostainingfor PGP9.5 is positive in both components of differ-entiated neuroblastoma tissue and is very helpful inconfirming their true nature.

In adults, examination of the bone marrow is nota routine staging procedure for most solid tumoursand, although it has been advocated for small celllung carcinoma and breast cancer, it is by no meanspractised universally, even for these tumours. Insmall cell carcinoma of the lung, bone marrowtrephine biopsy is positive in 25–30% of cases[10,62,63]; the aspirate is only slightly less sensitivefor detecting marrow involvement. It has been

suggested that bone marrow examination is indi-cated in patients with small cell lung carcinoma inorder to identify patients who may be suitable forattempted curative therapy [64]. However, somestudies have shown no difference in survival be-tween patients with and without marrow in-volvement [10,64], and the value of routine bonemarrow examination has therefore been ques-tioned [10]. Imaging techniques such as magneticresonance imaging have been proposed as moresensitive alternative staging procedures [65].

Several studies have evaluated the use of marrowaspiration and trephine biopsy in the staging ofbreast cancer. Detection of subclinical metastaticdisease may be useful in identifying patients with

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Fig. 10.36 BM trephine biopsysections, minimal infiltration byneuroblastoma shown using (a) H&E and (b) immuno-histochemistry for PGP9.5. Paraffin-embedded, ×376.

(a)

(b)


apparently localized disease who might benefitfrom adjuvant chemotherapy. Bone marrow biopsyis positive in 25–55% of patients with positiveradio-isotope bone imaging, but only 4–10% ofpatients with negative radio-isotopic imaging havetumours detected by bone marrow biopsy [8,66]. Inone reported series, 23% of all breast cancer pati-ents had positive bone marrow biopsies at the timeof first recurrence [66]. In an attempt to increasethe sensitivity of bone marrow examination as astaging procedure, some studies have used im-munocytochemical staining to identify micrometas-tases that would not be detected by conventionaltechniques. The method used has involved aspirat-ing bone marrow from multiple (up to eight) sitesunder general anaesthesia at the time of initialsurgery, pooling the material and preparing severalfilms. Tumour cells are then detected by immuno-staining with antibodies reactive with EMA [67,68],cytokeratins [69] or a cocktail of antibodies recog-nizing these antigens [70]. Using this approach,micrometastases have been found in 27–35% ofcases at the time of diagnosis and correlation hasbeen shown between their presence and the size of the primary tumour [67,68]. The presence ofbone marrow micrometastases is a predictor of earlyrelapse in bone. Despite these findings, applicationof immunocytochemistry to detect micrometastasesin bone marrow aspirates performed for staging ofbreast cancer has not yet become standard prac-tice. A similar approach has been suggested for:

(i) detection of oral, oesophageal and gastric cancers[71–74]; (ii) pancreatic cancer [75,76]; (iii) non-small cell lung cancer [77]; (iv) urological cancers[78]; and (v) malignant melanoma [79]. Use ofreverse transcriptase polymerase chain reaction(RT-PCR) directed at carcino-embryonic antigenmRNA to detect occult involvement of aspiratedbone marrow by carcinomas arising from breast orcolon [80,81], and directed at cytokeratin mRNA in cases of breast cancer [82], has also proved suc-cessful in initial studies. The clinical value of detect-ing occult bone marrow micrometastases in suchtumours by these techniques remains unproven[83]. In all cases, including breast carcinoma detec-tion, technical problems with the immunocyto-chemical approach, including false-positive resultsdue to skin contamination or non-specific haemo-poietic cell reactivity, limit its applicability in rou-tine practice [84,85].

Problems and pitfalls

Normal components of bone marrow may be mistaken for malignant non-haemopoietic cells inaspirate films and biopsy sections. These includemegakaryocytes, crushed erythroid cells, osteo-blasts (Fig. 10.38), osteoclasts, stromal macro-phages, endothelium and fibroblasts. Awareness ofthe appearances of stromal components in aspiratefilms is particularly important in avoiding confusionwith malignant cells.

Fig. 10.37 BM trephine biopsysection, post-treatment follow-upfor neuroblastoma. Cellular‘fibrous’ tissue remains and thecytoplasm of at least one largeganglion-like cell can be seen.Paraffin-embedded, H&E ×376.

Artefactual inclusions of extraneous tissue intrephine biopsy specimens (e.g. skin, sweat glands,hair follicles or skeletal muscle) may mimic malig-nant non-haemopoietic cells (Fig. 10.39 and seealso Figs 1.64, 1.66 and 1.67). Care should be takento avoid such inclusions by making a small skin in-cision prior to insertion of the biopsy needle andusing disposable needles to ensure a sharp cuttingedge. Carry-over from other specimens into theparaffin block during tissue processing should beavoided by good laboratory practice with regard topreparation of small or friable biopsy specimens;such specimens should be wrapped in tissue paperor sponge, or placed in a wire mesh insert, before

processing. If carry-over is suspected, it can beconfirmed by molecular analysis of major histocom-patibility antigen genes to demonstrate the differentpatient origins of separate tissue fragments in a single wax-embedded block [86]. However, formicacid decalcification limits the quality of DNAobtainable from trephine biopsy samples and thisapproach may therefore be unsuccessful in speci-mens decalcified in this way. Extraneous tissue mayalso appear to be present in histological sections due to contamination by a ‘floater’ from another tissue block as sections are cut, floated on a water-bath and picked up on individual glass slides (seeFig. 1.70). Good laboratory practice will ensure that

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Fig. 10.38 BM aspirate, showing acluster of osteoblasts mimickingcarcinoma cells. MGG ×970.

Fig. 10.39 BM trephine biopsysection, sweat gland interpositionmimicking adenocarcinoma.Paraffin-embedded, H&E ×376.


fragments of previous sections are not allowed tocontaminate the water-bath between cases. If suchcontamination is suspected, examination of thecomplete set of stained trephine biopsy sectionsshould show that other sections are free of extrane-ous material. If doubt persists, new sections cutfrom the trephine biopsy specimen will be free ofcontamination.

Other pathological components in trephinebiopsy samples may occasionally be mistaken formalignant non-haemopoietic cells. These includemacrophages (present singly or within granulomas),lymphoid cells in some types of NHL, Reed–Sternberg cells in Hodgkin’s disease, neoplastic mastcells in systemic mastocytosis and the cells of

Langerhans cell histiocytosis. Immunohistochem-istry will establish the nature of each of these typesof cell. Macrophages are best demonstrated by theCD68 antibody PGM1 and lymphoid cells by theirexpression of CD3 (T cells) or CD20 (B cells).Reed–Sternberg cells express CD30, mast celltryptase can be demonstrated with the monoclonalantibody AA1 and cells in Langerhans cell histiocy-tosis express CD1a.

Malignant cells may be confused with normalbone marrow constituents or with haemopoieticmalignancies. Examples include undifferentiatedcarcinomas that may infiltrate bone marrow with-out any stromal reaction, clear cell carcinoma,signet ring carcinoma (Fig. 10.40), malignant

Fig. 10.40 BM trephine biopsysection, signet ring carcinoma ofunknown primary origin. (a) InH&E-stained sections, malignantcells are indistinct and resemblemacrophages. (b) Immunostainingfor low molecular weightcytokeratins confirms theirepithelial nature. Paraffin-embedded, both ×376.

(a)

(b)

melanoma and the small cell solid tumours of child-hood. Deposits of metastatic carcinoma eliciting a fibrotic response may also be confused withmyelofibrosis, Hodgkin’s disease or NHL. Amongthe NHL, those of T-cell lineage are most likely toproduce significant stromal fibrosis. Immunohis-tochemistry will assist in making a correct diagnosis.Expression of low and high molecular weightcytokeratins is present in almost all carcinomas,S100 protein in melanomas and CD45 (plus CD3 or CD20) in T- or B-cell lymphomas, respectively.Neoplastic plasma cells frequently lack CD45 andCD20 expression but their nature can be confirmedby p63 expression (detected using monoclonal anti-body VS38c) in the absence of expression of cyto-keratins; CD79a is expressed in many cases, but notall, and a negative result can therefore be mislead-ing. The diagnosis of most solid small cell tumours ofchildhood can be confirmed on the basis of expres-sion of PGP9.5 (neuroblastoma), CD99 (Ewing’ssarcoma) or desmin (rhabdomyosarcoma).

Detection of malignant non-haemopoietic cells innecrotic deposits can be very difficult. Reticulinstaining may demonstrate a preserved pattern ofnested cells or gland formation despite loss of cellu-lar detail. Immunohistochemistry is often unhelpfulin necrotic tissue and can be misleading due to non-specific false-positive results, as well as loss of anti-gen expression by dead or dying cells [87].

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metastatic tumours marrow... · increasingly, bone marrow aspiration and trephine biopsy are being...

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