Leukaemia of platelet precursors: diverse features in four cases

British Joirrnnl oJHaematology, 1982, 51, 147-164

Leukaemia of platelet precursors: diverse features in four cases

DAVID BEVAN, MICHAEL ROSE A N D MELVYN GREAVES* HUematOlOgy Department, St James’ Hospital, Balham, and St George’s Hospital Medical School, London. and *Membrane Inimunology Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London

Received 15 July 1981; accepted for publication 1 7 November 1981

SUMMARY. Four patients are described with malignant cells of definite or probable megakaryocytic lineage in the bone marrow and blood. Megakaryocytic features were defined by morphological and cytochemical studies using light and electron microscopy, and in two cases by reaction with a monoclonal anti-platelet antibody, AN5 1. All patients had increased bone-marrow reticulin, which developed in less than 15 months in two cases. One of these fitted the clinical pattern of acute myelosclerosis of Lewis and Szur (now widely referred to as acute megakaryoblastic leukaemia). Three cases had unique clinical features which are described in detail. There may be several variants of megakaryocytic leukaemia, including chronic forms. One variant is associated with Down’s syndrome (trisomy 2 1).

Large mononuclear cells in the peripheral blood or bone marrow may be megakaryocytic precursors if they have abundant basophilic cytoplasm organized in buds, blunt pseudopodia or a platelet-like structure, or if they exhibit certain patterns of cytochemical reaction (Li e t al, 19 73). These light microscopic features are neither consistent within, nor specific to, the megakaryocytic lineage. Until recently this has led to under-recognition of megakaryocytic proliferations and an unwillingness to propose a separate category of megakaryocytic leukaemias. The French-American-British classification of acute leukaemias (Bennett et al, 19 76) does not include a megakaryoblastic/megakaryocytic identity despite distinguishing five separate types from the granulocytic-monocyte precursor. Where leukaemias composed of cells with convincing megakaryocytic features have been described, the authors have regarded them as rare variants of myeloblastic leukaemia (Chan et al, 1971; Habib et al, 1980).

Megakaryocytic lineage is now shown by specific platelet peroxidase activity on ultrastructural histochemistry and transmission electron microscopy in the perinuclear

Correspondence: Dr D. H. Bevan, Haematology Department, St George’s Hospital Medical School, Tooting. London, S.W.17. 000 7- 1048/8 2/0 500-0 147802 .00 @ 19 8 2 Blackwell Scientific Publications

147

148 David Bevan, Michael Ruse and Melvyn Greaves

cisternae of these cells, even where platelet-specific organelles such as a-granules and demarcation membranes are absent (Breton-Gorius & Guichard, 19 72; Breton-Gorius et al, 19 78a). Following these discoveries, circulating mononuclear cells of megakaryocytic origin have been described in acute myelosclerosis of Lewis and Szur (Breton-Gorius et al. 1973), blast crisis of chronic granulocytic leukaemia (Breton-Gorius et aZ, 1978b; Bain et al, 1977) and in acute leukaemia with blast cells otherwise indistinguishable from myeloblasts (Breton-Gorius et al, 1978a). Megakaryoblasts have also been demonstrated in congenital leukaemia in a child with Down’s syndrome (Cosson et al, 1974). Because this technique is at present restricted to few centres, and is inapplicable to aged or transported blood specimens (although frozen cells can be used), no clear idea of the prevalence of megakaryocytic proliferations can yet be formed. The addition of monoclonal antibody against megakaryocyte-platelet membrane determinants to the panel of antibodies used to provide a membrane phenotype of leukaemic blasts is now likely to provide this information. We report here four cases in which morphological features suggesting megakaryocytic lineage of the disordered cells have been confirmed by some or all of the methods available, and in two cases we have submitted circulating mononuclear cells to membrane analysis with a monoclonal anti-platelet antibody, anti-platelet glycoprotein I (McMichael et al, 198 1).

PATIENTS AND METHODS

The four patients presented to St James’ Hospital (cases 1-3) and St George’s Hospital (case 4) within a 5 year period (1975-80). This hospital group serves a population of roughly 300 000 people and is not a special referral centre for haematological malignancies.

The clinical and haematological data at presentation are shown in Table I.

Case histories

(1) E.V. This 58-year-old white housewife underwent radical surgery in February 1978 for a fungating carcinoma of the vulva. The tumour was locally invasive but had not spread to local lymph nodes and was completely excised. She recovered and had no radiotherapy or chemotherapy. Her pre-operative blood count was normal (Hb 1 5 . 1 g/dl; WBC 7.2 x 109//1, with normal differential count; platelet count normal).

Fifteen months Iater she was urgently admitted with a 2-week history of dyspnoea, epistaxis and productive cough. She was pale with widespread purpura but no lymphadeno- pathy, hepatosplenomegaly or recurrent carcinoma. She was pancytopenic and a small number of blast cells were seen on the film which had features suggestive of megakaryocytic proliferation. A bone marrow aspirate was non-particulate but contained enough blast cells to allow cytogenetic analysis and cytochemical studies. Marrow trephine biopsy contained sheets of megakaryocytic cells surrounded by dense reticulin. Megakaryoblastic leukaemia (acute myelosclerosis of Lewis and Szur) was diagnosed.

Radiographs and isotopic scanning confirmed the normal size of the spleen and liver. Skeletal radiographs showed a slight coarsening of the trabecular markings. Hepatic and renal function were normal. There was a mild hyperuricaemia (0.42 mmol/l).

Leukaernia of Platelet Precursors 149 Table I. Clinical and haematological data at presentation

Patient Age (years) Sex

E.V. P.P. R.G. A.C. 59 2 5 3 21 F M F M

Antecedent disorders

Major symptoms

Splenornegaly Haemoglobin (g/dl) MCV (f l ) Reticulocyte count (%)

White cell count ( x 109/1) Polymorphonuclear

leucocytes (%) Metamyelocytes (%) Myelocytes (%) Promyelocytes (%) Blasts (%) Monocytes (%) Lymphocytes (%) Platelets ( x 10y/I) Tear-drop poikilocytes Nucleated red cells Bone marrow, aspirate

Trephine biopsy Haematoxylin and eosin Silver stain

Ca vulva

Epistaxis; dyspnoea Absent 5.7 89 0.5 1 . 5

20 0 0 0 16 0 64 10 Nil Nil Hypocellular megakaryoblasts

Down's t hrombocytopenia Lethargy; purpura Present (2 cm) 3.6 93 0.5 6.3

40 0 0 0 10 7 4 3 1 2

Nil Nil Dry tap

Thyrotoxicosis Macrocytic anaemia

Night sweats; Dyspnoea; bruising anorexia Absent Present (3 cm) 12.1 7.0 89 106 2 0.3 16.5 6 '2

75 36 1 5 2 3 0 1 occ 24 2 4 20 27 20 1,720 Nil Nil Nil Nil Hypocellular Dry tap rnegakaryoblasts

Bizarre increased megakaryocytes Increased pericellular collagen

Red cell and platelet transfusions were given and folic acid, pyridoxine, allopurinol and tranexamic acid were commenced. Antileukaemic therapy was not given. Further supportive management was complicated by her resolute refusal to attend hospital. She was eventually re-admitted with deteriorating renal function, bleeding and infection and died whilst receiving parenteral antibiotics and platelet transfusion.

Microscopic examination of post-mortem sections of the spleen (260 g) and lymph nodes stained with haematoxylin and eosin, revealed a diffuse excess of large cells with abundant cytoplasm and multi-lobed nuclei (two to eight). There was no evidence of gross fibrosis in these tissues.

(2) P.P., a male infant with Down's syndrome, was born in July 1977. In infancy he had numerous infective episodes, including staphylococcal lymphadenitis a t age 3 weeks and

150 David Bevan, Michael Rose and Melvyn Greaves

pneumonia with radiographic consolidation at 7 months. At age 1 5 months widespread purpuric lesions were noted: he was otherwise well. A blood count showed haemoglobin 11.5 g/dl, WBC 8.3 x 109/1, with normal differential count, platelet count 28 x 109/l. Bone marrow aspiration was normocellular, with normal numbers of megakaryocytes, some with reduced nuclear segmentation. There was no evidence of incipient leukaemic change. Chromosomal analysis was not performed. Childhood idiopathic thrombocytopenia was diagnosed. The platelet count remained stable for 8 months: there were no serious bleeding manifestations and he was given no treatment.

At age 23 months he became acutely ill, with extreme lethargy and pallor. He had widespread purpura and palpable hepatosplenomegaly. He was profoundly anaemic and thrombocytopenic and blast cells could be seen on the blood film. These were large with features of megakaryocyte precursors (Fig 1). Cytochemical, ultrastructural and membrane phenotype studies were performed, and frozen cells were later studied for platelet peroxidase.

The blood blast count rose to 80 x lo9/] over a few weeks. Bone marrow aspiration yielded a dry tap; trephine biopsy showed increased reticulin surrounding abnormal megakaryocytoid cells (Figs 2 and 3). Other cell populations in the marrow were grossly subnormal.

In view of his Down's syndrome aggressive antileukaemic therapy was not contemplated. Two doses of vincristine 1 a4 mg/m2 and oral prenisolone 40 mg/m2 were given without effect on the blast count or clinical status. Maintenance therapy with 10 mg oral prednisolone daily controlled his epistaxis. He was admitted for periodic red cell and platelet infusions but otherwise remained at home.

Over the next 5 months there was a steadily progressive increase in hepatosplenomegaly. Abrupt and unpredictable swings occurred in the peripheral blood blast count: on one occasion from 99 x 109/1 to 8 x 109/1 in 24 h, with hyperuricaemia (1.46 mmol/l) and acute urinary tract obstruction with urate sludge.

Fig 1. Megakaryoblasts in blood: case 2 (P.P.) Jenner-Giemsa, x 1000.

Leukaemia of Platelet Precursors 151

Fig 2. Histology of bone marrow trephine biopsy: case 2 (P.P.). Silver stain, x 400.

Fig 3. Bone marrow trephine biopsy: case 2 (P.P.). Silver stain, x 400.

These episodes of massive leukaemic cell lysis were unassociated with any therapeutic, infective, traumatic or dietary event. Eventually there was almost complete disappearance of the megakaryoblasts from the peripheral blood, concurrent with a striking and progressive normoblastosis. Further bone-marrow studies were not undertaken in view of his age, clinical condition and prognosis. He died in January 1980 of cardiac failure, with massive splenomegaly and a peripheral WBC of 2.5 x 109/1 of which 10% were megakaryoblasts and 90% normoblastic erythroid precursors. Permission for autopsy was refused.


Fig 4. Megakaryoblast by electron microscopy displaying positive reaction for platelet peroxidase: case 2 (P.P.) x 6000. (Courtesy of Dr D. Catovsky.)

(3) R.G. This 53-year-old infant teacher was admitted to hospital in May 1976 for investigation of an obscure illness consisting of rigors and night sweats, easy bruising and paroxysmal attacks of visual and olfactory hallucinations with sensory dyaesthesia suggesting temporal lobe epilepsy. The symptoms were recent. Thirty years previously a hemithyroidectomy had been performed for Grave’s disease and recurrence in 1964 had led to the removal of the rest of the gland. She had been otherwise well. Clinical and neurological examination revealed only scattered bruises and a soft apical murmur: no splenic or hepatic enlargement was detected. Her symptoms subsided and did not recur in hospital but an abnormal blood count was detected.

Careful examination of the peripheral blood smears identified extremely small numbers of mononuclear cells which had morphological and cytochemical features of megakaryoblasts and micromegakaryocytes. A small amount of bloody fluid was aspirated from the bone marrow which contained megakaryocytes and blast-like mononuclear cells but few erythroid or granulocyte precursors. Trephine biopsy showed that the marrow had become replaced with sheets of megakaryocytic cells and increased reticulin. Megakaryocytic leukaemia was diagnosed.

She had minimal EEG evidence of left-sided temporal lobe epilepsy. Computerized axial tomography of the skull was normal. She experienced further seizures after discharge from hospital which were abolished by phenytoin 100 mg t.d.s. Other symptoms disappeared apart from a bruising tendency. No other therapy was given apart from folic acid 5 mg daily. Her clinical and haematological status has remained totally unchanged over the 5 years of observation.

Leukaemia of Platelet Preciirsors 1 5 3

Bone marrow biopsy in 1980 was unchanged and the number of megakaryocytic cells in the blood has remained constant.

(4) A.C. This 21-year-old white male underground railway guard was admitted to St George’s Hospital in January 1976. He gave a history of short and rapidly progressive malaise, anorexia and effort dyspnoea. There had been no known exposure to drugs, toxins or radiation. Anaemia and splenomegaly ( 3 cm below the costal margin) were the only clinical signs: a blood count showed a macrocytic anaemia and thrombocytosis. The blood film was leucoerythroblastic, with bizarre megathrombocytes and a population of unusual blast cells. There were no tear-drop poikilocytes. A mild macrocytic anaemia (Hb 10.5 g/dl; MCV 99 fl) with a platelet count of 569 x 109/1 had been detected 3 years before by his general practitioner. However, the thrombocytosis had not been confirmed on a subsequent sample and the anaemia was not followed up. Apart from this there was no history of illness or a significant family history. Attempted bone marrow aspiration yielded dry taps from sternum and iliac crest. Trephine biopsy showed that normal constituents had been replaced by bizarre megakaryocytes and heavy reticulin.

Radiographs of chest and abdomen confirmed the splenomegaly but were otherwise normal. There was hyperuricaemia (uric acid 0.50 mmol/l) and a polyclonal increase in IgM (1.57 g/l) and IgG (16.2 g/l) and alkaline phosphatase (106 IU/l) were also found but an isotopic liver scan was normal. Serum bilirubin, calcium, urea, iron, vitamin BI1 and folate were normal. No evidence of mycobacterial infection was found.

A diagnosis of myelofibrosis was made. Red cell transfusions were given and folic acid 5 mg daily commenced. The subsequent course was complicated by the rapid development of severe neutropenia and bacterial septicaemias (p-haemolytic streptococcus and Haeniophilus influenza). The spleen rapidly increased in size, as did his transfusion requirements. Isotope studies showed a 51Cr T: of 18.5 d and s9Fe studies virtually absent effective erythropoiesis. In August 19 76 he developed high fever and abdominal pain. Despite broad-spectrum antibiotics the fever persisted and signs of peritonitis became apparent. Laparotomy and splenectomy were performed, but he died following a post-operative cardiac arrest. The spleen showed numerous small infarcts from which a heavy growth of Pseudomonas aeruginosa was cultured.

Post mortem, the lymph nodes, liver, spleen and kidneys were extensively infiltrated by the malignant cells.

LABORATORY METHODS

Blood counts. Full blood counts were performed on electronic cell counters (Coulter Counters Model S and Coulter Thrombocounter C).

Blood and bone marrow smears. Cytological preparations were Romanowsky-stained with Jenner-Giesma stain.

Bone-marrow biopsies were obtained using Jamshidi-Swain biopsy needle, fixed in formal-saline, decalcified using sodium-EDTA and paraffin-embedded before staining with haematoxylin/eosin and for reticulin by the method of Gordon & Sweets (1936).

Cytogenetic studies. Chromosome studies on blood and/or bone marrow were performed at the Regional Cytogenetic Unit, Wandle Valley Hospital, London (case 1) and at the


Department of Cytogenetics and Immunogenetics at the Royal Marsden Hospital, Fulham Road (cases 2 and 3). Direct bone marrow preparations were studies from cases 1 and 3 and blood cultures with and without phytohaemagglutinin in cases 2 and 3. There was insufficient material for banding studies of bone-marrow karyotype in case 1.

Platelet antibodies (case 3). A direct platelet suspension immunofluorescence test (PFIST) was performed on paraformaldehyde-fixed platelets from case 3 following the method of von dem Borne et a1 (1980), and using FITC-labelled anti-IgG and broad spectrum antiglobulin reagent.

Quantitative 52Fe uptake was measured, using a rectilinear scanner as described by Lillecrap et a2 (1 9 76).

Membrane markers Ficoll-Isopaque separated blood mononuclear cells from patients P.P. and R.G. were tested with an extensive panel of immunological markers. These include (see Tables I11 and IV) 'rosette' assays for Fcy, Fcp, C3b, C3d, mouse and sheep erythrocyte binding sites (Bloom & David, 19 76) and indirect immunofluorescent assays with a variety of antisera detecting cell type or differentiation linked antigens. The latter include a series of murine monoclonal antibodies (Table 111, reviewed Greaves, 1981) made by hybridoma technique (Kohler & Milstein. 1975); one of these designates A N 5 1 is specific for platelet glycoprotein I (McMichael et al, 198 1). Binding ofmonoclonal antibodies was assessed by both fluorescence microscopy and with the Fluorescence Activated Cell Sorter (Greaves, 19 7 6 ) , using fluorescein labelled and affinity purified goat anti-mouse Ig. Nuclear terminal deoxynucleo- tidy1 transferase was assessed by indirect immunofluorescence using an affinity purified antibody to TdT (Bollum, 1975).

Cytochemical staining Light microscopy. Neutrophil alkaline phosphatase, periodic acid Schiff and Sudan Black B

staining were all performed by standard methods (Catovsky et al, 1975). a-Napthyl acetate esterase staining was performed after the method of Li et a1 (1973) at pH 6 .3 using hexazotized pararosanalin as coupler. a-Napthyl AS-D chloracetate esterase staining was performed as a double-esterase technique with hexazotized pararosanalin and Fast Blue BB as respective couplers.

Ultrastructural cytochemistry. The platelet-peroxidase reaction was carried out by the method of Breton-Gorius et aI (1 9 78a) on buffy coat cells, cryopreserved in liquid nitrogen. The cells were incubated unfixed in a medium containing diaminobenzidine in Ringer Tris buffer containing H202 (Roels et al, 1975). They were afterwards fixed in glutaraldehyde and processed as for routine electron microscopy. The sections were viewed unstained.

RESULTS

Cytology of peripheral blood and marrow aspirates

Red cell morphology. In cases 1 and 3 the red cells were essentially normocytic and normochromic without abnormalities. In case 2 there was anisopoikilocytosis, and in case 4

Leukaemia of Platelet Precursors 155

macro-ovalocytosis and poikilocytosis. No patient demonstrated large numbers of classical tear-drop poikilocytes.

Platelet morphology. Case 4 had thrombocythaemia at presentation, with large dysplastic forms. Marked thrombocytopenia was present in the other cases; giant dysplastic platelets with abnormal granulation could be found in all. Occasional circulating micro-megakaryocytes were present in case 3 .

White cell morphology. Granulocytic and lymphoid cells in peripheral blood and marrow aspirates appeared morphologically normal. Their numbers were much reduced, except in case 4.

Blast celZ morphology. Case 1. 30% of the nucleated cells in the blood and bone marrow were large (20-24 pm) oval blasts. Blast nuclei were uncleaved, non-folded, with fine chromatin and several small nucleoli. The cytoplasm was moderately abundant, grey-blue, and showed budding. The aparticulate material aspirated from the bone-marrow was enriched with blast cells, and also contained some micro-megakaryocytes.

Case 2. 70% of the nucleated cells were bizarre large blast cells (24-28 pm). Blast nuclei were irregular in outline with occasional folds and indentations, fine chromatin and small nucleoli. Some were binucleated. The cytoplasm was abundant, with amoeboid projections. Cytoplasmic granules were not prominent in Romanowsky stained cells.

Case 3 . Rare blast cells, amounting to 1% of the nucleated cell count, have been present on blood smears during the most recent year of follow-up. Aparticulate material aspirated from the marrow shows about 4% of abnormal blasts. The cytological features are identical to those in cases 1 and 4. In addition, about 2% of the nucleated cells have multiple long cytoplasmic projections and other features of micromegakaryocytes.

Case 4.40% of the nucleated cells in the peripheral blood, and 2 5% of those in the marrow aspirate, were large (20-25 pm), oval or elongated blasts with identical nuclear and cytoplasmic features to those of cases 1 and 3 .

Bone marrow biopsies

Case 1. The trephine biopsy showed areas of hypocellularity with increased fat spaces interspersed with areas of packed hypercellularity consisting of abnormal mononuclear cells, dysplastic megakaryocytes and intermediate forms. Erythroid precursors were present but reduced and granulocytic cells were greatly reduced. There was a heavy increase in reticulin staining. with many coarse fibrils, but no osteosclerosis.

Case 2. The biopsied marrow was grossly hypercellular with obliteration of fat spaces, and consisted of sheets of dysplastic megakaryocytes. No erythroid or granulocytic elements were identifiable. Reticulin staining was greatly increased. The fibrils formed coarse bundles surrounding the neoplastic megakaryocytes. Osteosclerosis was absent.

Case 3 . The biopsied marrow was hypercellular without fat spaces. All haemopoietic cell-lines were represented, but the megakaryocytic line was massively hypertrophied with many mononuclear and dysplastic forms. Reticulin was markedly increased, without osteosclerosis.

Case 4. Hypercellular bone marrow without fat spaces, consisting of sheets of

156 David Bevan, Michael Rose and M e l v y n Greaves

multinucleated cells with features of megakaryocytes, abnormal mononuclear cells and abnormal cells intermediate between the two. Erythroid and granulocytic elements were indentifiable but reduced. Reticulin was heavily increased and formed parallel arrays around megakaryocytes. There was no osteosclerosis.

Cytochemistry of blast cells

The reactions of the blast cells in all four cases are shown in Table 11. Blast cells in cases 2 and 3 showed the ‘megakaryocytic’ pattern of non-specific esterase staining predicted by Li et al (1 9 7 3 ) , but case 1 did not. Esterase studies were not performed on case 4.

Ultrastructural cytochemistry. Case 2 . A positive reaction for platelet peroxidase was seen in the nuclear envelope and endoplasmic reticulum of all the blast cells examined.

Cytogenetic studies

Case 1. The original bone-marrow aspirate yielded 27 mitoses suitable for evaluation in the direct preparation. Morphology was poor and banding studies could not be done. However, the majority of mitoses were hypodiploid (25/2 7 ) . Eleven mitoses (44XX) showed probable loss of two C group chromosomes; in a further nine (43XX) a number 1 chromosome was also missing. The Philadelphia (Ph) chromosome was not observed.

Case 2 . No bone marrow cells were available for study since marrow aspiration yielded a dry tap. Stimulated peripheral blood cultures yielded no mitoses on two occasions. Peripheral blood culture with phytohaemagglutinin confirmed the diagnosis of Down’s syndrome, trisomy 2 1.

Case 3 . Two bone marrow samples were examined. The yield from these was extremely poor but on the evidence available the karyotype of the cells in the marrow was normal 46XX. The patient also has normal lymphocytes.

Case 4. Cytogenetic analysis was not performed.

Table 11. Blast cell cytochemistry

Case 1 (E.V.) Case 2 (P.P.) Case 3 (R.G.) Case 4 (A.C.)

PAS Fine granular positivity

Sudan Black Negative Chloroacetate ASD esterase Negative a-Napthyl acetate Negative esterase

cc-Napthyl butyrate Negative

Granular positivity Negative

Negative Strongly positive in all blasts Weakly positive

Coarse granular Fine granular positivity positivity in cytoplasmic buds Negative Negative

Negative Negative Ranging from weakly Not done to strongly positive in all blasts Negative Not done

Leukaemia of Platelet Precursors Table 111. General marker character- istics of P.P. cells

157

1. Rosettes E (sheep) 10% E (mouse) 1% E (ox) A (M) C3b 0% E (ox) A (M) C3d 80% E (ox) A (GI 8 5%

Anti-AIL < 1% Anti-Ip 3%

3. TdT 0.1 units <0.5%

2. Antisera

Table IV. Cell surface phenotype of P.P. cells determined with a library of monoclonal antibodies

Specificity* Designation Result

1. HLA-associated i.

ii.

iii.

Monomorphic HLA-ABC

Monomorphic HLA-DR

bl microglobulin

2. Lymphoid cell associated i. T lineage

ii. B lineage

iii. Precursor 3 . Erythroid

i. Glycophorin ii. Band 111

4. Myeloid

5. Leucocyte 6. Platelet

W6/32 PA2.6 BB7.8

DA2 OKIa- 1

EC 3 BB 5

P17 F12 NA134 OKTl1 OKT6 OKT9

FMC 1 P1153/3

J- 5

28J10.1 1/6A OKM- 1

2D1 AN51

* Reviewed Greaves (1975).

158 David Bevan, Michael Rose and Melvyn Greaves Table V. Cell marker results on bone marrow cells from patient R.G.

cells staining

E (sheep) rosettes Anti-HLA-DR (DA-2) Anti-CALL (J-5) Anti-T (OKT11) Anti-monocyte (OKM-1) Anti-erythro (glycophorin-LICR/LON/K 10) Anti-platelet (AN51) TdT

4 5 40 <1 26 <1 <1 4 0 < I

Immunological marker studies

Blast cells from the blood of patient P.P. (case 2) were shown to have receptors for IgG (Fcy), C3b and C3d (Table IV) but not mouse or sheep erythrocytes. They were TdT negative and unreactive with monoclonal antibodies specific for cells of the lymphoid, granulocytic/ monocytic, and erythroid series. The majority (> 75%) of cells did, however, stain, albeit weakly, with monoclonal (AN5 1) anti-platelet glycoprotein I. The blast cells also stained for HLA-ABC (monomorphic determinants) and pz microglobulin but not for HLA-DR.

Bone marrow cells from patient R.G. (case 3) were tested with a smaller series of markers and 40% of mononuclear cells were found to be weakly stained with monoclonal AN51 anti-platelet. A relatively high number of T cells were also present.

To date (April 1981) five out of eight other ‘possible’ megakaryoblastic leukaemias (excluding the four patients reported here) have had cells reactive with monoclonal AN5 1. No other leukaemias have been reactive (out of 65 cases of AML or ALL tested) with one interesting exception. Four cases of CGL in blast crisis had minor populations (5-1 6%) of mononuclear bone marrow cells reactive with AN51 antibody-although none of the 25 so far tested have had a major population reactive with this platelet marker as anticipated from the study of Breton-Gorius et a1 (1978a) with platelet peroxidase.

Platelet surface immunoglobulin

No evidence of platelet associated immunoglobulin was detected using the direct platelet suspension immunofluorescence test on platelets from case 3.

52Fe scanning (case 3 )

There was no significant splenic uptake of the radioisotope. Bone marrow uptake was reduced: most of the isotope went to the liver.

Leukaeniia of Platelet Precursors 159

DISCUSSION

The clinical and pathological features of case 1 are identical with those of ‘malignant myelosclerosis’ (Lewis & Szur, 1963), which is currently thought to be a neoplastic proliferative disorder of megakaryocytes on the basis of ultrastructural studies (Breton- Gorius et al, 19 73; Den Ottolander et a I , 19 79). Critical analysis of reports pertaining to this disorder dismissed many as examples of other myeloproliferative diseases, but concluded that a core of typical cases remained which constitutes a unique disease entity (Bearman et al, 1979). Features common to case 1 and the aforementioned typical cases include pancytopenia, absence of leucoerythroblastosis or tear-drop poikilocytes, no enlargement of liver or spleen, increased bone marrow reticulin and an acute course: in this case normal blood findings 1 5 months earlier.

The biopsy appearances in case 1 were not of panmyelopathy described by Bearman et aI ( I 9 79); erythroid and granulocytic elements were remarkably few and the cellular component comprised the various stages of differentiation of the abnormal megakaryocytic lineage, as in a true leukaemic disorder. Bearman et al accept that this appearance may occur as a terminal event but the duration of our patient’s illness was so short that this pattern was probably present from the onset.

Mononuclear cells from case 1 did not display the non-specific esterase staining predicted by Li et aI(19 73) though absence of this pattern is seen (Den Ottolander et aI, 19 79) and may represent primitive blasts. The probable deletions in the C group chromosomes confirms an association previously noted by Nowell &I Finan (1978).

The clinico-pathological features in case 1 support the view of Den Ottolander et uZ(l9 79) that this disease be regarded as acute megakaryoblastic leukaemia.

There have been a handful of reports of acute fatal haematological disorders with marrow fibrosis and megakaryocytic proliferation in children with Down’s syndrome (Rosenberg & Taylor, 19 5 8; Hillman & Forrester. 1968; Cosson et al, 1 9 74; Evans, 19 7 5). In most of these cases blast cells were present in the blood at some time during the illness, but were not fully characterized except by Cosson et a1 (1 9 74), who demonstrated that in a case of congenital leukaemia with myelofibrosis and thrombocythaemia the circulating blast cells contained platelet-specific peroxidase.

There seems little doubt that the circulating blasts in P.P. were of megakaryocytic lineage, since in addition to their Romanowsky-stained appearance and megakaryocytic esterase pattern they possessed platelet-peroxidase activity on ultrastructural cytochemistry, and an unusual membrane phenotype when studied using monoclonal antibodies, including positive reaction with anti-platelet antibody.

Several other observations are of note. The existence of a virtually normal marrow aspirate 8 months prior to a dry tap and hypercellular fibrotic biopsy emphasize the speed with which reticulin deposition occurs in such a condition (cf. also case 1). There is good clinical and experimental evidence to suggest that increased fibroblastic activity in bone marrow disorders is not always a simple injury response to an infiltrative pathology, but may be a specific cellular interaction with megakaryocytes (Groopman, 1980), which participate via secretion of fibroblast-stimulating factors (Kaplan et al, 19 79; Castro-Malaspina et al,


1980). This interaction may be more pronounced in the setting of ineffective thrombopoiesis, where intense megakaryocytic proliferation is associated with impaired platelet output (H. Castro Malaspina, personal communication, 1980). Case 2 provides clinical evidence consistent with these hypotheses.

The spectacular, apparently spontaneous, phasic disappearance of the leukaemic megakaryocyte line from the blood, with good evidence of true cell destruction in the form of massive urate excretion, has a remarkable similarity to phenomena observed in other trisomy 2 1 haemopathies, such as the transient ‘leukamoid’ proliferations which occur both in Down’s syndrome (Ross et al, 1963; Engel et al, 1964) and in cell lines possessing extra no. 2 1 chromosomes in the absence of constitutional Down’s syndrome (Brodeur et al, 1980; Heaton et al, 1981).

Such observations suggest that abnormal clones of haemopoietic cells which possess trisomy 2 1 have a ‘proliferative advantage’ which may beinherently self-limitingorvulnerable to obscure regulatory influences (Killman, 19 72). The progression from a state resembling childhood immune thrombocytopenic purpura with a morphologically normal bone marrow to a megakaryocytic proliferation with rnyelofibrosis has remarkable similarity to the course of events described in murine leukaemias induced by Rauscher virus (Brodsky, 1973).

Our third case has a chronic intramedullary proliferation of megakaryocytes with increased reticulin, but without evidence of myeloid metaplasia in the form of clinical splenomegaly or 52Fe uptake in the spleen. Such a disorder might otherwise be regarded as an early stage of the disease known as myelofibrosis with myeloid metaplasia (MMM) in the United Kingdom and agnogenic myeloid metaplasia (AMM) in the United States, and also called ‘megakaryocytic myelosis with myelofibrosis’ (Hickling, 1968; Burkhardt et al, 19 75). The concept of primary proliferation of megakaryocytes inducing a secondary, paraneoplas- tic syndrome of osteomyelosclerosis is central to this terminology, and has not been widely adopted on the grounds that it is based on morphological evidence alone.

The appearance might be regarded as a variant of MMM/AMM rather than a stage of the disorder. However, thrombocytopenia is only seen terminally in MMM/AMM and would not be expected in the absence of splenomegaly (Ward & Block, 19 71). Some haemopathologists distinguish a subtype of chronic granulocytic leukaemia, in which megakaryocytic, in addition to granulocytic hypertrophy is identified (Georgii et al, 1980). Case 3 is dissimilar to those cases, since granulocytes are reduced in the marrow and the Philadelphia chromosome is not demonstrated. Where similar cells have been demonstrated in chronic myeloproliferative disorders, they have been associated with thrombocythaemia. and have been thought to be participating in intravascular (extramedullary) thrombopoiesis (Popescu, 19 72: Maldonado, 19 74), rather than thrombocytopenia with ineffective thrombopoiesis. Despite the lack of a demonstrable chromosome abnormality, the presence of a megakaryocytic proliferation with ineffective thrombopoiesis with thrombocytopenia suggests that this is a megakaryocytic leukaemia rather than a ‘myelosis’, analogous to chronic Di Guglielmo’s syndrome rather than to polycythaemia rubra Vera.

The overall pattern in case 4 was of massive proliferation of megakaryocytes. Since cytogenetic studies were not performed, megakaryocytic blast crisis of chronic granulocytic leukaemia superimposed on a short, undiagnosed chronic phase cannot be excluded.

Leukaemia 0s Platelet Precursors 161

The peripheral blood abnormalities (macrocytosis, mild anaemia and thrombocytosis) observed 4 years before the onset of severe symptoms, are of uncertain significance. They might suggest the (Sq-) syndrome (Sokal et al, 1975; Mahmood et al, 1979). Leukaemic transformation is not typical in this disorder but has been reported (Verhest et al, 1977).

The features in this patient bear similarities to ‘acute megakaryocytic myelosis’ (Rappaport, 1966; Fujinami et ul, 1961) and to case 2 of this series. All these had blast cells in the blood and/or bone marrow at some stage during the illness and could be designated megakaryocytic leukaemias.

In common with the malignant disorders of lymphocytic and myelocytic lineages, the cases in this series display a remarkable clinical diversity which contrasts with the uniformity observed amongst those described by Den Ottolander et al (1979). Though the cellular constituents identified in the bone-marrow left little doubt as to the main line of differentiation in the disturbed cell population, the process was represented in the peripheral blood by poorly differentiated cells. Such a reversal between the blood and the marrow of the mature and immature populations is seldom as striking in the other leukaemias. Unlike three of the four cases described by Bain et a1 (1 981) or the series of Sultan et al(1981), the features in our cases appear to be predominantly or exclusively those of a disorder effecting the megakaryocytic lineage. As the phenotype is correlated with maturation and may be weakly expressed, on the more primitive mononuclear megakaryoblasts, or not expressed at all, a combination of complementary markers is required to identify leukaemic clones at different stages of maturation arrest. The existence of these leukaemias could be used to advantage in developing and accumulating probes to identify and recognize new markers.

ACKNOWLEDGMENTS

Dr Oliver Brooke and Dr John Parker-Williams for permission to report details concerning patients under their care. Dr John Millard for referring case 3 to our care. Professor S. Lawler and Dr R. G. Chitham for cytogenetic analyses and for permission to quote work performed in their laboratories (Cytogenetic Unit, Institute of Cancer Research, Royal Marsden Hospital, Fulham Road, S.W.3; Regional Cytogenetic Laboratory, Wandle Valley Hospital, South London). Dr A. J. McMichael of the Radcliffe Infirmary, Oxford, for anti-platelet AN51. Dr D. A. Catovsky for ultrastructural demonstration of platelet peroxidase and permission to publish Fig 4. Ann Syred and Katherine Alexander for expert technical help. Dr H. Clink for making facilities available for j’Fe scanning and Mr Ken Maureemootoo for performing the scan. Janet Smith for typing the manuscript.

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