British Journal of Haematology, 1975, 30, 65.
Acute Leukaemia with Eosinophilia or Acute Eosinophilic Leukaemia: A Dilemma R. S.
WEINGER, JANINE AND&-SCHWARTZ,
JANE
F. DESFORGES AND
MARGARET BAKER Blood Research Laboratory, N e w England Medical Center Hospital, and Dtpartnzmt of Medicine, TuJs University School of Medicine, Boston, Massachttsetts (Received 5 August 1974; acceptedfor publication
25
October 1974)
A young malc patient is described with acute leukaemia whose bone marrow and peripheral blood contained abundant cells of the eosinophilic series in all stages of maturation. These cells, proven liistochemically to be true eosinophils, were abnormal in both maturation and proliferation. Upon electron microscopic study of bone marrow and peripheral blood, abnormalities in the eosinophilic series were identified as early as the promyelocytic stage as well as in the most mature eosiiiophil seen. The clinical and morphologic picture of this patient’s disease raises the possibility of this being an acute eosinophilic leukaemia. SUMMARY.
Eosiiiophilia in association with acute or chronic leukaemia, as well as other malignancies, is a well described entity (Hardy & Anderson, 1968). Controversy over tlie existcncc of an entity ‘acute eosinophilic leukaemia’ centres around the origin and significance of the eosinophilic cell. W e present a case of acute, nonlymplioblastic leukaemia, whose major morphologic abnormalities occurred in the eosinophilic granulocytic Iine and whose abnormalities could be traced as far as the promyelocytic stage. CASE REPORT
R.F., a 17-year-old malc, was in a car accident in November 1972, following which he was anticoagulated because of evidence of pulmonary emboli. In early February 1973, he complained of lethargy, anorexia, sore throat, fever and pain in his right arm. There was no past history of illness. According to the foster mother, lie was found to have an eosiiiophil count of 10% at age 3 years. Physical examination revealed an oral temperature of 37.s°C, an aciieiform eruption, dullness and decreased breath sounds at tlie right base, hepatomegaly, swelling and tenderness over his right deltoid muscle and palpable, non-tender anterior cervical nodes. Laboratory studies (see Table I) demonstrated a marked eosinopliilia, the cells having large eosinophilic granules scattered irregularly throughout pale blue-grey cytoplasm; iioiiseginented nuclei were conspicuous (Fig Ia). No blasts were seen. Bone-marrow examination revealed 90% blasts, each containing nuclei with one to three nucleoli, a thin rim of Correspondence: D r Janine AndrC-Schwartz, New England Medical Center Hospital, Department of Hematology, 171 Harrison Avenue, Boston, Massachusetts 02111, U.S.A.
65
R. S. Weinger et a1
66
pale, blue, granule-free cytoplasm and eosinophils at all levels of maturation (Fig I b and c). Peroxidase stain of the blasts was negative; PAS stain demonstrated a few PAS-positive blast cells as did O R 0 (oil-red-0) stain. The eosinophilic granules contained cyanideresistant peroxidase (Yam et d,1971). Chromosome studies failed to reveal a Ph' chromosome, although hypodiploidy consistent with a G group monosomy was found in 88% of the metaphases. Failure of his chest to clear clinically and by X-ray and persistent fever prompted an open lung biopsy. This showed vascular sclerosis, interstitial fibrosis, eosinophilia, and haemosiderin-laden macrophages, not considered to be a leukaemic infiltration. On 17 April 1973 the patient was begun on weekly vincristine (0.015 mg/kg) intravenously and daily oral prednisone (I mg/kg/24 h). However, symptoms progressed; he developed a severe exudative, sterile pharyngitis, smears of which revealed sheets of eosinophils and TABLE I. Haematological values 9 Feb. I973
Hb (g/dl) PCV WBC x 1o9/I. % PMN Bands Eos metamyelocytes Eos myelocytes Eosinophils Lymphocytes Monocytes Basophils Blasts
14.0 0.41 16.4 7.5 2.0
82.5 8.0
-
20
March I973
17 April I973
10.6
12.0
0.3 I 31.5
4.0 2.5 12.5
4.5 75.0 1-5 -
0.36 11.4 13.0 3.0
-
70.0 11.5 1.0
24 May I973
14Jttne I973
13.2 0.37 49.5 1.5 -
12.5 0.42 4.35 68.0
0.39 55.1
0
7.0
27.0
11.0
11.0
2.0
16.0
11.0
2.0
I .o
-
-
2.0
55.0
91.5 6.0
-
I973
4.0 -
-
-
14.0 0.40 23.4
1s Oct.
9.0 3.0 2.0
4.0 -
-
27JuIy I973
-
55.0 10.0
eosinophilic debris. Following the sixth dose of vincristine, bone marrow contained 30% blast forms with 29% eosinophils in all stages of maturation. His peripheral eosinophil count was 45.3 x 10~11. (24 May 1973). A 3 day course of Daunorubicin (60 mg/m2) was started. Within 72 h his pharyngitis cleared, he became afebrile and spleen size decreased. He achieved complete remission by 14 June 1973. Peripheral and bone marrow eosinophilia disappeared while in remission. In spite of maintenance therapy, he relapsed in 6 weeks. Eosinophilia returned with the onset of symptoms. Bone-marrow lysozyme level, measured by the method of Osserman (Osserman& Lawlor, 1966) was 22 mg/l. (normal 10-20 mg/l.) ; serum lysozyme was normal at 12 mg/l. Electrocardiogram and cardiac silhouette on chest X-ray were normal. Attempts to re-induce remission with Daunorubicin, POMP (prednisone, vincristine, methotrexate and 6-mercaptopurine) and a combination of cytosine arabinoside plus 6thioguanine failed. In early December 1973, chest X-rays showed bilateral pneumonia and he died in respiratory failure on 10 December 1973. Permission for autopsy was not granted.
Act& Eosinuphilic Leiikaeniin ?
FIG I. Light photomicrographs. (a) Peripheral blood eosinophils, oiic with ‘iioiiscgiiieiitcd’ iiucleus, but both having large eosinophilic granules and palc blue-grey cytoplasm (b) Bone marrow dciiioiistratitig inyeloblasts (thick arrows) surroundcd by cosinophils (thin arrows) in all stages of maturation. (c) Bone inarrow with large promyclocytc (thick arrow) containing large eosiiiophilic graiiulcs. Note that surrouiidirig cells are also eosinophils (thin arrows), whose granules arc similar to those sccn iii peripheral blood. Wright’s stain x 4500. (Fucbzg p 66)
R. S. Weinger et a1
FIG2 . Maturc polymorphonuclear neutrophil (blood). Note the 'beak' shape of the nucleus (N) (thick arrows). Thin splinter-shaped channels (thin arrows) are seen in the cytoplasm. Several azurophilic primary granules (pg) and neutrophilic secondary granules (sg) are also seen in the cytoplasm together with abundant glycogen (gl). x 20 700.
FIG 3 . Mature eosinophil (blood). Its nucleus (N) with clumped chromatin is mature. Its cytoplasm exhibits both signs of maturity: abundant glycogen (gl), and of immaturity: conspicuous Golgi apparatus (G) and numerous flattened RER cisternae (RER). The cytoplasm also contains immature eosinophilic granules (ig). x 17 100.
Acute Eosinophilic Leukaemia?
FIG 4. Eosiiiophilic granules (detail of Fig 3). Most of them arc not holnogcncously electron dcnse. One is aiigulatcd (double arrow) and cxhibits an irrcgular crystalline pattern (thin arrows). One granulc contains a crystal (thick arrow) whose lattice pattern is shown in the insert. x 80 ooo (insert x 120000).
R. S. Weinger et
a1
FIG 5 . Myeloblast (bonc marrow). Notc thc abnormal indented shape of its iiucleus (N) and its large nucleolus (n). Its cytoplasm packed with single ribosoincs contains niyelinic profiles (arrows) and abundant niitochondria (m). x 18 900. FIG 6. Promyelocytc eosinophil (bone marrow). This cell has a nucleus (N) with finely marginated chromatin. Its cytoplasm has nunierous electron light RER cisternae (RER) and a well-developed Golgi zone (G). No glycogen is seen. Numerous vacuolcs (V) are present. Abundant electron dense granules (9) are visible, one of them angulated (arrow). x 19 800.
Acute Eosinophilic Leirkaemia ?
67
METHODS Heparinized blood (or bone marrow) was centrifuged at 400 g for I 5 min at 4" C. Plasma was withdrawn and replaced by 2.5% glutaraldehyde (Leduc et a / , 1963) in 0.1 M phosphate buffer, pH 7.4. After I h, the buffy coat was lifted so that the fixative would be in direct contact with its undersurface and fixation was allowed to proceed for an additional hour. The buffy coat was then washed with multiple changes of 0.1 M phosphate buffer, pH 7.4, cut into tiny fragments which were fixed for another hour in 2.5% glutaraldehyde. After further washing in phosphate buffer, the pellet was post-fixed in I % tetroxide, dehydrated in graded alcohols and embedded in Epon (Luft, 1961). Thick sections (0.5 pm) were cut on a Porter Blum microtome and stained with azure blue and mcthylene blue (Richardson et al, 1960). Desired blocks were selected under the light microscope. Ultra-thin sections were mounted on uncoated grids and stained with 0.5% a!cohol solutions of uranyl acetate for 5 min, counterstained with 0.1% lead citrate (Venable & Coggershall, 1965) for 5 min. The sections were examined with HU-IIE Hitachi electron microscope (75 kV, objective aperture 50 pm). RESULTS
Electron Microscopic Findings Peripheral blood and bone marrow were studied during relapse (27 July 1973). Peripheral blood. Polymorphonuclear neutrophils and eosinophils were prominent. Lymphocytes, monocytes and basopliils appeared decreased in number but their ultrastructure was normal. A few polymorphoiiuclear neutropliils had atypical features both in their nucleus which presented a beak-like shape, and in their cytoplasm which contained splinterlike channels (Fig 2). Typical mature eosinophils were never seen. The cells observed, about 8-9 pm in diameter, exhibited nuclear and cytoplasmic asynchrony. Nuclear maturity was demonstrated by its multiple lobes and its condensed and marginated chromatin. The cytoplasm showed some signs of maturity: it was dense with single ribosomes and contained abundant particulate glycogen. It also exhibited features of an immature cell-numerous flattened rough endoplasmic reticulum (RER) cisternae and immature eosinophilic granules (Fig 3 ) . These granules were about 0.5-1.5 p m in maximum diameter, regularly dense and bound by a unit membrane; most appeared round or ova!-only a few were angulated. Although most of them had atypical crystalloid structures, a few contained crystals with a lattice pattern (Fig 4). Bone marrow. Three types of cells were prominent in the bone marrow: (I) undifferentiated blasts, (2) myeloblasts, and ( 3 ) abnormal eosinophilic myclocytes. The undifferentiated blasts were Unremarkable. Numerous myeloblasts had an irregularly shaped, deeply indented nucleus and myelinic profiles in their cytoplasm (Fig 5 ) . Only a few rnyelocytes and metamyelocytes of the neutrophilic and basophilic series were secn. Their ultrastructure was unremarkable. In contrast, all the cells of the eosinophilic type appeared abnormal. Nuclear-cytoplasmic asynchrony was a constant feature. The cell most often seen was one with the nucleus of a myelocyte and the cytoplasm of a youiigcr cell
68
R.S . Weinger et al
(promyelocyte) of the eosinophilic granulocytic line. This cell (Fig 6), about 8-10 p m in diameter, had a high N / P ratio. Its round or slightly indented nucleus had marginated chromatin and, often, a conspicuous nucleolus. Its cytoplasm had abundant RER with moderately distended cisternae and a well-developed Golgi apparatus which did not contain any dense material. Spherical and oval, membrane-limited vacuoles, 0.5-0.7 p m in diameter were present. Their content varied from a coarsely granular material to a fenestrated or homogeneous matrix. Such formations with a homogeneous clectron dense content were the most numerous and constituted granules. No glycogen, no sign of phagocytosis were seen in these cells. DISCUSSION This patient had a marked eosinophilia with what first seemed to be acute lymphoblastic leukaemia. Hisclinical features and morphological picture by light microscopy were reminiscent of those cases of ALL reported by Spitzer & Garson (1973). Treatment was directed towards that entity and failed until Daunorubicin was given and this successfully induced remission. His eosinophilia, both peripheral and marrow, disappeared. Upon relapse, further studies, including serum and bone marrow lysozyme determinations, were more compatible with a nonlymphoblastic leukaemia. By electron microscopy, the blast cells clearly fit the criteria for myeloblasts. Furthermore, the granulated cells seen by both light and electron microscopy were indeed positive for cyanide-resistant peroxidase. It is generally accepted that the multipotential stein cell or ‘undifferentiated blast’ gives rise to myeloblasts, which then mature into promyelocytes, myelocytes, metamyelocytes and mature granulocytes (Ford Bainton & Farquhar, 1966; Wetzel et a ] , 1967; Scott & Horn, 1970), whether neutrophilic, basophilic or eosinophilic. In the patient studied here, no morphological alteration of the undifferentiated blast was seen. The number of these cells was few in the bone marrow and they were not observed in the blood. Myeloblasts were increased in number in the bone marrow but not present in the blood until late in the disease. By electron microscopy, it is not possible to know what kind of promyelocytes-neutrophil, basophil, eosinophil-will arise from a given myeloblast because no granules are seen in that cell. In this patient the morphology of most myeloblasts was unremarkable. Some, however, had irregularly shaped nuclei and a few myelinic profiles; these cells might represent precursors of the abnormal eosinophils. The eosinophils were quantitatively and qualitatively abnormal. Quantitatively, there was an increased number of these cells both in the bone marrow and in the blood. Qualitatively, their maturation was ‘anarchic’ (Bessis & Breton-Gorius, 1969) in two respects: (I) nucleus and cytoplasm were not maturing at the same rate; (2) in the cytoplasm itself some organelles were more mature than others. In the bone marrow, the patient had promyelocytic eosinophils with a cytoplasm similar to that of the ‘early eosinophil’ described by electron microscopy by Scott & Horn (1970). As in the present case, cytoplasmic vacuoles were observed and it was concluded that ‘the progressive accumulation of dense material within the vacuoles caused them to be transformed into. . . granules.’ Thc authors named these ‘immature granules’ because, as shown by the study of the development of eosinophil granulocytes in human bone marrow, these granules later give rise to the elongated crystalloid-containing
Acute Eosinophilic Leukaemia?
69
granules of the mature eosiiiophil. In the blood of the patient, these cells contained some angulated granules. More important, a few granules presented a cubic lattice pattern previously shown to be typical of the core of eosiiiophil granules in man (Miller et al, 1966). Both the atypical cosinophils seen in the blood and tlie proniyclocytes in the bone marrow were eosinophils by light microscopy and chemical reactions. The unusual features observed in the mature polyniorphoiiuclear neutrophil are difficult to interpret. The myelinic profiles are not specific and have been described in myeloid cells in myeloproliferative disorders (Tavassoli & Weiss, 1973). The electron light, splintershaped channels do riot seem to represent extruded Auer bodies (Freeman, 1960); these would appear thicker aiid larger and were never seen in this patient. The beak-like appearance of their nuclei may be related to prior therapy. This patient’s blood aiid bone marrow contained numerous undifferentiated blasts and abundant abnormal eosinophilic granulocytes which could be recognized as early as the promyelocytic stage. Whether this picture represents an acute leukaemia with a prominent eosinophilic reaction, as in cases previously reported in the literature (Spitzer & Garson, 1973), or a true eosinophilic leukacmia is impossible to state at the present. It is only when new histochemical criteria might allow us to pinpoint differences between blasts giving rise to different lines of cells that such a diagnosis is possible. The scarcity of EM reports of cases such as tlie present one, make it worthwhile to add the present observation to the still coiitrovcrsial dossier of the ‘eosinophilic leukaemias’. ACKNOWLEDGMENTS
The authors wish to thank Dr Avrum Z . Bluiniiig for his assistance during this case study. This work was aided in part from the University Cancer Committee, American Cancer Society, Institutional Grant N-23-0. REFERENCES BESSIS,M. & BRETON-GORIUS, J. (1969) Pathologie ct asynchronisme de d6veloppement des organellcs cellulaires au cows des leucdmies aigues granulocytaircs. Norrvelle Reoiir Franfaise d’Hhatolo@r, 9, 24.5. FORDBAINTON,D. & FARQUHAR, M.G. (1966) Origin of granules in polymorphonuclear leukocytes. Two types derived from opposite faces of the Golgi complex in dcveloping granulocytes. journal (rf Cell Biology, 28, 277. FREEMAN, J.A. (1960) The ultrastructure and genesis of Auer bodies. Blood, 15,449. HARDY,W.R. & ANDERSON, R.E. (1968) The hypercosinophilic syndromes. Annals OJ’ Internal Medicine, 68,
1220.
LEDUC,E., MARINOZZI, V. & BERNHARD, W. (1963) The use of water-soluble glycol tnethacrylate i n ultrastructural cytochcmistry. joirmal of thr Roynl Micosropical Society, 81, I 19. LUFT, J.H. (1961) Improvements in epoxy resin
embedding methods. Joorrr~al of Biophysical and BiocRemical Cytology, 9, 409. MILLER,F., DE HARVEN,E. 81 PALADE,G.E. (1966) Thc structure of eosinophil leukocyte granules in rodents and in man. Journal of Cell Biology, 31, 349.
OSSERMAN, E.F. & LAWLOR,D.P. (1966) Scrum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukcmia. Journal of Experimental Medicine, 124,921. RICHARDSON, K.C., JARETT, L. & FINKE,E.H. (1960) Eni’ocdding in epoxy rcsins for ultrathin sectioning in electron microscopy. Stain Technology, 35, 313. SCOTT,R.E. & HORN,R.G. (1970) Fine structural features of eosinophile granulocyte development in human bone marrow. joumal of Ultrastrrtcture Research, 33, 16. SPITZER,G. & GARSON,O.M. (1973) Lymphoblastic leukemia with marked eosinophilia: a report of two cases. Blood, 42, 377.
70
R. 5’. Weinger et al
TAVASSOLI, M. & WEISS,L. (1973) An electron microscopic study of spleen in myelofibrosis with mycloid metaplasia. Blood, 42, 267. J.H. & COGGERSHALL, R. (1965) A simplified VENABLE, lead citrate stain for use in electron microscopy. Journal of Cell Biology, 24, 407. WETZEL,B.K., HORN, R.G. & SPICER,S.S. (1967)
Fine structural studies on the development of hcterophil, eosinophil, and basophil granulocytes in rabbits. Laboratory Inuestigation, 16,349. YAM, L.T., LI, C.Y. & CROSBY,W.H. (1971) Cytochemical identification of monocytes and granulocytes. American Journal of Clinical Pathology, 55, 283.
Acute Leukaemia with Eosinophilia or Acute Eosinophilic Leukaemia: A Dilemma R. S.
WEINGER, JANINE AND&-SCHWARTZ,
JANE
F. DESFORGES AND
MARGARET BAKER Blood Research Laboratory, N e w England Medical Center Hospital, and Dtpartnzmt of Medicine, TuJs University School of Medicine, Boston, Massachttsetts (Received 5 August 1974; acceptedfor publication
25
October 1974)
A young malc patient is described with acute leukaemia whose bone marrow and peripheral blood contained abundant cells of the eosinophilic series in all stages of maturation. These cells, proven liistochemically to be true eosinophils, were abnormal in both maturation and proliferation. Upon electron microscopic study of bone marrow and peripheral blood, abnormalities in the eosinophilic series were identified as early as the promyelocytic stage as well as in the most mature eosiiiophil seen. The clinical and morphologic picture of this patient’s disease raises the possibility of this being an acute eosinophilic leukaemia. SUMMARY.
Eosiiiophilia in association with acute or chronic leukaemia, as well as other malignancies, is a well described entity (Hardy & Anderson, 1968). Controversy over tlie existcncc of an entity ‘acute eosinophilic leukaemia’ centres around the origin and significance of the eosinophilic cell. W e present a case of acute, nonlymplioblastic leukaemia, whose major morphologic abnormalities occurred in the eosinophilic granulocytic Iine and whose abnormalities could be traced as far as the promyelocytic stage. CASE REPORT
R.F., a 17-year-old malc, was in a car accident in November 1972, following which he was anticoagulated because of evidence of pulmonary emboli. In early February 1973, he complained of lethargy, anorexia, sore throat, fever and pain in his right arm. There was no past history of illness. According to the foster mother, lie was found to have an eosiiiophil count of 10% at age 3 years. Physical examination revealed an oral temperature of 37.s°C, an aciieiform eruption, dullness and decreased breath sounds at tlie right base, hepatomegaly, swelling and tenderness over his right deltoid muscle and palpable, non-tender anterior cervical nodes. Laboratory studies (see Table I) demonstrated a marked eosinopliilia, the cells having large eosinophilic granules scattered irregularly throughout pale blue-grey cytoplasm; iioiiseginented nuclei were conspicuous (Fig Ia). No blasts were seen. Bone-marrow examination revealed 90% blasts, each containing nuclei with one to three nucleoli, a thin rim of Correspondence: D r Janine AndrC-Schwartz, New England Medical Center Hospital, Department of Hematology, 171 Harrison Avenue, Boston, Massachusetts 02111, U.S.A.
65
R. S. Weinger et a1
66
pale, blue, granule-free cytoplasm and eosinophils at all levels of maturation (Fig I b and c). Peroxidase stain of the blasts was negative; PAS stain demonstrated a few PAS-positive blast cells as did O R 0 (oil-red-0) stain. The eosinophilic granules contained cyanideresistant peroxidase (Yam et d,1971). Chromosome studies failed to reveal a Ph' chromosome, although hypodiploidy consistent with a G group monosomy was found in 88% of the metaphases. Failure of his chest to clear clinically and by X-ray and persistent fever prompted an open lung biopsy. This showed vascular sclerosis, interstitial fibrosis, eosinophilia, and haemosiderin-laden macrophages, not considered to be a leukaemic infiltration. On 17 April 1973 the patient was begun on weekly vincristine (0.015 mg/kg) intravenously and daily oral prednisone (I mg/kg/24 h). However, symptoms progressed; he developed a severe exudative, sterile pharyngitis, smears of which revealed sheets of eosinophils and TABLE I. Haematological values 9 Feb. I973
Hb (g/dl) PCV WBC x 1o9/I. % PMN Bands Eos metamyelocytes Eos myelocytes Eosinophils Lymphocytes Monocytes Basophils Blasts
14.0 0.41 16.4 7.5 2.0
82.5 8.0
-
20
March I973
17 April I973
10.6
12.0
0.3 I 31.5
4.0 2.5 12.5
4.5 75.0 1-5 -
0.36 11.4 13.0 3.0
-
70.0 11.5 1.0
24 May I973
14Jttne I973
13.2 0.37 49.5 1.5 -
12.5 0.42 4.35 68.0
0.39 55.1
0
7.0
27.0
11.0
11.0
2.0
16.0
11.0
2.0
I .o
-
-
2.0
55.0
91.5 6.0
-
I973
4.0 -
-
-
14.0 0.40 23.4
1s Oct.
9.0 3.0 2.0
4.0 -
-
27JuIy I973
-
55.0 10.0
eosinophilic debris. Following the sixth dose of vincristine, bone marrow contained 30% blast forms with 29% eosinophils in all stages of maturation. His peripheral eosinophil count was 45.3 x 10~11. (24 May 1973). A 3 day course of Daunorubicin (60 mg/m2) was started. Within 72 h his pharyngitis cleared, he became afebrile and spleen size decreased. He achieved complete remission by 14 June 1973. Peripheral and bone marrow eosinophilia disappeared while in remission. In spite of maintenance therapy, he relapsed in 6 weeks. Eosinophilia returned with the onset of symptoms. Bone-marrow lysozyme level, measured by the method of Osserman (Osserman& Lawlor, 1966) was 22 mg/l. (normal 10-20 mg/l.) ; serum lysozyme was normal at 12 mg/l. Electrocardiogram and cardiac silhouette on chest X-ray were normal. Attempts to re-induce remission with Daunorubicin, POMP (prednisone, vincristine, methotrexate and 6-mercaptopurine) and a combination of cytosine arabinoside plus 6thioguanine failed. In early December 1973, chest X-rays showed bilateral pneumonia and he died in respiratory failure on 10 December 1973. Permission for autopsy was not granted.
Act& Eosinuphilic Leiikaeniin ?
FIG I. Light photomicrographs. (a) Peripheral blood eosinophils, oiic with ‘iioiiscgiiieiitcd’ iiucleus, but both having large eosinophilic granules and palc blue-grey cytoplasm (b) Bone marrow dciiioiistratitig inyeloblasts (thick arrows) surroundcd by cosinophils (thin arrows) in all stages of maturation. (c) Bone inarrow with large promyclocytc (thick arrow) containing large eosiiiophilic graiiulcs. Note that surrouiidirig cells are also eosinophils (thin arrows), whose granules arc similar to those sccn iii peripheral blood. Wright’s stain x 4500. (Fucbzg p 66)
R. S. Weinger et a1
FIG2 . Maturc polymorphonuclear neutrophil (blood). Note the 'beak' shape of the nucleus (N) (thick arrows). Thin splinter-shaped channels (thin arrows) are seen in the cytoplasm. Several azurophilic primary granules (pg) and neutrophilic secondary granules (sg) are also seen in the cytoplasm together with abundant glycogen (gl). x 20 700.
FIG 3 . Mature eosinophil (blood). Its nucleus (N) with clumped chromatin is mature. Its cytoplasm exhibits both signs of maturity: abundant glycogen (gl), and of immaturity: conspicuous Golgi apparatus (G) and numerous flattened RER cisternae (RER). The cytoplasm also contains immature eosinophilic granules (ig). x 17 100.
Acute Eosinophilic Leukaemia?
FIG 4. Eosiiiophilic granules (detail of Fig 3). Most of them arc not holnogcncously electron dcnse. One is aiigulatcd (double arrow) and cxhibits an irrcgular crystalline pattern (thin arrows). One granulc contains a crystal (thick arrow) whose lattice pattern is shown in the insert. x 80 ooo (insert x 120000).
R. S. Weinger et
a1
FIG 5 . Myeloblast (bonc marrow). Notc thc abnormal indented shape of its iiucleus (N) and its large nucleolus (n). Its cytoplasm packed with single ribosoincs contains niyelinic profiles (arrows) and abundant niitochondria (m). x 18 900. FIG 6. Promyelocytc eosinophil (bone marrow). This cell has a nucleus (N) with finely marginated chromatin. Its cytoplasm has nunierous electron light RER cisternae (RER) and a well-developed Golgi zone (G). No glycogen is seen. Numerous vacuolcs (V) are present. Abundant electron dense granules (9) are visible, one of them angulated (arrow). x 19 800.
Acute Eosinophilic Leirkaemia ?
67
METHODS Heparinized blood (or bone marrow) was centrifuged at 400 g for I 5 min at 4" C. Plasma was withdrawn and replaced by 2.5% glutaraldehyde (Leduc et a / , 1963) in 0.1 M phosphate buffer, pH 7.4. After I h, the buffy coat was lifted so that the fixative would be in direct contact with its undersurface and fixation was allowed to proceed for an additional hour. The buffy coat was then washed with multiple changes of 0.1 M phosphate buffer, pH 7.4, cut into tiny fragments which were fixed for another hour in 2.5% glutaraldehyde. After further washing in phosphate buffer, the pellet was post-fixed in I % tetroxide, dehydrated in graded alcohols and embedded in Epon (Luft, 1961). Thick sections (0.5 pm) were cut on a Porter Blum microtome and stained with azure blue and mcthylene blue (Richardson et al, 1960). Desired blocks were selected under the light microscope. Ultra-thin sections were mounted on uncoated grids and stained with 0.5% a!cohol solutions of uranyl acetate for 5 min, counterstained with 0.1% lead citrate (Venable & Coggershall, 1965) for 5 min. The sections were examined with HU-IIE Hitachi electron microscope (75 kV, objective aperture 50 pm). RESULTS
Electron Microscopic Findings Peripheral blood and bone marrow were studied during relapse (27 July 1973). Peripheral blood. Polymorphonuclear neutrophils and eosinophils were prominent. Lymphocytes, monocytes and basopliils appeared decreased in number but their ultrastructure was normal. A few polymorphoiiuclear neutropliils had atypical features both in their nucleus which presented a beak-like shape, and in their cytoplasm which contained splinterlike channels (Fig 2). Typical mature eosinophils were never seen. The cells observed, about 8-9 pm in diameter, exhibited nuclear and cytoplasmic asynchrony. Nuclear maturity was demonstrated by its multiple lobes and its condensed and marginated chromatin. The cytoplasm showed some signs of maturity: it was dense with single ribosomes and contained abundant particulate glycogen. It also exhibited features of an immature cell-numerous flattened rough endoplasmic reticulum (RER) cisternae and immature eosinophilic granules (Fig 3 ) . These granules were about 0.5-1.5 p m in maximum diameter, regularly dense and bound by a unit membrane; most appeared round or ova!-only a few were angulated. Although most of them had atypical crystalloid structures, a few contained crystals with a lattice pattern (Fig 4). Bone marrow. Three types of cells were prominent in the bone marrow: (I) undifferentiated blasts, (2) myeloblasts, and ( 3 ) abnormal eosinophilic myclocytes. The undifferentiated blasts were Unremarkable. Numerous myeloblasts had an irregularly shaped, deeply indented nucleus and myelinic profiles in their cytoplasm (Fig 5 ) . Only a few rnyelocytes and metamyelocytes of the neutrophilic and basophilic series were secn. Their ultrastructure was unremarkable. In contrast, all the cells of the eosinophilic type appeared abnormal. Nuclear-cytoplasmic asynchrony was a constant feature. The cell most often seen was one with the nucleus of a myelocyte and the cytoplasm of a youiigcr cell
68
R.S . Weinger et al
(promyelocyte) of the eosinophilic granulocytic line. This cell (Fig 6), about 8-10 p m in diameter, had a high N / P ratio. Its round or slightly indented nucleus had marginated chromatin and, often, a conspicuous nucleolus. Its cytoplasm had abundant RER with moderately distended cisternae and a well-developed Golgi apparatus which did not contain any dense material. Spherical and oval, membrane-limited vacuoles, 0.5-0.7 p m in diameter were present. Their content varied from a coarsely granular material to a fenestrated or homogeneous matrix. Such formations with a homogeneous clectron dense content were the most numerous and constituted granules. No glycogen, no sign of phagocytosis were seen in these cells. DISCUSSION This patient had a marked eosinophilia with what first seemed to be acute lymphoblastic leukaemia. Hisclinical features and morphological picture by light microscopy were reminiscent of those cases of ALL reported by Spitzer & Garson (1973). Treatment was directed towards that entity and failed until Daunorubicin was given and this successfully induced remission. His eosinophilia, both peripheral and marrow, disappeared. Upon relapse, further studies, including serum and bone marrow lysozyme determinations, were more compatible with a nonlymphoblastic leukaemia. By electron microscopy, the blast cells clearly fit the criteria for myeloblasts. Furthermore, the granulated cells seen by both light and electron microscopy were indeed positive for cyanide-resistant peroxidase. It is generally accepted that the multipotential stein cell or ‘undifferentiated blast’ gives rise to myeloblasts, which then mature into promyelocytes, myelocytes, metamyelocytes and mature granulocytes (Ford Bainton & Farquhar, 1966; Wetzel et a ] , 1967; Scott & Horn, 1970), whether neutrophilic, basophilic or eosinophilic. In the patient studied here, no morphological alteration of the undifferentiated blast was seen. The number of these cells was few in the bone marrow and they were not observed in the blood. Myeloblasts were increased in number in the bone marrow but not present in the blood until late in the disease. By electron microscopy, it is not possible to know what kind of promyelocytes-neutrophil, basophil, eosinophil-will arise from a given myeloblast because no granules are seen in that cell. In this patient the morphology of most myeloblasts was unremarkable. Some, however, had irregularly shaped nuclei and a few myelinic profiles; these cells might represent precursors of the abnormal eosinophils. The eosinophils were quantitatively and qualitatively abnormal. Quantitatively, there was an increased number of these cells both in the bone marrow and in the blood. Qualitatively, their maturation was ‘anarchic’ (Bessis & Breton-Gorius, 1969) in two respects: (I) nucleus and cytoplasm were not maturing at the same rate; (2) in the cytoplasm itself some organelles were more mature than others. In the bone marrow, the patient had promyelocytic eosinophils with a cytoplasm similar to that of the ‘early eosinophil’ described by electron microscopy by Scott & Horn (1970). As in the present case, cytoplasmic vacuoles were observed and it was concluded that ‘the progressive accumulation of dense material within the vacuoles caused them to be transformed into. . . granules.’ Thc authors named these ‘immature granules’ because, as shown by the study of the development of eosinophil granulocytes in human bone marrow, these granules later give rise to the elongated crystalloid-containing
Acute Eosinophilic Leukaemia?
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granules of the mature eosiiiophil. In the blood of the patient, these cells contained some angulated granules. More important, a few granules presented a cubic lattice pattern previously shown to be typical of the core of eosiiiophil granules in man (Miller et al, 1966). Both the atypical cosinophils seen in the blood and tlie proniyclocytes in the bone marrow were eosinophils by light microscopy and chemical reactions. The unusual features observed in the mature polyniorphoiiuclear neutrophil are difficult to interpret. The myelinic profiles are not specific and have been described in myeloid cells in myeloproliferative disorders (Tavassoli & Weiss, 1973). The electron light, splintershaped channels do riot seem to represent extruded Auer bodies (Freeman, 1960); these would appear thicker aiid larger and were never seen in this patient. The beak-like appearance of their nuclei may be related to prior therapy. This patient’s blood aiid bone marrow contained numerous undifferentiated blasts and abundant abnormal eosinophilic granulocytes which could be recognized as early as the promyelocytic stage. Whether this picture represents an acute leukaemia with a prominent eosinophilic reaction, as in cases previously reported in the literature (Spitzer & Garson, 1973), or a true eosinophilic leukacmia is impossible to state at the present. It is only when new histochemical criteria might allow us to pinpoint differences between blasts giving rise to different lines of cells that such a diagnosis is possible. The scarcity of EM reports of cases such as tlie present one, make it worthwhile to add the present observation to the still coiitrovcrsial dossier of the ‘eosinophilic leukaemias’. ACKNOWLEDGMENTS
The authors wish to thank Dr Avrum Z . Bluiniiig for his assistance during this case study. This work was aided in part from the University Cancer Committee, American Cancer Society, Institutional Grant N-23-0. REFERENCES BESSIS,M. & BRETON-GORIUS, J. (1969) Pathologie ct asynchronisme de d6veloppement des organellcs cellulaires au cows des leucdmies aigues granulocytaircs. Norrvelle Reoiir Franfaise d’Hhatolo@r, 9, 24.5. FORDBAINTON,D. & FARQUHAR, M.G. (1966) Origin of granules in polymorphonuclear leukocytes. Two types derived from opposite faces of the Golgi complex in dcveloping granulocytes. journal (rf Cell Biology, 28, 277. FREEMAN, J.A. (1960) The ultrastructure and genesis of Auer bodies. Blood, 15,449. HARDY,W.R. & ANDERSON, R.E. (1968) The hypercosinophilic syndromes. Annals OJ’ Internal Medicine, 68,
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LEDUC,E., MARINOZZI, V. & BERNHARD, W. (1963) The use of water-soluble glycol tnethacrylate i n ultrastructural cytochcmistry. joirmal of thr Roynl Micosropical Society, 81, I 19. LUFT, J.H. (1961) Improvements in epoxy resin
embedding methods. Joorrr~al of Biophysical and BiocRemical Cytology, 9, 409. MILLER,F., DE HARVEN,E. 81 PALADE,G.E. (1966) Thc structure of eosinophil leukocyte granules in rodents and in man. Journal of Cell Biology, 31, 349.
OSSERMAN, E.F. & LAWLOR,D.P. (1966) Scrum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukcmia. Journal of Experimental Medicine, 124,921. RICHARDSON, K.C., JARETT, L. & FINKE,E.H. (1960) Eni’ocdding in epoxy rcsins for ultrathin sectioning in electron microscopy. Stain Technology, 35, 313. SCOTT,R.E. & HORN,R.G. (1970) Fine structural features of eosinophile granulocyte development in human bone marrow. joumal of Ultrastrrtcture Research, 33, 16. SPITZER,G. & GARSON,O.M. (1973) Lymphoblastic leukemia with marked eosinophilia: a report of two cases. Blood, 42, 377.
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TAVASSOLI, M. & WEISS,L. (1973) An electron microscopic study of spleen in myelofibrosis with mycloid metaplasia. Blood, 42, 267. J.H. & COGGERSHALL, R. (1965) A simplified VENABLE, lead citrate stain for use in electron microscopy. Journal of Cell Biology, 24, 407. WETZEL,B.K., HORN, R.G. & SPICER,S.S. (1967)
Fine structural studies on the development of hcterophil, eosinophil, and basophil granulocytes in rabbits. Laboratory Inuestigation, 16,349. YAM, L.T., LI, C.Y. & CROSBY,W.H. (1971) Cytochemical identification of monocytes and granulocytes. American Journal of Clinical Pathology, 55, 283.
