Eritirhjournal of Haematology, 1980, 45, 535540.

Histological Study of Bone Marrow Regeneration following Chemotherapy for Acute Myeloid Leukaemia

and Chronic Granulocytic Leukaemia in Blast Transformation

ANWARUL ISLAM, DANIEL CATOVSKY AND DAVID A. G. GALTON

MRC Leukaemia Uni t , Royal Postgraduate Medical School, London

(Received 18 December 1979; accepted f o r publication 20January 1980)

SUMMARY. Haemopoietic regeneration following chemotherapy was studied on serial bone-marrow trephine specimens from 10 patients with acute myeloid leukae- mia (AML), and seven with chronic granulocytic leukaemia (CGL) in blast transfor- mation. In AML following the stage of treatment-induced hypoplasia during which the marrow was extremely hypocellular, oedematous, and contained widely dilated sinuses, areas of large, uniform, unilocular fat cells, designated ‘structured fat’ developed from multilocular precursor fat cells. Early foci of haemopoietic regene- ration were present almost exclusively in areas of structured fat: only scattered $mall erythropoietic foci were seen in the earliest specimens taken 1 week after the completion of treatment; small granulopoietic foci were first seen 2 weeks later, arid the first megakaryocytcs were not seen until the third week. Transient regeneration was observed in two patients who subsequently failed to enter remission and died.

The pattern of haemopoietic regeneration in CGL in blast transformation treated by intensive therapy and autografting with cryopreserved buffy-coat cells differed from that seen in AML. Bone marrow regeneration occurred earlier than in AML and appeared to be independent of the presence of structured fat. Regeneration in all three cell lines appeared simultaneously within 2 weeks of autografting. Regene- ration tended to be focal, each aggregate consisting almost exclusively of erythroid, granulocytic or megakaryocytic cells.

The proximity of regenerating haemopoietic foci to structured fat in AML suggests that, in some circumstances, haemopoietic stem cells require the presence of fat cells in their immediate environment before they proliferate.

Microenvironmental influences within the bone marrow and spleen are critical for haemo- poietic stem cell proliferation and differentiation in experimental animals (McCulloch et al , 1965; Trentin, 1971; Chamberlin et al, 1974; Cline et al, 1977). One in vitro culture technique for haemopoietic stem cells and their committed progenitors has shown that a bone-marrow-

Correspondence: Dr Anwarul Islam, MRC Leukaemia Unit, Royal Postgraduate Medical School, Uucane Road, London W12 OHS.

0007-1048/80/0800-0535$02.00 0 1980 Blackwell Scientific Publications

535

536 A. Islam, D. Catovsky and D. A. G. Galton

derived adherent-cell layer must be established before stem cells will proliferate, differentiate and undergo maturation (Dexter et al, 1977). The bone-marrow-derived adherent layer contains several cell types, phagocytic mononuclear cells, ‘epithelial’ cells and giant fat-con- taining cells, all of which appear to have counterparts in vivo (Allen, 1977). These cells apparently provide an in vitro microenvironment necessary for pluripotential stem cell renewal and differentiation (Allen & Dexter, 1976). Among the various cell types in the adherent layer, fat cells appear to be particularly important for the maintenance of the haemopoietic stem cells and their growth in the mouse marrow stromal cultures (Dexter et al, 1977). The only information available in the literature about the relationship of fat cells to haemopoiesis are those obtained from in vitro culture systems. We have found no documentary evidence of the role of these cells in haemopoietic regeneration in man.

We have obtained serial bone-marrow trephine specimens following the completion of remission-induction chemotherapy from patients with acute myeloid leukaemia (AML) and chronic granulocytic leukaemia (CGL) in blast transformation and studied the sequence of events during marrow regeneration. Our study suggests that in vivo as well as in vitro, fat cells may play an important role in haemopoietic regeneration.

MATERIALS AND METHODS

Bone-marrow trephine specimens were obtained from 10 AML patients at diagnosis and at different time intervals during the course of intensive remission-induction chemotherapy and after its completion. One to three specimens were obtained from each patient during the first 8 weeks of observation and in one patient additional specimens were obtained at weeks 10 and 12. Specimens were also obtained from seven CGL patients in blast transformation at the time of diagnosis and after marrow reconstitution by infusion of cryopreserved autologous buffy- coat cells following intensive therapy (Goldman et a/, 1979). One or two specimens were obtained from each patient during the first 4 weeks after autografting. AML patients were treated with one to four courses (two in seven cases) of the RATE protocol (daunorubicin, cytosine arabinoside, 6-thioguanine and the epipodophyllotoxin (VP16-213) (Goldman er al, 1979) and CGL patients in blast transformation were treated with the RATE protocol followed in some instances by cyclophosphamide at high dosage and whole body irradiation (600,800 or 1000 rads) (Goldman et al, 1979).

The bone marrow specimens were taken with an 11-gauge Jamshidi needle from the posterior iliac crest and were processed in methyl-methacrylate (Burkhardt, 1971) from which 3 pm sections were cut, without decalcification, with a tungsten carbide knife in a Jung’s high performance microtome (Autocut). Sections were softened in 96% ethanol and were then attached to gelatinized slides by gentle brushng with a very soft brush. The sections were stained with May-Griinwald Giemsa (MGG) and the methacrylate was dissolved from the sections by immersion in benzene before staining.

RESULTS

Following intensive chemotherapy for both AML and CGL in blast transformation the bone-marrow appearance can be seen to go through three clearly defined stages: first, is the

A4arrow Regetreration in Leukaemia

FIG 1. BM section from case 3 (Table 11) 1 d after the completion of intensive therapy. Acute oedematous stage, showing extremely hypocellular marrow, gross oedema and widely dilated sinuses (MGG Ftain, x 320).

FIG 2. BM section from case 4 (Table I) 1 week after thc completion of treatment showing the appearance of young multilocular fat cells (arrow) (MGG stain, x 480).

(Facing p. 536)

A. Islam, D. Catovsky arid D. A. G. Galtcn

FIG 3. Section from case 4 (Table I) 1 week after the completion of treatment showitig two young multilocular fat cells (MGG btain, x 1500).

Marrow Regeneration in Leukaernia 537

acute oedematous stage when the marrow becomes extremely hypocellular, grossly oedcma- tous with widely dilated sinuses. This stage is more pronounced in the specimens obtained immediately after therapy from patients with CGL probably because of the greater intensity of the treatment administered (Fig 1). In the second stage, young multilocular fat cells containing varying numbers of discrete fat droplets of varying size are present (Figs 2 and 3) . A few large unilocular fat cells may be seen. The bone marrow is still oedematous, hypocellular, and shows dilated sinuses. In the third stage, the oedema and vascular dilatation of the preceding stages have largely disappeared, and the bone marrow is occupied by relatively uniform large unilocular fat cells in close apposition between which islands of regenerating haemopoietic cells are prescnt (Figs 4 and 5). We use the term ‘structured fat’ to describe areas of tissue consisting of ncwly formed large unilocular fat cells in close apposition. The features of the second and third Ftages were more clearly defined in AML following the cessation of chemotherapy, as endogenous marrow recovery took place, than in the CGL patients who received autografts. The features of the second stage overlap with those of the first and third stages in varying degree and this can be seen in different areas of the same section. In order to draw attention to the close relationship between the appearance of structured fat and haemo- poietic regeneration, we have recorded the amount of structured fat in Tables I and 11.

Pattern of Haemopoietic Regeneration in AML (Table I ) Sections of trephine specimens taken 1 week after the end of chemotherapy (cases 4 and 6)

showed numerous multilocular fat cells and small areas of structured fat. Sections of specimens from seven patients 2 weeks after the end of Chemotherapy showed the presence of moderately abundant structured fat. Abundant structured fat was present in specimens taken 3, 4 and 8 weeks after cessation of therapy. Foci of haemopoietic regeneration were always closely

TABLE 1. Results of serial bone-marrow biopsies in patients with AML

Weeks a j e r cessation of chemotherapy Response to

c a s e 1 2 3 4 8 treatment

+ + + (egm)

+ + + (c) NR NR CR + + + (egm) CR CR CR +++ +++ NR NR + + + (egm) CR + + + (egm) CR

+ =Small areas of structured fat; + + =moderately abundant struc- tured fat; + + + =abundant structured fat. NR=no remission; CR=com- plete remission. (e) =Regeneratioti mainly erythroid; (eg) =regeneration mainly erythroid and granulocytic; (egm) = good regeneration consisting of erythroid, granulocytic and megakaryocytic cells.

538 A. Islam, D. Catovsky and D. A. G. GaIton TABLE 11. Haemopoietic regeneration in CGL in BC following intensive therapy and marrow

reconstitution by autograft -

Weeks after autograj Therapy before Establishment of

Case autografi I 2 3 4 chronic phase CCL

1 RATE x 1 +HD Cyclo + (egm) Died

3 RATEx2+HD Cyclo+ + (egm) + + (egm) Yes

4 RATEx2 + (egm) Yes

2 RATE x 2+HD Cyclo + (egm) YeS

TBI (lo00 rads)

5 RATE x 2 + HD Cyclo + + (egm) + + + (egm) Yes TBI (800 rads)

TBI (1200 rads)

TBI (1200 rads)

6 RATE x 1 +HD Cyclo + (egm) Died

7 RATE x 1 + HD Cyclo + + (eem) Yes

RATE=daunorubicin, cytosine arabinoside, Gthioguanine and epipodophyllotoxin (VP16-213). HD Cyclo =high dose cyclophosphamide. TBI = whole body irradiaton. Abbreviations and signs as in Table I.

associated with areas of structured fat. One week after the end oftreatment almost all foci were erythropoietic; granulopoietic foci were also present in four of the five specimens taken at the end of week 2, but megakaryocytic foci were seen only in samples taken at the end of week 3 and later. Four patients did not enter complete remission; two of them (cases 2 and 8) died of recurrent infections after 6 and 4 weeks without sufficient haemopoietic regeneration to maintain normal neutrophil and platelet counts. Nevertheless, in both cases the sections showed moderately abundant structured fat with some erythropoietic and granulopoietic foci at the end of the second week. Adequate bone-marrow regeneration did not occur in the other two patients who failed to enter complete remission (cases 1 and 7) . The marrow sections showed severe hypoplasia, but moderately abundant structured fat was present at the end of the fourth week. Some evidence of erythropoietic regeneration was seen in case 1 at the end of the fourth week, but this patient relapsed 10 weeks later. No evidence of haemopoietic regene- ration was seen in the marrow sections in case 7 (Fig 6) at the end of weeks 4,s and 10. He died of intercurrent infection 12 weeks after treatment was stopped without evidence of regene- ration in the specimens obtained at post mortem.

Pattern of Haemopoietic Regeneration in CCL in Blast Transformation (Table 11) In CGL the pattern of haemopoietic regeneration differed from that seen in AML. Evidence

of haemopoietic regeneration with cells of all the three cell lines was seen within 2 weeks of autograft when the structured fat was only just beginning to appear; the latter was not seen abundantly before the end of the fourth week. In addition, marrow regeneration was noted to be more discretely focal than in AML, each focus consisting almost entirely of erythroid (Fig 7), granulocytic (Fig 8) or megakaryocytic cells (Fig 9) and mitotic figures were frequently observed among the cluster of cells (Fig 7) . Five out of seven patients in this group reverted to

Marrow Regeneration in Leukaenria

FIG 4. Section from case 5 (Table I) 3 weeks after the completion of treatment. Abundant structured fat with some oedema between the fat cells. Erythroblasts (c), granulopoistic cells (g) and two megakaryocytes (m) are present (arrows) (MGG stain, x 320).

FIG 5. Section from case 4 (Table I) 4 weeks after the complction oftrcatmcnt. Active haemopoictic regeneration is seen in association with structured fat (MGG stain, ~ 3 2 0 ) .

(Facing p . 538)

A. Islam, D. Catovsky and D. A. G. Gulton

FIG 6. BM section from case 7 (Table I) 8 weeks post-therapy. Abundant structured fat with some oedema between the fat cells but no evidence of haemopoietic regeneration (MGG stain, x 320).

FIG 7. BM scction from case 7 (Table Jl) 2 weeks after the autograft showing a focus consisting almost entirely of erythropoietic precursor cells among which mitotic figures remain prominent (arrows) (MGG stain, x 1200).

Marrow Regeneratiori in Leukaemia

FIG 8. BM section from case 3 (Table 11) 3 weeks after the autograft, showing a focus consisting entirely of granulopoietic prccursor cells (MGG stain, x 480).

Fit; 9. BM section from case 5 (Table 11) 2 weeks after the autograft, showing a focus consisting almost entirely ofjuvcnile and mature rncgakaryocytes (MGG stain, x 480).

Marrow Regeneration in Letikaernia 539

chronic-phase CGL after receiving the autograft. Cases 1 and 6 died of intercurrent infection shortly after regeneration was observed.

DISCUSSION

It is generally accepted that normal haemopoiesis is influenced to a considerable extent by the stromal component of the marrow microenvironment. Marrow stromal cells in culture have also been found to enhance the survival and proliferation of haemopoietic stem cells by means of diffusable factors and probably by direct cell contact (Blackburn & Patt, 1977, 1980).

I n an in vitra culture system for mouse haemopoietic cells, Dexter et af (1977) have shown that CFUs (pluripotent stem cells that form colonies in the spleen of lethally irradiated mice) CFUc (granulocyte-monocyte precursor cells that form colonies in culture) and BFUe (erythrocyte precursor cells that form colonies in culture) all show enhanced proliferation and survival when the mouse marrow cells are added to the cultures of mouqe marrow stromal cells. These stromal cell cultures contained epithelial cells, monocytes and fat cells. The fat cells appear to be important for the maintenance of the haemopoietic stem cells and their growth in the mouse marrow stromal cultures, although under different culture conditions they may not be essential (Greenberger et af , 1980). In Dexter’s culture system granulocyte proliferation seems to take place preferentially in regions containing fat cells, particularly on the periphery of such areas where cells are in an early stage of lipid accumulation (Allen, 1977). Our observation in vivo also suggests that fat cells may play an essential role in human haemopoietic regeneration.

In AML the appearance of young multilocular fat cells followed by the establishment of areas of tissue consisting of large uniform unilocular fat cell? in close aposition which we have designated ‘structured fat’ appeared to be a regular occurrence after the elimination of the leukaemic cells by therapy. Haemopoietic regeneration was seen to occur only within areas of structured fat. However, although in AML haemopoietic regeneration was not observed except within areas of structured fat, the establishment of structured fat was not invariably followed by haemopoietic regeneration. Patient 7 (Table I) showed no evidence of marrow regeneration in trephine sections taken 2, 4, 8, 10 and 12 weeks after the completion of the second course of treatment. All the sections showed moderate to abundant structured fat. There was clinical, haematological and cytomorphological evidence in case 7 that the AML had arisen on a background of myelodysplasia, and it is possible that normal stem cells were not present at all in this patient’s bone marrow and that the intensive therapy he had received had destroyed the myelodysplastic stem cells as well as the leukaemic cells.

In contrast to the pattern of regeneration seen in AML, the association of haemopoietic foci with areas of structured fat was not obvious in blast transformation of CGL. In these cases the hypoplastic stage, characterized by grossly dilated sinuses and oedema, was succeeded by the early and simultaneous appearance of individual foci of erythropoietic, granulopoietic and megakaryocytic cells which were not clearly anatomically related to areas of structured fat and which were sometimes at a relatively advanced stage when only small areas of structured fiat were present. Frequent mitotic figures among the homogeneous cell clusters suggest that these foci arose from the local proliferation of precursor cells. Discrete clustering of haemopoietic

540 A. Islam, D. Cafovsky and D. A. G. Galton cells has also been observed in marrow specimens of aplastic anaemia patients and in leukaemic patients after bone marrow transplantation (Cline et al, 1977).

It is not possible to explain the difference between the patterns of haemopoietic regeneration in AML and CGL. Perhaps CGL haemopoietic stem cells are less dependent on the proximity of fat cells than the regenerating stem cells in AML, which are believed to be essentially normal. Alternatively, the difference may reflect different growth requirements of the endo- genous stem cells in the AML patients, and the transfused autologous stem cells among the cryopreserved chronic-phase peripheral blood leucocytes from which the early haemopoietic regeneration in the CGL patients is believed to have taken place (Goldman el al, 1979).

In spite of the different patterns ofhaemopoietic regeneration observed in our two groups of patients, the close association of regenerative haemopoietic foci with structured fat in the AML suggests that the fat cells play some part in haemopoietic regeneration in some circumstances. From the evidence so far available, further speculation on the role of the fat cells in haemo- poiesis is not warranted.

ACKNOWLEDGMENTS

We wish to thank Mr W. Hinks for taking all the photomicrographs, and Mrs D. Haysome and Miss S. Masterson for typing the manuscript. A.I. is a Research Fellow and grant holder of the Leukaemia Research Fund.

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