CELLULAR DYSMATURITY AS A PROGNOSTIC INDEX IN ACUTE MYELOID LEUKAEMIA Liam G. O'Connell, Angela Gorman, Thomas J. Kea.ne, James J. Fennelly

Departments of Haematology and Oncolcgy, St. Vincent's Hospital, Elm Park, Dublin 4. Summary HE bone marrow and peripheral blood findings of 22 selected patients with acute myeloid leukaemia were studied. A correlation was detected between the incidence of attainment of complete remission and the balance of nucleo-cytoplasmic differentiation in the neutrophil series. The prognostic significance of this finding and its relevance to choice of therapy is discussed, as are the possible underlying causes for the phenomenon.

T

Introduction The human acute leukaemic process is characterised by two striking cytological qualities. Firstly, there is virtually uncontrolled mitotic activity on the part of the leukaemic stem cells. Secondly, there is a tendancy for the maturation process to become arrested at a particular morphological stage. A third, less constant feature, is the presence of abnormal patterns of maturation. For example, abnormal granulation may develop in the cytoplasm, or normal granulation may be diminished or completely absent. These maturation aberrations present in peripheral blood or bone marrow smears stained by the Romanowski method have been recognised for a very long time. (Hayhoe and Cawley, 1972; Kass, 1973; Bessis, 1956.) More recently, the application of a wide variety of cytochemical procedures and the use of electron-microscopy have permitted some refinement in the classification anl quantitation of many of these abnormalities (Cawley and Hayhoe, 1973; 8essis, 1973). Very recently, the application of immunological markers in the study of the lymphatic group of diseases seems certain to result in a very significant advance. To date, however, these maturation aberrations have been used mainly for diagnostic purposes. While the intimacy of the link between maturation defects and the basic leukaemic process is as yet unclear, evidence is accumulating which suggests that those cases of acute myeloid leukaemia (AML) which supervene on a "pre-leukaemic" state respond unfavourably to cytotoxic drug therapy. One of the striking features of many of these "pre-leukaemic" states is a cellular marrow displaying multiple maturation defects. We considered the possibility that the presence of similar maturation defects in the bone-marrows {before treatment) of patients presenting with "classical" AML, influences the prognosis when a recognised and effective cytotoxic drug routine was used in their treatment. The study reported here was intended as a pilot study only, but the results were so striking that their publication at this time seemed justified. 35

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Materials and Methods The study is based on the examination of specimens from 22 patients with AML who were admitted consecutively to our unit during the years 1971 and 1972. In all, the diagnosis of AML was substantiated by the absence of granular, P.A.S.-reacting material in the primitive forms, and the presence of Sudanophilic and/or myelopercxidase-positive material in over 5 per cent of these cells (Hayhoe and Cawley, 1972).

The treatment involved the use of daunorubican, cytosine arabinosicle and vincristine, given in a series of five-day courses interupted by suitable recovery periods, averaging seven days in length (Gorman eta/., 1972). All patients who achieved complete remission (CR) did so after one to four such courses, and were then given one further course. Supportive therapy throughout was standard. The overall CR rate was 50 per cent. The data was examined in two ways. Firstly, all initial pre-treatment bone marrow samples were examined "blindly" by three of the authors (L.O'C., T.K. and A.G.) and assessed for (1) the overall balance of nucleo-cytoplasmic differentiation in the neutrophil series, with particular reference to the quality and quantity of cytoplasmic granulation. The marrow smears were all made by the slide technique and stained by the Wright-Giemsa procedure. When all these observers reached a common grade for each specimen, the code was broken, and the grading compared with the course of the disease. Examples of the criteria on which the assessment was made are shown in Figs. 1-6. Bearing in mind the subjective element involved, it is remarkable that complete agreement was reached initially in the grading of 15 of the 22 marrows. In the remaining seven, minor disagreement was resolved on review before the code was broken. The second assessment involved serial examination of all the peripheral blood smears available for each patient. These had also been prepared by the slide technique and stained by the Wright-Giemsa procedure. It was not

Fig. 1--Myeloblastic Leukaemia. Uniform cell type with minimal but normal maturation. (Wright-Giemsa X 1200).

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Fig. 2--PromyelocytiC Leukaemia. Uniform cell type, with normal maturation (Wright-Giemsa X 1200).

possible to perform this assessment blindly because the number of smears available was directly related to the success of the treatment regime. Nonetheless, a clear cut pattern of neutrophil granulation emerged, based on the following standard : (1) If more than 80 per cent of the "neutrophils" present in the peripheral blood consistently showed normal granulation, the patient was rated "positive"; while (2) If more than 80 per cent of the "neutrophils" consistently displayed diminished or absent granulation, the patient was

Fig. 3--Myelocytic Leukaemia, with normal maturation (Wright-Giemsa X 1200).

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Fig. 4--Myelocytic Leukaemia, with abnormal maturation (Wright-Giemsa X 1200).

Fig. 5--Myelocytic Leukaemia, with abnormal maturation (Note also abnormal erythroblast) (Wright-Giemsa X 1200).

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Fig. 6--Myelocytic Leukaemia, with grossly abnormal granulation, of the Chediak-Higashi type (Wright-Giemsa X 1500).

rated "negative". The term "neutrophil" was applied to both morphologically typical neutrophiEs, and also to those cells whose nucleus and cytoplasm resembled those of the classical neutrophit in all features, except in the lack of the characteristic secondary granulation. We have also included in this classification, cells which fit Hayhoe's description of the para-neutrophil (Hayhoe and Cawley, 1972). Examples of the criteria on which this assessment was based are shown in Figs. 7 and 8. In practice, the divergence between the "positive" and the "negative" groups was such that no equivocation in this assessment arose.

Fig. 7--Neutroph'il granulocyte, with normal granulation. (Wright-Giemsa .X 1400).

Fig. 8--" Granulocyte ", with absent granulaton (Wright-Giemsa X 1400).

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Results The results of the first assessment are shown in Table I. Two points emerge. Firstly there is no correlation between the level at which maturation arrest occurred, and the atainment of a complete remission. Secondly, 11 patients attained complete remission, and all were adjudged to have balanced or symmetrical maturation of the neutrophil series. On the other hand, 11 failed to attain CR, and of these, nine were adjudged to have unbalanced or asymmetrical maturation characteristics. TABLE I Bone Marrows: Assessment of Nucleo-Cytoplasmic Maturation Complete Remission Predominant Cell Form Normal Myeloblast Promyelocyte Myelocyte.

No Complete Remission Abnormal

Normal

Abnormal

0 0 0

1 0 1

4 2 3

3 5 3

The results of the second assessment are presented in Table I1. Of the 11 patients who attained CR nine showed consistently normal or increased neutrophil granulation. On the other hand, eight of the 11 patients who failed to attain a CR showed consistenly diminished or absent neutrophil granulation. One of the patients who attained a CR, but later relapsed after three months is of special interest. Following CR, her neutrophils displayed normal " s e c o n d a r y " granulation, but immediately preceeding clinically detectible relapse, a pattern of reduced granulation appeared, Figs. 7 and 8. TABLE II Peripheral Blood : Assessment of Neutrophil Cytoplasmic Granulation Complete Remission Positive

Negative

9

2

No Complete Remission Positive 3

Negative 8

Discussion From a practical point of view, the results of our study suggest that the presence of a defect in nucleo-cytoplasmic balance or of cytoplasmic granulation in the neutrophil series in a patient with AML is associated with a very reduced possibility of attaining a complete remission. We suggest therefore, that in these patients at the present time, the aim should be control of, rather than complete remission in, their disease, at least until more effective drug combinations are available. In this way, iatrogenic damage to the

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patient's natural defence mechanisms against both the disease process and against opportunistic infections would be minimised. Many of the miseries of intensive therapy would thus be avoided. A surprising number of these patients do in fact manage to return to their own homes to live in reasonable comfort for a number of months. On the other hand, we feel that a sustained attempt should be made to attain a CR in those patients who show no significant morphological abnormality in the neutrophil series. These results also have some theoretical significance. The criteria of a CR emphasise the importance of both major components in this remission : that of the destruction of the leukaemic tissue, and that of the repopulation of haemopoietic organs with normal tissue. The relationship of maturation defects to prognosis may be to either or both of these factors. The action of the drugs used in this regime seems to be exerted mainly on DNA and RNA synthesis, and on cell mitosis (Krakoff, 1971; Jacobs, 1974; Wolfe, 1972). The selectivity of ths therapy would obviously be greatly enhanced if drug-affected pathways specific to the leukaemic cells existed. To date, no such pathway has been demonstrated. In fact, the results of the present study could be interpreted as indicating that in at least 50 per cent of cases, leukaemic stem cells (LSC) had lost dependance on pathways which remained essential for normal stem cells. It seems not unreasonable to postulate that an LSC which generated progeny displaying such gross defects in structure as are discussed above, would be itself defective in other respects; such defects could be expressed as reversion to more primitive metabolic pathways less sensitive to current cytotoxic therapy (Black and Speer, 1957). Such a reversion is well recognised in many solid tumours with the production of foetal substances, eg., production of alpha-feto-protein by hepatic tumours. In view of the specific or optimal activity of these three drugs on particular phases of the cell cycle (Clarkson and Fried, 1971; Jacobs, 1974; Krakoff, 1971; Bernard et al., 1969), it seems clear that the larger the proportion of the target cell population in a sensitive phase of the cycle at the time of drug administration, then the greater will be the cell kill. Thus, the synchronisation of a large proportion of the LSC in germinal cycle, with the bulk of the remaining normal stem cells being "dormant", would confer considerable selectivity on a drug regime. It could therefore be argued as follows, that in those cases of AML which present a gross abnormality in the process of maturation (as opposed to a simple "maturation block" with normal morphology) there is also a defect at stem cell level which introduces an element of unpredictability in response to maturation inducers. This in turn renders remote the possibility of significant synchronisation. In fact, such a state would render the LSC population less vulnerable to these drugs than the normal, non-target tissue. While destruction of the leukaemic population is the first condition for the achievement of a CR, the second, and equally important condition is repopulation of the haemopoietic organs with normal tissue. This latter condition tends to follow readily after initial cell reduction in cases of acute lymphatic leukaemia, but only in about 50 per cent of patients with AML. It has been suggested that depression of the normal stem cell lines is due to a "negative feed-back" effect of the large mass of malignant tissue present.

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If this were the sole explanation, then simple reduction in the numbers of leukaemic cells would b9 followed automatically by return of normal tissue to the bone marrow and blood. Since this occurred in only half of the patients in the present series, an explanation involving the response of normal as well as leukaemic tissue was sought. Firstly, the virtual or complete lack of normally maturing cells of the neutrophil series in eight of the 11 "non-remitters" may simply have been a reflection of the profundity of the marrow involvement by the malignancy; with so little normal tissue remaining that the achievement of the appropriate n o r m a l : leukaemic ratio is impossible, at least with the present relatively ineffective cytotoxic regimes. Secondly, the mature neutrophil may itself play a more fundamental role in the maturation of the dormant normal stem cell population than has been hitherto realised. Heit et al. (1974) demonstrated that the growth of colonies of mature myeloid elements from peripheral blood stem cells jn artificial culture is dependant on the presence of mature mononuclear and neutrophilic forms in the culture medium. Barak et al. (1974) suggested that it is possible to induce A M L stem cells to mature in vitro to apparently normal forms if mature leucocytes are also present in the medium. If substantiated, these results would imply that the ability to mature and the realisation of this ability are essential features of a normal marrow and blood population. It could therefore be argued that those of our patients who achieved CR did so because they retained sufficient maturation capacity (even after the massive cell reduction caused by the induction procedure) to permit the restoration of the normal balance between mature and immature forms. In those of our patients who achieved a CR, over 80 per cent of the polymorph neutrophils were m o r p h o l o g i c a l l y normal; while nine of the 11 who did not remit consistently showed over 80 per cent abnormal polymorph neutrophils. Apart from the prognostic value of these observations, they raise the possibility of the use of granulocyte infusions as maturation inducers in addition to their current use as anti-infective agents. The methods of assessment of maturation employed in this study are crude, and contain a large subjective element. Since 1973, we have applied a wide variety of cytochemical and immunological procedures to pre-treatment bl:ood and bone marrow specimens from all patients with AML and acute m o n o c y t i c leukaemia, in an effort to quantitate maturation abnormalities more accurately. This is time-consuming and some years will elapse before data becomes available. References

Barak, Y., Shore, N. A., Higgins, G. R. Vadakan, V. V. 1974. Functional cellular maturation in cultures of human haemopoietic cells, erit. J. Haemat. 27, 543. Bernard, J., Paul, R., Boiron, M., Jacquillat, J. ~nd Maral, R. 1969. Rec~nt results in cancer research : rubidomycin. Springer-Veriag, Berlin, Heidelberg, New Yor. p. 46. Bessis, M. (1956. Cytology of the blood ~nd blood-forming organs. Grune and Stratton, New York, London. p. 418. Bessis, M. (1973). Living blood cells and their ultrastructure. Springer-Verlag, Berlin, Heidelberg, New York. p. 595.

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Black, M. M. and Speer, F. D. 1957. Biochemistry of cancer with reference to chemotherapy. Abs. Surg. 104, 1. Cawley, J. C. ~nd Hayhoe, F. G. J. 1973. Ultrastructure of haemic cells. W. B. Saunders, London, Philadelphia, Toronto. p. 156. Clarkson, B. D. ~nd Fried, J. 1971. Changing concepts of treatment in acute leukaemia. Med. Clin N. Amer. 55, 572. Gorman, A., Fennslly, J. J., O'Connell, L. G. 1972. Intensive chemotherapy of acute myelogcnous leukaemia. J. Irish Med. Assn. 65, 509. Hayhoe, F. G. J. End Cawley, J. C. 1972. Acute leukaemia : cellular morphology, cytochemistry and fine structure, in "Clinics in Haematology : Acute Leukaemia. 1". W. B. Saunders, London, Philadelphia, Toronto. Heit, W,, Kern, P., Kubanek, B., Heimphel, H. 1974. Some factors influencing granulocytio colony formation in vitro by human white blood cells. Blood. 44, 511. Jacobs, P. 1974. Some implications of current therapy in leukaemia. S. Aft. Med. J. 48, 1573. Kass, L. 1973, Bone marrow interpretation. Charles C. Thomas, Springfield, Illinois. pp. 16; 48-49; 56. Krakoff, 1. H. 1971. The present status of cancer chemotherapy. Med. Clin. N. Amer. 55, 690. Wolfe, J. A. 1972. Acute leukaemia in children; in "Clinics in Haematology; Acute Leukaemia. 1", W. B. Saunders, Philadelphia, London, Toronto. pp. 215, 217.