514 We thank the members of the Vegetarian Society and other volunfor their participation, Dr G. M. Ardran, Dr G. Fowler, and Dr N. MacLennan for their help, staff of the Oxford University Press and W. Lucy and Co, Engineers, who participated in the pilot study, Dr K. McPherson and Dr D. Yates for statistical and computing assistance, Mrs A. Reeve and Miss V. Williams for secretarial help, and the Medical Research Council for financial support. teers
Requests for reprints should be addressed to J. 1. M., Department of Social and Community Medicine, 8 Keble Road, Oxford OX1 3QN. REFERENCES 1.
Painter, N. S., Burkitt, D. P. in Refined Carbohydrate Foods and Disease (edited by D. P. Burkitt and H. C. Trowell); p. 99. London, 1975. 2. Mendeloff, A. I. New Engl. J. Med. 1977, 297, 811. 3. Brodribb, A. J. M., Humphreys, D. M. Br. med. J. 1976, i, 421. 4. Burke, B. S. J. Am. diet. Ass. 1947, 23, 1041. 5. McCance and Widdowson in The Composition of Foods (edited by A. A. Paul and D. A. T. Southgate). H. M. Stationery Office, 1978. 6. Ardran, G. M., Nolan, D. J., Gear, J. S. S., Fursdon, P. S., Brodribb, A. J. M. Br. J. Radiol, 1978, 51, 472. 7. Irradiation of Human Subjects for Medical Research (based on a W.H.O.
working document). Br. Inst. Radiol. Bull 1975, 1, 4. J., Hougaard-Enevddsen, O., Dinesen, K. Scand. J. Gastroent. 1976, 11, 86. 9. Manousos, O. N., Truelove, S. C., Lumsden, K. Br. med. J. 1967, iii, 760.
8. Weinreich,
10. Van Soest, P. J., Robertson, J. B. Nutr. Rev. 1977, 35, 12. 11. Cummings, J. H., Southgate, D. A. T., Branch, W., Houston, H., Jenkins, D. J. A., James, W. P. T. Lancet, 1978, i, 5. 12. Findlay, J. M., Smith, A. N., Mitchell, W. D., Anderson, A. J. B., Eastwood, M. A. ibid. 1974, i, 146. 13. Brodribb, A. J. M. ibid. 1977, i, 664.
AUTOLOGOUS BONE-MARROW TRANSPLANTATION IN RELAPSED ADULT ACUTE LEUKÆMIA A. ZANDER D. S. VERMA L. VELLEKOOP
K. A. DICKE
G. SPITZER L. PETERS K. B. MCCREDIE
host disease and a lower incidence of interstitial pneumonia.7 Syngeneic bone-marrow transplantation, however, is limited by the very low incidence of identical to syngeneic and allogeneic bonetransplantation, we have transplanted the leuksemic patient’s own cryopreserved remission bonemarrow after high-dose chemotherapy and irradiation.8 In some cases we attempted to reduce possible leukamiccell contamination of the marrow-cell suspension collected during remission by discontinuous albumin density gradient centrifugation of the cells.
As
Houston, Texas 77030, U.S.A. 24 cases of adult acute leukæmia, of which 21 were evaluable, were treated in irreversible relapse with high-dose piperazinedione and supralethal total-body irradiation (T.B.I.) in conjunction with autologous marrow transplantation (A.B.M.T.). The grafted marrow cells had been collected and stored in liquid nitrogen at the time of remission. In 12 patients the marrow cells were fractionated on discontinuous albumin gradients in an attempt to separate normal cells from residual leukæmic cells. 11 patients achieved complete remission (C.R.); 7 other patients had signs of engraftment but died before C.R. The median remission duration was 4 months (2-14). 6 of 9 acute myeloblastic leukæmia patients, in whom bone-marrow transplantation was the first treatment of relapse, achieved C.R. 4 of 5 patients with acute lymphoblastic leukæmia, whose bone-marrow cells were collected during first remission, reached C.R. Autologous bone-marrow transplantation is a valuable first treatment for acute myeloblastic leukæmia in relapse and acute lymphoblastic leukæmia in second relapse.
Summary
Introduction of adult
prognosis of adult acute myeloblastic leukaemia (A.M.L.) after the first relapse is dismal. Only 30% achieve a second remission, which is of short duration, and overall survival is only 18 weeks.3 The survival of patients with acute lymphoblastic leukaemia (A.L.L.) and undifferentiated leukaemia (A.u.L.) in their second relapse equals that of patients with A.M.L. in their first relapse.4 The resistance of relapsed leukxmia could be overcome with high-dose chemotherapy plus total body irradiation (T.B.I.) followed by allogeneic bone-marrow transplantation from HLA-identical, mixed-lymphocyteculture-negative donors. However, allogeneic bone-marrow transplantation is limited by the scarcity of suitable donors: only 30% of our leukaemia patients have HLAidentical donors. There is a large early mortality, up to 75%, within 3 months of transplantation because of infectious complications, interstitial pneumonia, and graftversus-host disease resulting from histoincompatibility. Only up to 17% of the transplanted patients survive 2 years after transplantation in complete remission (C.R.) .1 6Patients treated by the Seattle group with syngeneic bone-marrow transplantation-i.e., transplantation of marrow from identical twins-showed longer term survival because they had no fatal graft-versusThe
an
alternative
marrow
Departments of Developmental Therapeutics and Radiotherapy, University of Texas System Cancer Center, M. D. Anderson Hospital, and Tumor Institute, 6723 Bertner,
treatment
onset.
twins.
J. HESTER
,RECENT advances in the
leukxmia have improved rates of complete remission and lengthened survival. 12 However, despite this progress the majority of adult acute leukaemia patients die of recurrence of leukxmia within 2 years of disease
acute
Patients 14
patients had
median age
was
A.M.L., 8 had A.L.L., and 2 had A.U.L. The 28 (18-48). All except 5 bone-marrows
(patients 1, 7, 10, 12, 15) were aspirated during first remission, A.L.L. and A.u.L. entered the transplantation proin second, third, or fourth relapse; patients with gramme A.M.L. in first and second relapse. All patients had received
Patients with
extensive induction, consolidation, maintenance, and late intensification therapy.’ 15 patients had received second-line chemotherapy, 2 even investigational chemotherapy; and were considered failures. 9 patients (8, 11, 13, 14, 16, 18-21) were grafted without receiving second-line chemotherapy. 3 patients 2 A.M.L., 1 A.L.L.) could not be evaluated because they died within 5 days of transplantation of pre-existing conditions such as fungaemia, liver failure, and Pseudomonas septicaemia. 12 of the 24 patients, 10 of the 21 evaluable cases, received fractionated marrow. The decision to perform marrow fractionation before storage depended on the work load of the laboratory, namely, on the number of other marrows simultaneously being frozen, an important determinant in qualm control of freezing and thawing procedures.
515 TABLE I-BONE-MARROW GRAFT CHARACTERISTICS
Methods
(n=21)
Preparation of Bone-marrow Cell Suspensions Cells were collected by multiple bone-marrow aspirations under general anaathesia from the posterior iliac crest. 1.5-2.5 1 of bone-marrow cells was centrifuged in Hanks’ balanced salt solution with preservative-free heparin. Nucleated cells were harvested from the buffy coat and prepared either for fractionation over an albumin gradient or for immediate storage according to the method described previously.9 Of the 18 evaluable patients, unfractionated bone-marrow was stored in 9 cases, and in 9 patients separation of leukxmic from normal stem cells by albumin gradient was attempted. Discontinuous Albumin Gradients Erythrocyte-poor bone-marrow cells were suspended in 17% bovine serum albumin solution (B.S.A. fraction V, Sigma) layers (21%, 23%, and 25% solutions of defined osmolarity) and pipetted on top of denser albumin. After centrifugation at 100g, the fractions of cells visible near the interfaces between the albumin layers were collected and diluted with Hanks’ balanced salt solution.1O Fraction 1+2 lay between the 17 and 21% B.S.A. layers, fraction 3 between 21 and 23% B.s.A., and Fractionation by
so on to
fraction 5 at the bottom of the tube.
Bone-marrow Storage and Thawing
Bone-marrow cells were stored as described by Schaefer et al." After suspension in Hanks’ balanced salt solution (305 mosmol/1) containing 10% dimethyl sulphoxide and 20% calf serum, the cells (2-20xlOVmt) were transferred to 2 or 5 ml polypropylene ampoules and cooled at 1°C min to -40°C in a Cryoson automatic controlled freezer. After rapid cooling from -40°C to -80°C, the cells were stored in liquid nitrogen at -192°C. Cell viability on thawing was tested immediately after storage and compared with unfrozen cells by the in-vitro colony-forming assay for myeloid progenitor cells (c.F.u.-C
assay) (see below). At the time of
transplantation the ampoules were thawed 50°C and the cells were immediately slowly diluted with Hanks’ balanced salt solution (by dropwise addition of 1/50, 1/25, 1/12, and so on, of the ampoule volume) to a 10-fold dilution. Between each dilution step, the suspension rapidly
at
carefully mixed for 3 to 5 min. Deoxyribonuclease (D.N.ase Sigma) 4 mg/1 (7600 u/l collected marrow) was added to avoid clump formation by extracellular D.N.A.l0 After stepwise dilution, the cells were centrifuged, resuspended in Hanks’ solution, and filtered through G, glass filters (Jena glass, pore size 40-80 p.m). Viability of the cells was again tested by the
was
c.F.u.-c
In-vitro Assay for Ha’mopoietic Stem Cells (C..F. {7.-C assay) 2x 105 cells were suspended in 1 ml of agar medium (0-3% agar, a -M.E.M., and 15% fetal calf serum) and pipetted on to previously prepared human peripheral-blood leucocyte (1 x 106/ml) underlayers in agar medium (0.5% agar, rJ.-M.E.M., and 15% fetal calf serum). After gelling, cultures were incubated for 7 days and colonies larger than 40 cells were counted under an inverted or dissecting microscope. 12
Conditioni’}g Patients for Bone-marrow Transplantation Before transplantation, the patients were given piperazinedione (N.S.C. 135785), 25 mg/m2 intravenously, on days 6 and 5 before T.B.I. The first 17 patients had 850-950 rad calculated at the mid-abdominal plane at approximately 12-14 rad/ min from a 25 MV linear accelerator. The last 6 patients had 750 rad. The patients were treated horizontally with 4of the total dose being delivered from the anterior, posterior, right, and left lateral aspects to minimise dose non-homogeneity. Nucleated cells of fractions 3, 4, and 5 were infused within 24 h ofT.B.I.
TABLE II-RESPONSE AND H1P.MATOPOIETIC RECOVERY
’In penpheral-blood granulocytes U D
>1000/1,1,
platelets > 50
s.=adult respiratory-distress syndrome;
000/1.
c.M.v.=cytomegalovirus.
assay.
(LATEST
FOLLOW-UP
DEC.,
1978)
516 TABLE IV-INFLUENCE OF DIAGNOSIS AND PRETREATMENT ON
Results
OUTCOME OF BONE-MARROW TRANSPLANTATION
The number of cells infused/kg body-weight differed in the groups given fractionated and unfractionated cells (median 0-96x10 vs. median 3x 108) (table I) but there was no significant difference in the total number of c.F.u.-c infused. 50-75% of the cells were recovered after freezing; the number of c.F.u.-c per 105 plated cells was not altered by freezing, confirming previous data. 13 Cells from the 2 patients who did not show engraftment had no colony growth when thawed. The c.F.u-c number per 105 plated cells in these patients was low (before freezing less than 0-3, which is at least 20 times less than normal). The median time interval between storage and subsequent relapse was 9 months. All 21 patients showed clearing of bone-marrow from leukxinic cells. 19 of 21 had evidence of marrow engraftment-i.e., nucleated cells appeared in the bonemarrow within 7 days after transplantation (table II). 11 patients achieved c.R., defined as absence of leukaemia for more than 1 month, and haemopoietic recovery withgranulocytes >1000/µl and platelets >50 000/µl in the peripheral blood. 9 patients achieved granulocyte counts of 15001[il and platelet counts of 100 000/1 peripheral blood (table III). But in 1 of the 9 patients (no. 6), these TABLE III-HAEMATOPOIETIC RECOVERY OF THE ELEVEN PATIENTS ACHIEVING COMPLETE
REMISSION*
with fractionated marrow cells (table 11). 5 achieved c.R. with durations of 2, 2, 3, 5+, and 14 months. These data are not statistieally different from the results in 11 patients transplanted with unfractionated bone marrow: 6 achieved c.R. with a duration of 2, 3, 4, 5+, 5+, 8+ months. The haemopoietic recovery in the two groups was similar. All 11 patients who achieved C.R. had bone-marrow aspirated during the first remission (table iv). None of the 5 patients whose bone-marrow was aspirated during the second remission achieved C.R. although in 4/5 patients signs of engraftment were evident. When bonemarrow transplantation was the first treatment after a relapse, 10 of 14 (71%) patients achieved c.R. compared with a 14% remission rate (1 of 7) when second-line chemotherapy was given for A.M.L. in first relapse or A.L.L. in second relapse before bone-marrow transplantation was done. Discussion
*Patients >
no.
2 and
no.
19 did
achieve a granulocyte level of in the peripheral blood.
not
1 5001P and platelets >100 000/1
values
were reached only after 42 and 60 days, respectively. This patient had received a low number of cells (6-8 x106 cells/kg body-weight) of fraction 3, which had a six-fold enrichment of c.F.u.-C. In patient 2 the granulocytes and platelets increased to 1200/1 and 60 000/.1, respectively, before relapse at day 60. Patient19 reached granulocyte levels of > 5 00 by day 35, > 1000 by day 80, and platelet counts of >20 000 by day 80 and >30 000 by day 114 without recurrence of leukaemia more than 5 months after transplantation. Of the 11 pa-
tients who reached c.R., 3 are still alive in unmaintained remission: 4+, 4+, 7+ months. 1 patient relapsed after 2 months and is alive 5 months after relapse with a slow growing leukaemic infiltrate, not requiring chemotherapy. 7 patients died: 5 of recurrent leukaemia and 2 of infections. Of the 21 patients listed in table 11, 10 did not achieve c.R. Signs of take were evident in 19 patients, but 7 of these died before achieving c.R. The causes of death were acute respiratory distress syndrome (3), cardiomyopathy with intractible congestive heart-failure (2), infections (2), and recurrent leukaemia (3). 10 of the 21 evaluable patients were transplanted
Case
reports
regimens and
show that
high-dose cytoreductive
autologous remission bone-marrow can in-
duce C.R. in relapsed acute leukaemia.14-17 Our study evaluated autologous transplantation after high-dose chemotherapy and supralethal T.B.I. as an integral part of anti-leukaemia therapy for A.M.L. in first relapse and A.L.L. and A.U.L. in second or third relapse. 201 remission bone-marrows have been stored during the past 3 years. In this
study we were concerned with the survival of pluripotent stem cells during prolonged cryopreservation, the transplantation potential of bone-marrow stem cells exposed to continuous chemotherapy, the persistof leukxmic cells in the remission bone-marrow and possible removal by albumin density-gradient centrifugation, and the development of an optimal cytoreductive regimen with limited nonhaematopoietic toxicity. Bone-marrow stored for up to 30 months in liquid nitrogen maintained its transplantation potential and allowed for full haemopoietic recovery after piperazinedione and T.B.I. The pattern of haemopoietic recovery with granulocyte levels above 500/[LI and platelet levels above 20000/[Ll by 3 weeks appeared similar to that reported for syngeneic bone-marrow transplantation by the Seattle group.7 Failure of bone-marrow take correlated with absence of colonies in the c.F.u.-c assays in patients 7 and 9. 1
ence
their
517
patient died at day 30 of fungaemia and intractable congestive heart-failure with no evidence of a take by bonemarrow examination and peripheral blood-count, the second patient died on day 56 of disseminated cytomegalovirus infection without evidence of haemopoietic recovery. This experience suggests that the C.F.u.-c assay might be valuable in monitoring quality and viability of cryopreserved bone-marrow. We have shown that there is a significant correlation between the number of the c.F.u.-c infused and the time of recovery of granulo-
cytes.l8 The discontinuous albumin gradient technique successfully separated normal from leukaemic marrow cells in patients with low but detectable leukxmic infiltrates’9 and the leuksemic cell population appeared predominantly lighter than the normal c.F.u.-c population. These results’9 confirmed the data of Moore et al.20 In 10 patients fractionated cells were used for transplantation. The effect of marrow-cell fractionation on the relapse-rate after transplantation is uncertain because of the small number of cases in each leukaemia subclassification and because of the different radiation doses. However, haematopoietic recovery was much the same in patients who received unfractionated bone-marrow and in those who received gradient-separated bone-marrow. It is impossible to assess the degree of separation between leukaemic cells and normal remission cells in any individual patient. Because there are differences in the densities of leuksemic cells it should be possible to compare the distribution of c.F.u.-c in leukaemic cells at first presentation with that of c.F.u.-c in remission to see if separation is possible. In patient 1 leukaemia recurred rapidly after initial clearing of the bone-marrow (30 days after transplantation). In this case the marrow had been aspirated in second remission, 2 weeks before overt relapse and was transplanted unfractionated after all other means of therapy were exhausted. Leukaemia may have been transplanted.8
chemotherapy alone as a cytoreductive in bone-marrow transplantation has led to remission of limited duration.21 The combination of chemotherapy and irradiation yielded a higher percentage of patients with long-term survival. 21 The actuarial relapse-rate of patients treated with the S.C.A.R.I. regimen (a highly toxic chemo-irradiation treatment consisting of 6-thioguanin, cyclophosphamide, cytarabine, daunorubicin, and irradiation) is lower than the actuarial relapse-rate with cyclophosphamide/T.B.I. but the percentage of patients surviving more than 2 years is not significantly different.22 For our conditioning regimen we selected a combination of piperazinedione, a fermentation product of a Streptomyces species with alkylating activity,23 and T.B.I. In all evaluable cases, leukaemic cells became morphologically undetectable but follow-up after transplantation was too short to be certain about this regimen’s efficiency in eradicating leukaemic cells compared with other regimens. Clinical studies showed that piperazinedione had good anti-leukasmic activity but its general use was abandoned because of excessive myelotoxicity.24 Non-haematopoietic toxicity of this drug is restricted to mild nausea and vomiting. Piperazinedione and T.B.I. were given 5 days apart because their close proximity led to excessive gastrointestinal toxicity in the mouse. 21 T.B.I.
regimen
The toxicity noted in this study is in part related to cumulative effect of preceding chemotherapy. The two patients who had intractable congestive heart-failure with cardiomyopathy at necropsy had received large amounts of anthracyclines-500 mg/m2 doxorubicin in one case and 1750 mg/m2 of rubidazone in the other. The 3 patients who had acute pulmonary toxicity had had more than average amounts of chemotherapy before transplantation and high-dose irradiation at the time of transplantation (950, 900, and 850 rad). In an attempt to decrease the acute radiation toxicity we reduced T.B.I. to 750 rad. 4 of 6 patients achieved c.R. (patients 16, 17, 19, and 21) and 2 patients had recurrent leukaemia (patients 18 and 20). There was less acute toxicity with this regimen. Piperazinedione and T.B.I. followed by autologous bone-marrow transplantation is effective when employed early in relapse: 10 of 14 patients who had earlier bonemarrow transplantation achieved C.R. 10 patients did not achieve c.R. because of cumulative drug toxicity, decreased transplantation potential of marrow stored after extensive chemotherapy, and poor clinical condition after failing reinduction with chemotherapy. The treatment leads to C.R. in about 50% of cases of relapsed acute leukaemia. The quality of life after transplantation is good and patients achieving haemopoietic recovery are kept in hospital for a short time (median 26 days). More patients treated earlier in relapse are needed to compare this treatment with conventional chemotherapy. We thank Mrs Sharon Thomson, Ms Danita Stewart, and Mrs Donna Wampler for assistance in preparing the bone-marrow cells for fractionation, storage, and transplantation. We also thank Ms Barbara Fisher and Ms Helen Goldberg for analysing the charts, and Ms Lee Foster for secretarial assistance.
Supported by
N.I.H. grants
CA-11520, CA-14528, and CA-19586.
Reprint requests should be addressed to K.A.D.
or
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9.
Freireich, E. J. and others. Cancer, 1978, 42, 874. Gale, R. P., Cline, M. J. Lancet, 1977, i, 497. Benjamin, R. S., Keating, M. J., McCredie, K. B., Bodey, G. P., Freireich, E. J. Cancer Res. 1977, 37, 4623. McCredie, K. B., Hewlet, J., Gehan, E. Abstracts vol. I, XVIIth Congress of the International Society of Hæmatology; p. 231, Paris, 1978. Thomas, E. D., and others. Blood, 1977,49, 511. U.C.L.A. Bone-marrow Transplantation Team. Lancet, 1977, i, 1197. Fefer, A., and others. Sem. Hemat. 1974, 11, 353. Dicke, K. A., and others. Transplant. Proc. 1977, 9, 193. Dicke, K. A., Tridente, G., van Bekkum, D. W. Transplantation, 1969, 8,
422. 10. Dicke, K. A., and others. Blood, 1973, 42, 195. 11. Schaefer, U. W., Dicke, K. A., van Bekkum, D. W. Rev. Europ. Etud. Clin. Biol. 1972, 17, 483. 12. Pike, B. L., Robinson, W. A. J. Cell. Physiol. 1979, 76, 77. 13. Schaefer, U. W., Dicke, K. A., van Bekkum, D. W. Proceedings of the 3rd Conference on Experimental Medical Surgery. Primates Part II; p. 142. Basle, 1972. 14. McGovern, J. J., and others. New Engl. J. Med. 1959, 260, 675. 15. Herzig, G. P. Unpublished. 16. Gorin, M. C., Najman, A., Duhamel, G. Lancet, 1977, i, 1050. 17. Schaefer, U. W., and others. Exp. Hemat. 1977, 5, 101. 18. Spitzer, G., and others. Blood, 1978, 52, 232. 19. Dicke, K. A., and others. Transplant. Proc. (in the press). 20. Moore, M. A. S., Williams, N., Metcalf, D. J. natn. Cancer Inst. 1973, 50, 591. 21. Thomas, E. D., and others. New Engl. J. Med. 1975, 292, 832. 22. Gale, R. P., for the U.C.L.A. Bone-marrow Transplantation Team. Transplant. Proc. 1978, 10, 1. 23. Gitterman, C. D., and others. J. Antibiot. 1970, 23, 305. 24. Benjamin, R. S., and others. Unpublished. 25. Dicke, K. A., and others. Proceedings of International Society for Experimental Hæmatology Fourth Annual Conference Yugoslavia, September
21-24,1975 (abstr).
Requests for reprints should be addressed to J. 1. M., Department of Social and Community Medicine, 8 Keble Road, Oxford OX1 3QN. REFERENCES 1.
Painter, N. S., Burkitt, D. P. in Refined Carbohydrate Foods and Disease (edited by D. P. Burkitt and H. C. Trowell); p. 99. London, 1975. 2. Mendeloff, A. I. New Engl. J. Med. 1977, 297, 811. 3. Brodribb, A. J. M., Humphreys, D. M. Br. med. J. 1976, i, 421. 4. Burke, B. S. J. Am. diet. Ass. 1947, 23, 1041. 5. McCance and Widdowson in The Composition of Foods (edited by A. A. Paul and D. A. T. Southgate). H. M. Stationery Office, 1978. 6. Ardran, G. M., Nolan, D. J., Gear, J. S. S., Fursdon, P. S., Brodribb, A. J. M. Br. J. Radiol, 1978, 51, 472. 7. Irradiation of Human Subjects for Medical Research (based on a W.H.O.
working document). Br. Inst. Radiol. Bull 1975, 1, 4. J., Hougaard-Enevddsen, O., Dinesen, K. Scand. J. Gastroent. 1976, 11, 86. 9. Manousos, O. N., Truelove, S. C., Lumsden, K. Br. med. J. 1967, iii, 760.
8. Weinreich,
10. Van Soest, P. J., Robertson, J. B. Nutr. Rev. 1977, 35, 12. 11. Cummings, J. H., Southgate, D. A. T., Branch, W., Houston, H., Jenkins, D. J. A., James, W. P. T. Lancet, 1978, i, 5. 12. Findlay, J. M., Smith, A. N., Mitchell, W. D., Anderson, A. J. B., Eastwood, M. A. ibid. 1974, i, 146. 13. Brodribb, A. J. M. ibid. 1977, i, 664.
AUTOLOGOUS BONE-MARROW TRANSPLANTATION IN RELAPSED ADULT ACUTE LEUKÆMIA A. ZANDER D. S. VERMA L. VELLEKOOP
K. A. DICKE
G. SPITZER L. PETERS K. B. MCCREDIE
host disease and a lower incidence of interstitial pneumonia.7 Syngeneic bone-marrow transplantation, however, is limited by the very low incidence of identical to syngeneic and allogeneic bonetransplantation, we have transplanted the leuksemic patient’s own cryopreserved remission bonemarrow after high-dose chemotherapy and irradiation.8 In some cases we attempted to reduce possible leukamiccell contamination of the marrow-cell suspension collected during remission by discontinuous albumin density gradient centrifugation of the cells.
As
Houston, Texas 77030, U.S.A. 24 cases of adult acute leukæmia, of which 21 were evaluable, were treated in irreversible relapse with high-dose piperazinedione and supralethal total-body irradiation (T.B.I.) in conjunction with autologous marrow transplantation (A.B.M.T.). The grafted marrow cells had been collected and stored in liquid nitrogen at the time of remission. In 12 patients the marrow cells were fractionated on discontinuous albumin gradients in an attempt to separate normal cells from residual leukæmic cells. 11 patients achieved complete remission (C.R.); 7 other patients had signs of engraftment but died before C.R. The median remission duration was 4 months (2-14). 6 of 9 acute myeloblastic leukæmia patients, in whom bone-marrow transplantation was the first treatment of relapse, achieved C.R. 4 of 5 patients with acute lymphoblastic leukæmia, whose bone-marrow cells were collected during first remission, reached C.R. Autologous bone-marrow transplantation is a valuable first treatment for acute myeloblastic leukæmia in relapse and acute lymphoblastic leukæmia in second relapse.
Summary
Introduction of adult
prognosis of adult acute myeloblastic leukaemia (A.M.L.) after the first relapse is dismal. Only 30% achieve a second remission, which is of short duration, and overall survival is only 18 weeks.3 The survival of patients with acute lymphoblastic leukaemia (A.L.L.) and undifferentiated leukaemia (A.u.L.) in their second relapse equals that of patients with A.M.L. in their first relapse.4 The resistance of relapsed leukxmia could be overcome with high-dose chemotherapy plus total body irradiation (T.B.I.) followed by allogeneic bone-marrow transplantation from HLA-identical, mixed-lymphocyteculture-negative donors. However, allogeneic bone-marrow transplantation is limited by the scarcity of suitable donors: only 30% of our leukaemia patients have HLAidentical donors. There is a large early mortality, up to 75%, within 3 months of transplantation because of infectious complications, interstitial pneumonia, and graftversus-host disease resulting from histoincompatibility. Only up to 17% of the transplanted patients survive 2 years after transplantation in complete remission (C.R.) .1 6Patients treated by the Seattle group with syngeneic bone-marrow transplantation-i.e., transplantation of marrow from identical twins-showed longer term survival because they had no fatal graft-versusThe
an
alternative
marrow
Departments of Developmental Therapeutics and Radiotherapy, University of Texas System Cancer Center, M. D. Anderson Hospital, and Tumor Institute, 6723 Bertner,
treatment
onset.
twins.
J. HESTER
,RECENT advances in the
leukxmia have improved rates of complete remission and lengthened survival. 12 However, despite this progress the majority of adult acute leukaemia patients die of recurrence of leukxmia within 2 years of disease
acute
Patients 14
patients had
median age
was
A.M.L., 8 had A.L.L., and 2 had A.U.L. The 28 (18-48). All except 5 bone-marrows
(patients 1, 7, 10, 12, 15) were aspirated during first remission, A.L.L. and A.u.L. entered the transplantation proin second, third, or fourth relapse; patients with gramme A.M.L. in first and second relapse. All patients had received
Patients with
extensive induction, consolidation, maintenance, and late intensification therapy.’ 15 patients had received second-line chemotherapy, 2 even investigational chemotherapy; and were considered failures. 9 patients (8, 11, 13, 14, 16, 18-21) were grafted without receiving second-line chemotherapy. 3 patients 2 A.M.L., 1 A.L.L.) could not be evaluated because they died within 5 days of transplantation of pre-existing conditions such as fungaemia, liver failure, and Pseudomonas septicaemia. 12 of the 24 patients, 10 of the 21 evaluable cases, received fractionated marrow. The decision to perform marrow fractionation before storage depended on the work load of the laboratory, namely, on the number of other marrows simultaneously being frozen, an important determinant in qualm control of freezing and thawing procedures.
515 TABLE I-BONE-MARROW GRAFT CHARACTERISTICS
Methods
(n=21)
Preparation of Bone-marrow Cell Suspensions Cells were collected by multiple bone-marrow aspirations under general anaathesia from the posterior iliac crest. 1.5-2.5 1 of bone-marrow cells was centrifuged in Hanks’ balanced salt solution with preservative-free heparin. Nucleated cells were harvested from the buffy coat and prepared either for fractionation over an albumin gradient or for immediate storage according to the method described previously.9 Of the 18 evaluable patients, unfractionated bone-marrow was stored in 9 cases, and in 9 patients separation of leukxmic from normal stem cells by albumin gradient was attempted. Discontinuous Albumin Gradients Erythrocyte-poor bone-marrow cells were suspended in 17% bovine serum albumin solution (B.S.A. fraction V, Sigma) layers (21%, 23%, and 25% solutions of defined osmolarity) and pipetted on top of denser albumin. After centrifugation at 100g, the fractions of cells visible near the interfaces between the albumin layers were collected and diluted with Hanks’ balanced salt solution.1O Fraction 1+2 lay between the 17 and 21% B.S.A. layers, fraction 3 between 21 and 23% B.s.A., and Fractionation by
so on to
fraction 5 at the bottom of the tube.
Bone-marrow Storage and Thawing
Bone-marrow cells were stored as described by Schaefer et al." After suspension in Hanks’ balanced salt solution (305 mosmol/1) containing 10% dimethyl sulphoxide and 20% calf serum, the cells (2-20xlOVmt) were transferred to 2 or 5 ml polypropylene ampoules and cooled at 1°C min to -40°C in a Cryoson automatic controlled freezer. After rapid cooling from -40°C to -80°C, the cells were stored in liquid nitrogen at -192°C. Cell viability on thawing was tested immediately after storage and compared with unfrozen cells by the in-vitro colony-forming assay for myeloid progenitor cells (c.F.u.-C
assay) (see below). At the time of
transplantation the ampoules were thawed 50°C and the cells were immediately slowly diluted with Hanks’ balanced salt solution (by dropwise addition of 1/50, 1/25, 1/12, and so on, of the ampoule volume) to a 10-fold dilution. Between each dilution step, the suspension rapidly
at
carefully mixed for 3 to 5 min. Deoxyribonuclease (D.N.ase Sigma) 4 mg/1 (7600 u/l collected marrow) was added to avoid clump formation by extracellular D.N.A.l0 After stepwise dilution, the cells were centrifuged, resuspended in Hanks’ solution, and filtered through G, glass filters (Jena glass, pore size 40-80 p.m). Viability of the cells was again tested by the
was
c.F.u.-c
In-vitro Assay for Ha’mopoietic Stem Cells (C..F. {7.-C assay) 2x 105 cells were suspended in 1 ml of agar medium (0-3% agar, a -M.E.M., and 15% fetal calf serum) and pipetted on to previously prepared human peripheral-blood leucocyte (1 x 106/ml) underlayers in agar medium (0.5% agar, rJ.-M.E.M., and 15% fetal calf serum). After gelling, cultures were incubated for 7 days and colonies larger than 40 cells were counted under an inverted or dissecting microscope. 12
Conditioni’}g Patients for Bone-marrow Transplantation Before transplantation, the patients were given piperazinedione (N.S.C. 135785), 25 mg/m2 intravenously, on days 6 and 5 before T.B.I. The first 17 patients had 850-950 rad calculated at the mid-abdominal plane at approximately 12-14 rad/ min from a 25 MV linear accelerator. The last 6 patients had 750 rad. The patients were treated horizontally with 4of the total dose being delivered from the anterior, posterior, right, and left lateral aspects to minimise dose non-homogeneity. Nucleated cells of fractions 3, 4, and 5 were infused within 24 h ofT.B.I.
TABLE II-RESPONSE AND H1P.MATOPOIETIC RECOVERY
’In penpheral-blood granulocytes U D
>1000/1,1,
platelets > 50
s.=adult respiratory-distress syndrome;
000/1.
c.M.v.=cytomegalovirus.
assay.
(LATEST
FOLLOW-UP
DEC.,
1978)
516 TABLE IV-INFLUENCE OF DIAGNOSIS AND PRETREATMENT ON
Results
OUTCOME OF BONE-MARROW TRANSPLANTATION
The number of cells infused/kg body-weight differed in the groups given fractionated and unfractionated cells (median 0-96x10 vs. median 3x 108) (table I) but there was no significant difference in the total number of c.F.u.-c infused. 50-75% of the cells were recovered after freezing; the number of c.F.u.-c per 105 plated cells was not altered by freezing, confirming previous data. 13 Cells from the 2 patients who did not show engraftment had no colony growth when thawed. The c.F.u-c number per 105 plated cells in these patients was low (before freezing less than 0-3, which is at least 20 times less than normal). The median time interval between storage and subsequent relapse was 9 months. All 21 patients showed clearing of bone-marrow from leukxinic cells. 19 of 21 had evidence of marrow engraftment-i.e., nucleated cells appeared in the bonemarrow within 7 days after transplantation (table II). 11 patients achieved c.R., defined as absence of leukaemia for more than 1 month, and haemopoietic recovery withgranulocytes >1000/µl and platelets >50 000/µl in the peripheral blood. 9 patients achieved granulocyte counts of 15001[il and platelet counts of 100 000/1 peripheral blood (table III). But in 1 of the 9 patients (no. 6), these TABLE III-HAEMATOPOIETIC RECOVERY OF THE ELEVEN PATIENTS ACHIEVING COMPLETE
REMISSION*
with fractionated marrow cells (table 11). 5 achieved c.R. with durations of 2, 2, 3, 5+, and 14 months. These data are not statistieally different from the results in 11 patients transplanted with unfractionated bone marrow: 6 achieved c.R. with a duration of 2, 3, 4, 5+, 5+, 8+ months. The haemopoietic recovery in the two groups was similar. All 11 patients who achieved C.R. had bone-marrow aspirated during the first remission (table iv). None of the 5 patients whose bone-marrow was aspirated during the second remission achieved C.R. although in 4/5 patients signs of engraftment were evident. When bonemarrow transplantation was the first treatment after a relapse, 10 of 14 (71%) patients achieved c.R. compared with a 14% remission rate (1 of 7) when second-line chemotherapy was given for A.M.L. in first relapse or A.L.L. in second relapse before bone-marrow transplantation was done. Discussion
*Patients >
no.
2 and
no.
19 did
achieve a granulocyte level of in the peripheral blood.
not
1 5001P and platelets >100 000/1
values
were reached only after 42 and 60 days, respectively. This patient had received a low number of cells (6-8 x106 cells/kg body-weight) of fraction 3, which had a six-fold enrichment of c.F.u.-C. In patient 2 the granulocytes and platelets increased to 1200/1 and 60 000/.1, respectively, before relapse at day 60. Patient19 reached granulocyte levels of > 5 00 by day 35, > 1000 by day 80, and platelet counts of >20 000 by day 80 and >30 000 by day 114 without recurrence of leukaemia more than 5 months after transplantation. Of the 11 pa-
tients who reached c.R., 3 are still alive in unmaintained remission: 4+, 4+, 7+ months. 1 patient relapsed after 2 months and is alive 5 months after relapse with a slow growing leukaemic infiltrate, not requiring chemotherapy. 7 patients died: 5 of recurrent leukaemia and 2 of infections. Of the 21 patients listed in table 11, 10 did not achieve c.R. Signs of take were evident in 19 patients, but 7 of these died before achieving c.R. The causes of death were acute respiratory distress syndrome (3), cardiomyopathy with intractible congestive heart-failure (2), infections (2), and recurrent leukaemia (3). 10 of the 21 evaluable patients were transplanted
Case
reports
regimens and
show that
high-dose cytoreductive
autologous remission bone-marrow can in-
duce C.R. in relapsed acute leukaemia.14-17 Our study evaluated autologous transplantation after high-dose chemotherapy and supralethal T.B.I. as an integral part of anti-leukaemia therapy for A.M.L. in first relapse and A.L.L. and A.U.L. in second or third relapse. 201 remission bone-marrows have been stored during the past 3 years. In this
study we were concerned with the survival of pluripotent stem cells during prolonged cryopreservation, the transplantation potential of bone-marrow stem cells exposed to continuous chemotherapy, the persistof leukxmic cells in the remission bone-marrow and possible removal by albumin density-gradient centrifugation, and the development of an optimal cytoreductive regimen with limited nonhaematopoietic toxicity. Bone-marrow stored for up to 30 months in liquid nitrogen maintained its transplantation potential and allowed for full haemopoietic recovery after piperazinedione and T.B.I. The pattern of haemopoietic recovery with granulocyte levels above 500/[LI and platelet levels above 20000/[Ll by 3 weeks appeared similar to that reported for syngeneic bone-marrow transplantation by the Seattle group.7 Failure of bone-marrow take correlated with absence of colonies in the c.F.u.-c assays in patients 7 and 9. 1
ence
their
517
patient died at day 30 of fungaemia and intractable congestive heart-failure with no evidence of a take by bonemarrow examination and peripheral blood-count, the second patient died on day 56 of disseminated cytomegalovirus infection without evidence of haemopoietic recovery. This experience suggests that the C.F.u.-c assay might be valuable in monitoring quality and viability of cryopreserved bone-marrow. We have shown that there is a significant correlation between the number of the c.F.u.-c infused and the time of recovery of granulo-
cytes.l8 The discontinuous albumin gradient technique successfully separated normal from leukaemic marrow cells in patients with low but detectable leukxmic infiltrates’9 and the leuksemic cell population appeared predominantly lighter than the normal c.F.u.-c population. These results’9 confirmed the data of Moore et al.20 In 10 patients fractionated cells were used for transplantation. The effect of marrow-cell fractionation on the relapse-rate after transplantation is uncertain because of the small number of cases in each leukaemia subclassification and because of the different radiation doses. However, haematopoietic recovery was much the same in patients who received unfractionated bone-marrow and in those who received gradient-separated bone-marrow. It is impossible to assess the degree of separation between leukaemic cells and normal remission cells in any individual patient. Because there are differences in the densities of leuksemic cells it should be possible to compare the distribution of c.F.u.-c in leukaemic cells at first presentation with that of c.F.u.-c in remission to see if separation is possible. In patient 1 leukaemia recurred rapidly after initial clearing of the bone-marrow (30 days after transplantation). In this case the marrow had been aspirated in second remission, 2 weeks before overt relapse and was transplanted unfractionated after all other means of therapy were exhausted. Leukaemia may have been transplanted.8
chemotherapy alone as a cytoreductive in bone-marrow transplantation has led to remission of limited duration.21 The combination of chemotherapy and irradiation yielded a higher percentage of patients with long-term survival. 21 The actuarial relapse-rate of patients treated with the S.C.A.R.I. regimen (a highly toxic chemo-irradiation treatment consisting of 6-thioguanin, cyclophosphamide, cytarabine, daunorubicin, and irradiation) is lower than the actuarial relapse-rate with cyclophosphamide/T.B.I. but the percentage of patients surviving more than 2 years is not significantly different.22 For our conditioning regimen we selected a combination of piperazinedione, a fermentation product of a Streptomyces species with alkylating activity,23 and T.B.I. In all evaluable cases, leukaemic cells became morphologically undetectable but follow-up after transplantation was too short to be certain about this regimen’s efficiency in eradicating leukaemic cells compared with other regimens. Clinical studies showed that piperazinedione had good anti-leukasmic activity but its general use was abandoned because of excessive myelotoxicity.24 Non-haematopoietic toxicity of this drug is restricted to mild nausea and vomiting. Piperazinedione and T.B.I. were given 5 days apart because their close proximity led to excessive gastrointestinal toxicity in the mouse. 21 T.B.I.
regimen
The toxicity noted in this study is in part related to cumulative effect of preceding chemotherapy. The two patients who had intractable congestive heart-failure with cardiomyopathy at necropsy had received large amounts of anthracyclines-500 mg/m2 doxorubicin in one case and 1750 mg/m2 of rubidazone in the other. The 3 patients who had acute pulmonary toxicity had had more than average amounts of chemotherapy before transplantation and high-dose irradiation at the time of transplantation (950, 900, and 850 rad). In an attempt to decrease the acute radiation toxicity we reduced T.B.I. to 750 rad. 4 of 6 patients achieved c.R. (patients 16, 17, 19, and 21) and 2 patients had recurrent leukaemia (patients 18 and 20). There was less acute toxicity with this regimen. Piperazinedione and T.B.I. followed by autologous bone-marrow transplantation is effective when employed early in relapse: 10 of 14 patients who had earlier bonemarrow transplantation achieved C.R. 10 patients did not achieve c.R. because of cumulative drug toxicity, decreased transplantation potential of marrow stored after extensive chemotherapy, and poor clinical condition after failing reinduction with chemotherapy. The treatment leads to C.R. in about 50% of cases of relapsed acute leukaemia. The quality of life after transplantation is good and patients achieving haemopoietic recovery are kept in hospital for a short time (median 26 days). More patients treated earlier in relapse are needed to compare this treatment with conventional chemotherapy. We thank Mrs Sharon Thomson, Ms Danita Stewart, and Mrs Donna Wampler for assistance in preparing the bone-marrow cells for fractionation, storage, and transplantation. We also thank Ms Barbara Fisher and Ms Helen Goldberg for analysing the charts, and Ms Lee Foster for secretarial assistance.
Supported by
N.I.H. grants
CA-11520, CA-14528, and CA-19586.
Reprint requests should be addressed to K.A.D.
or
REFERENCES
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