British Iournal of Haematology. 1985, 60, 245-251

Lack of correlation between nucleated bone marrow cell dose, marrow CFU-GM dose or marrow CFU-E dose and the rate of HLA-identical sibling marrow engraftment

KERRY ATKINSON, SUSAN NORRIE, PIK C H A N , KATE DOWNS A N D J A M E S BIGGS Department of Haematology, St Vincent’s Hospital, Sydney, Australia

Received 14 May 1984; accepted for publication 22 October 1984

SUMMARY. There was no correlation between the rate of marrow engraftment and the number of nucleated bone marrow cells infused into 50 HLA-identical sibling marrow graft recipients with haematological malignancy conditioned with cyclo- phosphamide and fractionated total body irradiation, and immunosuppressed with either cyclosporin (42 patients) or methotrexate (eight patients). Similarly, there was no correlation between the number of marrow CFU-GM or CFU-e infused into recipients immunosuppressed with cyclosporin. The data show that recipients of HLA-identical sibling marrow allografts conditioned with cyclophosphamide and total body irradiation require less than 3 x lox nucleated bone marrow cells/kg recipient weight to ensure engraftment, and throw doubt on the relevance of measuring committed progenitor cells in the donor marrow to assess the likelihood of subsequent haemopoietic reconstitution after matched sibling transplantation.

HLA-identical sibling bone marrow transplantation is an important treatment option for patients with haematological malignancy or severe aplastic anaemia (Thomas, 198 3: Powles et al, 1980; Biggs et al. 1983: Storb et al, 1982: Hows et al, 1982). Based on a multifactorial analysis of factors predisposing to marrow graft rejection in patients with severe aplastic anaemia conditioned with cyclophosphamide and immunosuppressed with methotrexate (Storb et al, 1977), it has become common practice to attempt to obtain 3 x lox nucleated bone marrow cells/kg recipient weight at the time of marrow harvest. In this study we investigated the influence of the nucleated bone marrow cell dose and the dose of marrow CFU-GM and CFU-e infused into the recipient on the rate of engraftment in 50 HLA-identical sibling marrow graft recipients with haematological malignancy conditioned with cyclo-

Correspondence: Dr K. Atkinson, Department of Haematology, St Vincent’s Hospital. Sydney, N.S.W. 2010, Australia.

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246 Krrrg Atkinson et a1

phosphamide and total body irradiation and immunosuppressed with cyclosporin or niethotrexate.

MATERIAIS A N D METHODS

Putients. Fifty patients with haematological malignancy were conditioned for HI,A-identi- cal sibling marrow transplantation with cyclophosphamide 60 mg/kg on two consccutive days followed by fractionated tot.al body irradiation 12-14 Gy over 3 or 3 . 5 d. Patients with acute non-lymphoblastic leukaemia (ANL) or acute lymphoblastic leukaemia (ATJ,) in Erst remission and patients with chronic myelogenous leukaemia (CML) were given 12 Gy: all others received 14 Gy. Forty-two of these patients received cyclosporin and eight mcthotrexate as immunosuppression to minimize graft-versus-host disease post transplant as described previously (Biggs ct (11. 198 3 ) . Methotrexate was given as previously described (Thomas P t al, 1975). No donor huffy coat cells were infused in any patient group. The day of marrow transplantation was designated day 0. Patients had daily blood counts post transplant until neutrophils were 1 ~0 x 109/l. Ungraftmeiit was assessed by rising blood counts and increasing cellularity on marrow aspiration. The number of days post transplant taken to reach a neutrophil count of 0 . 5 x 10y/l was taken as an arbitrary measure of the rate ot cngraftmcnt. Evidence for the origin of engraftment was sought whenever a genetic marker (chromosomal karyotype or red cell antigen) difference existed pre transplant bctwecn recipient and donor.

Murrow st tidies. Donor bone marrow was aspirated under general anaesthesia from the anterior and posterior iliac crests its previously described (Thomas & Storb. 1970) . The bone niarrow nucleated cell count was obtained using a Coulter S + counter (Coulter Electronics, Hialeah, Florida) and the calculation included subtraction of the donor's concurrent white blood cell count. Donor marrow CFIT-GM and CI:IJ-e colonies were cultured as follows: a known volume of marrow ( 1 4 ml) was diluted 1 : 1 in Iscove's medium (Medos, Sydney. N.S.W.) containing 2%) heat-inactivated fetal calf serum (Flow Laboratories, Sydney, N.S.W.), and loaded on to Ficoll-Paque gradients (Pharmacia, IJppsala, Sweden). The total number of cells present in the interface and pellet fractions was counted. Both a proportion of cells from the resulting mononuclear cell interface ( 1 O 5 cells) and the pellet (4 x 10j cells) fractions from these gradients were assayed for mycloid and erythroid stem cell growth. Prior to plating, the red cells in the pellct fraction were lysed with ammonium chloride lysing reagent. For CFIJ-GM assays three I ml aliquots from a 10 ml culture containing 4 ml agar, I . 5 ml fetal calf serum, 1.9 ml Alpha medium (Flow), 1 - 6 ml sterile distilled water and 1.0 ml bone marrow cell suspension were placed in sterile plastic petri dishes containing 0.1 5 ml leucocyte-conditioned medium. Thc lcucocyte-conditioned medium was the supernatant from 7 and 14 d pokeweed mitogen cultures of peripheral blood leucocytes from patients with polycythaemia rubra Vera. It was standardized against the growth of CFIJ-GM rells from normal human marrow. Bone marrow cells were cultured at 1 x lo5 nucleated cells/nil culture. The plates wcrc incubated in a humidified incubator at 3 7 O C and continually tlushcd with 5% COr in air. CFU-CM colonies were scored at day 7. Colonies consisted of at least 5 0 rells. For CFU-e assays. marrow nucleated cells were added to a culture containing 0.1 5 1111

Marrow Engraftrnent 24 7

CaC12, 0.1 5 ml thrombin, 0.1 5 ml bovine serum albumin, 0.3 ml heat-inactivated fetal calf serum, 0.3 ml citrated AB plasma and sheep erythropoietin step I11 (Connaught 1,aboratories. Willowdale, Ontario) at 2 u/ml final concentration. Cells were plated at 1 x 1 Oi ml plasma clot and incubated as for CFIJ-GM colonies. Colonies were scored after benzidine staining and had eight or more cells. Results were expressed as the number of stem cells/ml of bone marrow, and took into account the results of both the interface and pellet cultures, although, in fact, no stem cells were obtained from the pellet cultures in these normal marrows. The total number of colony cells infused into the patient was calculated as follows: CFU-GM or CFIJ-e infused/kg recipient weight =

CFIJ-GM or CFU-e/ml bone marrow x volume bone marrow infused patient weight

Statistical analysis. The influence of the number of cells infused on the rate of engraftment was analysed using a standard linear regression model and the correlation coefficient between the two variables.

HES U TIT S

Engraftment

All 42 patients with haematological malignancy immunosuppressed with cyclosporin post transplant engrafted, although one patient did not reach 0.5 x 109/1 neutrophils until ti d after a booster marrow infusion given 33 d after the initial transplant; this patient was excluded from analysis of the rate of engraftment. All eight patients with haematological malignancy immunosuppressed with methotrexate engrafted. Engraftment was shown to be of donor origin in 17/17 evaluable patients in whom a chromosomal difference existed pre transplant between donor and recipient, and in 19/19 evaluable patients in whom a red cell antigen difference existed.

lnfluence of nucleated bone marrow cell dose, CFU-GM dose, and CFU-e dose on rate ofengraftrnent

The numbers of nucleated marrow cells, CFU-GM and CFU-e infused into each of the patient groups together with the number of days taken to reach a neutrophil count of 0.5 x 109/1 are shown in Table I.

There was no correlation between the rate of engraftment and the nucleated bone marrow cell dose in either of the two patient groups. This is shown in patients with haematological malignancy immunosuppressed with cyclosporin in Fig 1, For patients with haematological malignancy immunosuppressed with methotrexate, the slope of the linear regression was -0.59 (P>0 .2 ) and the correlation coefficient was 0.1. Furthermore, in patients with haematological malignancy immunosuppressed with cyclosporin, there was no

248 Kc.rr!j Atkinsorr et a1

Tilhlr I . I h e of niiclealed marrow crlls. CFlI-CM, CFU-c and rate of engraftmcnt*

Ihys post NIIclrilled bone transplant for

marrow ntw trophils

Patient group rccipicnt wcight rccipicnt weight recipient weight 0 . 5 x 1 ()"/I cells x 10K/kg CFU-GM x 105/kg CFU-c x 105/kg to rcach

-

I I;~emittol~gical malignancy. cyclosporin 2.7fO.Y 1 . 7 k 2 . 1 7 . 3 * 11.1 15i-4 t liirrniitological malignancy. incthotrexatc 2 . h * 0 . 9 1 * 5 & 0 4 , 3 .8*2 .5 20f 5

* Values represent mean :k 1 SD.

301

.. ...

..

I 0 0 5 1 1.5 2 2 5 3 3.5 4 4.5 5 55 6

BONE MARROW NUCLEATED CELLS (x 10' ) INFUSED I kg RECIPIENT WEIGHT

Fig 1, Rclationship bctwcen ratc of cngraftment and the nucleated bone marrow ccll number infuscd in to 4 2 marrow graft recipicnts with haematological malignancy immunosuppressed with cyclosporin. Slope of line (-0.7) not significant ( / '>0.2, supported by low correlation coefficient r=O.17.

correlation between the rate of engraftment and either the number of CFU-GM infused (Fig 2 ) or the number of CFU-e infused (Fig 3 ) . Insufficient donor marrows were cultured for CE'U-GM and CFIJ-e colonies in the methotrexate patient group to allow meaningful analysis.

DISCIJSSION

Marrow graft rejection was a common, and often fatal, complication in multiply-transfused patients receiving HLA-identical sibling grafts for severe aplastic anaemia conditioned with cyclophosphamide, given donor niarrow and immunosuppressed with methotrexate (Storb

Marrow Engraftnient

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Fig 2. The relationship between the rate of engraftment and the number of CFU-GM infused into 2 3 marrow graft recipients with haematological malignancy immunosuppressed with cyclosporin. Slope of line ( -0.07) not significant ( P > 0 . 2 ) . supported by low correlation coefficient r = 0 4 4 .

. .. 0 . .

. 0

w 0 . . . 0 0 0 . . ..

4

Fig 3. The relationship between the rate of engraftment and the number of CFU-e infused into 21 marrow graft recipients with haematological malignancy immunosuppressed with cyclosporin. Slope of line ( - 0.04) not significant ( P > 0.2) , supported by low correlation coefficient r = 0.14.

2 50 Kerry Atkinson et a1

( r t n l , 1974). Analysis of factors predisposing to marrow graft rejection in such patients revealed two major risk factors (Storb ut d , 1977): these were positive in vitro tests of sensitization of the patient's cells against donor cells, and a donor marrow nucleated cell dose ot less than 3 x 1 O8 nuclcatcd marrow cclls/kg recipient weight. As a result of this study, an effort is commonly made to obtain 3 x 1 Ox nucleated marrow cells/kg recipient weight for all recipients of HLA-identical sibling marrow allografts. However. the graft rejection rate in patients with haematological malignancy conditioned with cyclophosphamide and 10-14 r;y total body irradiation prior to an HLA-identical sibling marrow graft is very low (none of 5 0 in this study).

These findings suggcst that fcwcr marrow cclls may be nccded to ensure engraftment in such patients and this is borne out by the data recorded here. We could detect no intluencc on the rate otengraftment ofthe bone marrow nucleated cell dose in either ofthe patient groups, nor indeed could we detect any intlucnce on the rate of engraftment by the number of I'FU-GM or CFU-e colony forming cells infused into patients immunosuppressed with cyclosporin. The finding tor nucleated marrow cells contirms that of Thomas ~t a/ ( 1 977) in a group of HLA-identical sibling graft recipients giveti methotrexate. Our findings with the ('FIT-GM assay in the patients immunosuppressed with cyclosporin are in contrast to those of Spiker ct c d (1980) who found ii significant relationship between the number of CFIJ-GMs infused and the rate of engraftment in patients given autologous marrow grafts as Lreatment for acute leukaemia or solid tumours. They arc also different from those of Faille c't a/ ( 1 98 1 ) who found a relationship between the number of CFU-GM infused and neutrophil recovery post transplant in 10 recipients of HLA-identical sibling grafts for severe aplastic anaemia conditioned with cyclophospharnide and total body irradiation and immunosuppressed with methotrcxatc. Comparison is difticult. however. since a number of factors including the riature of the reconstituting inoculum (in Spitzcr's report), the conditioning regimens, the post transplant imniunosuppression (in Faille's report) and the source of colony-stimulating factor used for the CFU-GM assay differed between the studies.

Our data throw into doubt the value of measuring the CFIJ-GM and CFIJ-e content of donor marrow to assess the likelihood of subsequent haemopoietic engraftment after matched sibling transplantatioti. This is relevant to current efforts to dcplcte donor marrow of T cells prior to infusion (Prentice rt ( I J . 1984; Filipovich ct al, 1984), in which it will be important to monitor multipotent, rather than unipotent, stem cell activity.

However, in recipients with haematological malignancy who are conditioned with cyclophosphamide and total body irradiation and who receive unmanipulated HLA-identical sibling marrow transplants. the data reported suggest that a target of less than 3/10x nucleated marrow cells/kg recipient weight is acceptable sincc engraftment occurred rcgularly at doses lower than this in this study.

ACKNOWI.ED(;MENTS

We thank Deirdre Nix for typing the manuscript and the Audiovisual Department of St Vincent's Hospital for help with the preparation of the figures.

Supported by grants from the National Health and Medical Research Council of Australia and the New South Wales State Cancer Council.

Marrow Engruftment 251

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