Cord blood stem cell transplants Cord blood transplants: early ... - Nature

Bone Marrow Transplantation (2001) 28, 355–363  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt

Cord blood stem cell transplants Cord blood transplants: early recovery of neutrophils from co-transplanted sibling haploidentical progenitor cells and lack of engraftment of cultured cord blood cells, as ascertained by analysis of DNA polymorphisms MN Fernańdez1, C Regidor1, R Cabrera1, J Garcıá-Marco1, M Briz1, R Fore´s1, I Sanjuań1, A McWhinnie3, S Querol2, J Garcıá2 and A Madrigal3 1

Servicio de Hematologıá, Clıńica Puerta de Hierro, Universidad Autońoma de Madrid; 2Banco de Sangre de Cordoń de Barcelona (IRO), Spain; and 3The Anthony Nolan Research Institute and Royal Free Hospital School of Medicine, London, UK

Summary: The number of infused cells is a very important factor in cord blood transplant (CBT) engraftment. Prior ex vivo expansion of aliquots of transplanted cord blood (CB) units is being investigated as a procedure to increase engraftment potential, but results are difficult to evaluate due to a lack of markers for assessing the contribution of expanded cells. We transplanted five patients, infusing the best available CB unit and cells from a second donor simultaneously. In two patients, these cells were obtained from another frozen CB unit by CD34+ positive selection and culture expansion; the other three patients received uncultured highly purified haploidentical CD34+ cells. The first two patients had DNA from the culture expanded CB cells detected only for a few days around day +11 when the absolute neutrophil count (ANC) was ⬍200/␮l; thereafter and when the ANC was ⬎500/␮l, only donor DNA from the uncultured CB was detected. For the other three patients, DNA analysis showed early and transient granulocyte engraftment of haploidentical cells, progressively replaced by the CB-derived granulocytes. We concluded that: (1) simultaneous infusion of lymphocyte-depleted HLA highly mismatched haematopoietic progenitor cells has not produced unfavourable effects for CBT; (2) the double transplant model is suitable for evaluating the engraftment potential of ex vivo cultured CB cells in the clinical setting; (3) the culture conditions used did not result in early recovery of ANC; and (4) co-transplantation of purified uncultured HLA haploidentical CD34+ cells may reduce the time of neutropenia following CBT. Bone Marrow Transplantation (2001) 28, 355–363. Keywords: cord blood transplants; stem cell expansion; haploidentical haematopoietic stem cell transplants Correspondence: Dr MN Fernańdez, Servicio de Hematologıá, Hospital Universitario ‘Clıńica Puerta de Hierro’, San Martıń de Porres, 4, 28035 Madrid, Spain Received 30 November 2000; accepted 12 May 2001

Cord blood (CB) is well established as a source of haematopoietic stem cells for allogeneic transplantation, with more than 2000 unrelated cord blood transplants (CBT) performed in the last 12 years. Although it has several advantages over bone marrow transplantation (BMT), for example, rapid availability, lower risk of transmission of viral disease, and less GVHD,1–12 several controversial issues still remain. While a number of adult transplants have been performed successfully, CBT has been used mostly for paediatric patients and analysis of clinical results has shown that the number of nucleated cells infused is the most important factor in predicting the outcome of the transplant.1–14 A matter of concern is whether the number of cells required for an adult transplant are present in a typical CB unit, which contains a limited number of nucleated cells that range from about 1.30 to more than 37 × 108. The average (12 × 108) represents 0.6 × 108/kg for a 20 kg pediatric recipient but only 0.17 × 108/kg for a 70 kg patient (respectively, around 1/5 and 1/16 of the usual total cell dose for BMTs). Several groups have evaluated CBT of units partially subjected to previous culture, trying to increase the number of haematopoietic progenitor cells and, thus, to enhance engraftment.15–18 The reported engraftment times, however, are similar to those of CBTs with units without any previous culture.1–6,19 Unfortunately, due to a lack of genetic markers for differentiating between cells of different origin, it has not been possible to assess to what extent the expanded cells contributed to engraftment. Expanded cells might not engraft as well as non-cultured ones. We have recently shown, using xenogeneic transplant of human CB cells into SCID/NOD mice, that ex vivo expansion of haematopoietic CB progenitors may result in the loss of capacity for early engraftment. This could be attributed to reduced capacity for post-transplant homing and/or proliferative growth.20 Similar and also contradictory observations have been made by others.21,22 Simultaneous transplantation of several unexpanded CB units has also been attempted to enhance engraftment.23 We theorized that simultaneous transplantation of an unexpanded CB unit, together with one previously subjected to

CBT with infusion of HPC from a different donor MN Ferna´ ndez et al

356

culture, could provide valuable information about the relative engraftment kinetics of cultured and uncultured CB cells in human recipients, since the two units could have differing genetic markers.19 We also theorized that double transplants, employing non-expanded CB units and highly T cell-depleted uncultured haploidentical stem cells from a family donor, could result in an earlier increase of granulocytes that could provide antimicrobial protection during the longer engraftment period of the CB stem cells. These transplants would also provide comparative data to evaluate the engraftment kinetics of the culture-expanded CB cells in human recipients receiving double CBTs. This approach could have other unknown unfavourable or favourable effects. The former could include higher risks of GVHD, given the dual stem cell populations of different histocompatibility or lack of engraftment due to immunoreactivity among the transplanted units. Favourable effects could include induction of immunotolerance that could reduce risks of rejection and/or GVHD.24 In the Hospital P de Hierro, Universidad Auto´ noma de Madrid, we have transplanted five refractory acute leukaemia patients with the best available CB units (in terms of both HLA compatibility and cell content) in conjunction with cells from a different donor. In two cases, these were obtained by culture expansion of all the CD34+ cells that were recovered from a different CB unit. For the other three, they were purified and uncultured PB CD34+ cells obtained from a haploidentical sibling. In all cases, these cells were highly devoid of contaminating lymphocytes. Our primary objective has been to obtain shortened intervals to granulocyte recovery; a secondary objective has been to investigate both the engraftment potential of culture expanded CB cells and the engraftment kinetics of cotransplanted cells of different genetic origin. Patients and methods Patients

Patient clinical features, pre-transplant Patient 1 Patient 2 Patient 3

Age Weight (kg) Gender Diagnosis Status

The double transplant strategies are outlined in a phase I– II protocol. This is offered to eligible patients as compassionate treatment with specific consent from the Hospital Ethical Committee for each individual case. Signed consent from each patient is obtained after providing appropriate information, according to national and international regulations for research on human subjects. In patients Nos 1 and 2, the strategy was to successively infuse the best available CB unit immediately after thawing and the cells resulting from the ex vivo culture of all the CD34+ cells recovered from the second best available unit, with no more than four HLA-A, -B, -DR Ag differences. These CD34+ cells were obtained by positive immunomagnetic (Isolex 50; Baxter, Deerfield, IL, USA) selection and ex vivo static culture for 6 days in a serum-free, stromafree, medium containing SCF, FLT3, TPO and IL3 at final concentrations of 100 ng/ml, as previously described.25 For the other three patients, Nos 3, 4 and 5, instead of using cultured CB cells, the strategy differed in the use of highly purified PB CD34+ haploidentical cells obtained from a sibling by apheresis after 4 days of G-CSF given subcutaneously every 12 h. Purification was performed by positive/negative immunomagnetic selection using the Isolex 300i 2.0 Nexel (Irvine, CA, USA) system for patients Nos 3 and 4, and by positive selection using the CliniMACS (Miltenyi-Biotec, Bergisch Gladbach, Germany) system for patient No. 5. Antibodies used in the negative selection step were anti-CD4 and anti-CD8 (Nexell) for patient No. 3, and these plus anti-CD3 (Ortho, Raritan, NJ, USA) for patient No. 4. Our aim was to procure final products containing 1–2.5 × 106/kg CD34+ cells (2 to 5 times the final number of CD34+ cells in the unexpanded CB products) with the lowest possible number, to a maximum of 1 × 104/kg, of contaminating CD3+ cells. Haploidentical donors were also 2 to 3 HLA Ag mismatched with the transplanted CB units. Pre-transplant conditioning

Pre-transplant clinical features of the patients are shown in Table 1. All patients were adults aged between 25 to 47 years, weighing 66 to 92 kg, with a diagnosis of high risk acute leukaemia. Patients were in relatively good clinical condition despite their poor prognosis and did not have potential donors with acceptable HLA compatibility. All had positive CMV serology. Table 1

Transplantation strategies

43 92

25 70

40 70

F M M AML ALL ALL CR-2 PR CR-2 high risk 20% blasts high risk in BM

Bone Marrow Transplantation

Patient 4

Patient 5

28 66

47 80

M M ALL AML (M-7) PR PR mediastinal residual blasts and pleural in BM residual tumour

The regimen consisted of TBI, 12 cGy divided into six doses, given over 3 days with lung shielding at 800 cGy, cyclophosphamide 120 mg/kg, and ATG 75 mg/kg. CsA was initiated at −7 to −1 days and continued after the transplant at a dose adjusted to maintain the plasma level in the range of 180–200 ng/ml, unless a lower level was required because of toxic manifestations. Prednisone was given during the period of ATG treatment and after the transplant at a dose of 1 mg/kg until day +30 when it was discontinued unless required for treatment of manifestations of GVHD. Prior to transplantation, patients received a course of trimetoprin-sulphamethoxazol as prophylaxis for Pneumocistis carinii, and for herpes virus prophylaxis, gancyclovir from day −7 to −1 and acyclovir from day +1 to +28 were given. Patients were nursed in single rooms with filtered air at positive pressure and only foods of low microbiological content were provided. Prophylactic oral antibiotics (norfloxacin, ketokenazol and nystatin) were given, to be discontinued when intravenous antibiotic therapy was required for treatment of infections or fever. G-CSF was given from day +1 until engraftment.


Diagnostic methods For pre-transplant HLA typing, serological methods were used for class I and molecular methods for class II loci, as these are methods used in CB banking. Trypan blue exclusion and/or 7-AAD flow cytometry were used to evaluate viability of the cellularity in the transplanted products. Analysis of DNA polymorphisms was used to identify the derivation of cells present in PB and BM samples after the transplant. In each case, this was carried out on the basis of the most appropriate microsatellite markers and of the HLA differentiating antigens using the RSCA method.26 This method allows a relative quantification of the DNA from different possible origins (patient, purified CD34+ cells, and unexpanded CB, as well as contaminating maternal cells in the CB units) with a sensitivity in the order of 3–5%.

planted without previous culture had no more than two HLA-A, -B, -DR antigen disparities with the recipient. Based on post-thaw counts, the infused TNC of these CB units were 1.56 to 2.7 × 107/kg. The CB cells that were transplanted in patients Nos 1 and 2 after CD34+ selection and culture had two and four major HLA Ag disparities with the recipient and also had HLA differences with the transplanted uncultured CB. The number of CD34+ expanded cells that were infused into these patients were 0.57 and 0.45 × 106/kg, respectively, virtually free of contaminating CD3+ cells. Their expansion factors were 11.4 and 24.3. The numbers of CD34+ uncultured haploidentical cells transplanted in patients Nos 3, 4 and 5 were 1.22, 1.55 and 2.43 × 106/kg, respectively, and the number of contaminating CD3+ cells, 0.7, 0.3 and 0.22 × 104/kg.

357

Engraftment Results Transplanted products Table 2 shows histocompatibility, cell content and cell viability data for the transplanted products. Cell viability was 70–95% (median 84%). All CB units that were trans-

Table 2

As may be seen in Table 3, all five patients achieved haematopoietic reconstitution after the transplant. The two patients (Nos 1 and 2) who received cells of two CB units (uncultured and cultured) had sustained ANC ⬎500/ml from, days +27 and +17 respectively. Patient No. 1 had a sustained platelet count of ⬎20 000/ml without transfusion

HLA compatibility and cell content data of transplanted products Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

24, 30 18, 49 *0301, *1303

3, 29 7, 49 *0101, *1103

*02, *03 *58, *51 *1114, *1302

24, 28 27, 57 *07, *1601

1, 2 41, 57 *1303, *0701

1, 30 18, 49 *0301, *1302 2/6

3, 29 7, 35 *0101, *1101 2/6

*02, *33 *58, *51 *1101, *1302 2/6

0101, − 2702, 5701 *0701, *1601 2/6

1, 2 41, 57 *1303, *0701 0/6

1.85 0.1 239 1.67 95%

2.55 0.03 342 2.61 70%

1.56 0.11 ND 1.57 94%

2.70 0.26 ND 2.6 92%

2.38 0.107 ND 1.12 70%

Transplanted purified CD34+ cells HLA A B DR Major HLA Ag mm with patient Source

24, 30 18, 7 *0301, *1302 2/6 unrel UCB

2, 32 49, 61 *0101, *1101 4/6 unrel UCB

*02, − 45, 51 11, 10 3/6 haploid sib PB cells

2, 24 51, 57 *1601, − 2/6 haploid sib PB cells

1, 29 44, 41 *07, *13 2/6 haploid sib PB cells

Infused cells (Culture exp. factor) TNC (×106/kg) CD34+ (×106/kg) CD3+ (×104/kg) CFU (×104/kg) Cell viability

0.6 (×10.2) 0.57 (×11.4) 0.058 16.5 (×13.2) 89%

0.51 (×14.2) 0.45 (×24.3) 0.026 2.11 (×12.3) 90%

1.25 1.22a 0.7 4.53d 83%

1.59 1.55b 0.3 6.6d 70%

2, 43 2, 41c 0.22 27.48d 88%

Patient HLA A B DR Transplanted uncultured UCB HLA A B DR Major HLA Ag mm Infused cells TNC (×107/kg) CD34+ (×106/kg) CD3+ (×104/kg) CFU (×104/kg) Cell viability

0.4 CD38− and 0.79 CD38+. 0.2 CD38− and 1.36 CD38+. c 0.7 CD38− and 1.77 CD38+. d CFU GM + E + Meg (Only CFU-GM counted in patients Nos 1 and 2). ND = not done. a

b



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Table 3

Patients’ post-transplant course

Days with ANC ⬎500/␮l

Patient 1

Patient 2

from +27 onwards

from +17 onwards

Patient 3

from +12 to +16 and from +24 onwards +72 NA +54 +45 +30 +87 full uncultured CB mixed uncultured CB full CB and patient CMV Ag CMV Ag CMV Ag CMV pneumonia recurrent focal pneumonia very mild 0 grade I ⬎22 months 63 days ⬎19 months alive dead alive

Days to platelets ⬎20 000/␮l Hospital discharge Final chimerism Major later infections GVHD Survival Status

Patient 4

Patient 5

from +17 onwards

from +10 onwards

+41 — full CB

NA +31 mixed patient, haplo and CB (CB predominant)

CMV Ag recurrent focal pneumonia 0 71 days dead

CMV pneumonia grade 1 56 days dead

NA = not achieved.

support from day +72 onwards; this was not achieved by patient No. 2. Of the other three patients receiving CB and haploidentical CD34+ cells, patient No. 3 had a clearly biphasic recovery, with an initial wave of ANC ⬎500/ml from days +12 to +16, reaching a maximum of 2040 on day +14. This was followed by lower counts from day +17 to day +26, and then by a long-lasting recovery. Patients Nos 4 and 5 had a continued ANC recovery, passing the 500/ml threshold on days +17 and +10, respectively. Post-transplant chimerism In the two patients who received cells from two CB units, DNA of the cultured cells was detected in BM and PB samples for a few days (1–3) around day +11, when the total leukocyte count was ⬍200/␮l, but not in later samples. Thereafter, and from several days before the ANC increased to ⬎500/␮l, the only donor DNA detected belonged to the uncultured CB units. Patient No. 1 reached full chimerism and patient No. 2 was in mixed (patient/uncultured CB) chimerism at the time of his death on day + 63 (Figures 1 and 2). In the three patients co-transplanted with CB and hap-

loidentical CD34+ cells, DNA of this phenotype was detected, also transiently, in BM and PB samples starting on day +10 or +11. In patient No. 3 it was detected until day +17, coinciding with the first wave of granulocyte recovery, a cell population in which the haploidentical phenotype peaked at ⬎90% of all DNA on day +14 (Figures 3, 4 and 5). In patient No. 4 the haploidentical phenotype was detected until day +24 and was the predominant fraction in the DNA extracted from granulocytes on day +19, when ANC reached ⬎500/␮l (Figure 6). These two patients remained in full chimerism beyond day +24. In patient No. 5, haploidentical DNA was initially the predominant but then the declining fraction, while CB DNA was detected in increasing proportions from day +12 onwards, becoming the predominant fraction both in granulocytes and mononuclear cells at the time of death on day +56 (Figure 7). This patient had persistent marrow blasts, and traces of autologous DNA also were detected after the transplant. In the two longest-surviving patients (Nos 1 and 3) there has been no reappearance of DNA belonging to the transplanted CD34+ cells (CB or haploidentical), which is consistent with no late development of lymphocytes from these cells.

Neg control

Post-transplant clinical course

Lane

1

2

3

4

5

6

7

8

9

10 11 12 13

Figure 1 Patient 2: double transplant of unexpanded and expanded CB cells. Microsatellite analysis of DNA chimerism (INZ 22 marker). Transient engraftment of expanded cord blood detected on day +11 sample. Samples in lanes are as follows: 1: expanded cord blood; 2: unexpanded cord blood; 3: patient’s cells pre-transplant; 4: day +11 bone marrow mononuclear cells; 5: day +17 peripheral blood mononuclear cells; 6: day +17 peripheral blood granulocytes; 7: day +18 bone marrow mononuclear cells; 8: day +18 bone marrow granulocytes; 9: day +25 peripheral blood mononuclear cells; 10: day +25 peripheral blood granulocytes; 11: day +25 bone marrow mononuclear cells; 12: day +25 bone marrow granulocytes; 13: negative control. Bone Marrow Transplantation

The patients did not have any major complications, infectious or otherwise, during the period of post-transplant neutropenia. Following engraftment, three patients had manifestations of acute GVHD that were only cutaneous: very mild and very quickly subsiding without specific treatment in patients Nos 1 and 5; grade I and promptly responding to steroid treatment in patient No. 3 (Table 3). Analysis of the DNA extracted from cutaneous biopsies of patients Nos 3 and 5 did not show evidence of infiltration by haploidentical cells. In all cases, this lack of major manifestations of acute GVHD allowed for rapid tapering of steroids and CsA in an attempt to minimize risks related to post-CB transplant immune deficiency, and to hasten possible GVT effects. After engraftment and initial hospital discharge, the main clinical problems in four patients have been related to


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a 0.5

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B*0702

B*4002

0.5

B*0705

0.5

B*35

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Exp CB

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0.16

B*4901 393

1300 1400 1500 1600

ai

Upper marker

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394

1900 2000 2100

2200 2300 2400

B*0705 B*35

Unexp CB

395

0.14

1300 1400 1500 1600

0.12 0.10 0.08

0.5

B*0702

0.06 0.04

B*0705/0702 (shoulder)

B*4002 (trace)

0.4 0.2

1900 2000 2100

2200 2300 2400 B*4901

BM day + 11 1300 1400 1500 1600

0.02

1700 1800

B*4002 (trace amounts) (see 4a-1)

1700 1800

396

1900 2000 2100

2200 2300 2400

B*0705 then B*0702 distinct peaks

PB PMN day + 17

398

1220123012401250126012701280 1290130013101320133013401350136013701380

1300 1400 1500 1600

1700 1800

1900 2000 2100

2200 2300 2400

Mobility values Figure 2 Patient 2: double transplant of unexpanded and expanded CB cells. RSCA analysis of chimerism. (a) HLA-B locus RSCA electropherograms of pre- and post-transplant DNA. RSCA was performed as described by Arguello et al.26 HLA-B mismatches are seen as different peak mobilities after polyacrylamide gel electrophoresis, the method having capacity to detect DNA of minor populations above a threshold of 3–5%. Lanes 393, 394 and 395 demonstrate HLA type of pre-transplant samples. Lane 393: patient 2 alleles B*0702 and B*4901. Lane 394: expanded CB unit tissue typed as B*4002 allele (serological specificity B61(40)) with the shared B*4901. Lane 395: non-expanded CB unit confirms the B antigen mismatch B35/49 and an additional B*0705/0702 incompatibility with the patient. Lane 396: 11 days post-transplant BM total cell-derived DNA exhibiting HLA-B chimerism with transient engraftment of CB (expanded) detectable from trace amounts of B*4002 (not evident on panel A electropherogram, although visible in the amplified tracing shown in panel ai). The derivation of low levels of B*4901 may be the patient or CB (expanded). B*0702 from the patient is detected as a ‘shoulder’ present on the B*0705 peak from the CB (not expanded). Lane 398: PB granulocytes 17 days post transplant demonstrating patient and CB (not expanded) HLA-B alleles.

G-CSF DNA in PMN

10000

day CB Brother

9000

Leukocytes/ml

8000

+13

+17

6% 91% 94% 9%

+29 100% 0%

7000

WBC

6000

ANC

5000

Platelets

4000 3000 2000

Platelets 20000

1000 0

ANC 500 0

2

5

8

11 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56

Days Figure 3 Patient 3: double transplant of unexpanded CB cells and purified PB CD34+ cells. Biphasic raise of ANC. Predominance of haploidentical cells in the initial wave.

CMV, for which they received therapy with gancyclovir and/or foscarnet. Patients Nos 1 to 4 received pre-emptive courses upon detection of CMV antigenemia. The evolution has been favourable in patients Nos 1 and 3, whose survival at the time of writing is, respectively, more than 22 and 19 months; both remain in CR. In patients Nos 2, 4 and 5, rapidly evolving CMV infection resulted in death (+63, +71 and +56 days).

Discussion The relatively narrow HLA restriction that, compared to solid organ transplants, is required for BM or PB stem cell transplantation is related more to the risks inherent to the immune reaction of the transplanted cells against the host than to graft rejection. Thus, due to the relative naivety of its lymphoid cell content, the use of CB as a source of Bone Marrow Transplantation


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a B*51011

0.5

Upper marker

B*5801

Patient

6262 1750 1800 B*51011

1850

1900

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2050

2100 B*4501

0.5

Haplo

6254 1750

1800

1850

b51 variam

0.5

1900

1950

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B*5801

CB

6268 1750

1800

1850

1900

0.5

1950

6275B 1750

1800

1850

1900

0.5

1950

1750

1800

1850

1900

1800

1850

2100

+11 2000

2050

2100

+19

1950

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2050

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+21

PB

6294 1750

2050

PB

6289

0.5

2000

BM gr

1900

1950

2000

2050

2100

Mobility values b Upper marker 0.5

6448A

1650

+123 1700

1750

1800

1850

1900

1950

2000

2050

2100

6448B PB gr

1600

0.5

B*5801

PB mn

1600

0.5

b51 variam

6313

1600

1650

Skin

1650

+123 1700

1750

1800

1850

1900

1950

2000

2050

2100

1750

1800

1850

1900

1950

2000

2050

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B*51011

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Mobility values Figure 4 Patient 3: double transplant of unexpanded CB cells and purified peripheral blood haploidentical CD34+ cells. RSCA analysis of chimerism. (a) HLA-B locus RSCA electropherograms of pre- and post-transplant DNA. Lanes 6262, 6254 and 6268 demonstrate HLA type of patient and donors pre-transplant. Lane 6262: Patient 3 alleles B*51011 and B*5801. Lane 6254: haploidentical sibling DNA exhibits the shared B*51011 with B*4501. Lane 6268: CB donor has B*5801 in common with the patient but possesses a B51 variant with a mobility value different from that observed for B*51011. Sequence-based typing (SBT) has detected a new HLA-B allele with a single nucleotide substitution, with respect to B*51011, at a known polymorphic site (McWhinnie et al, unpublished data). Lane 6275B: DNA derived from BM granulocytes 11 days post transplant shows mixed chimerism with CB and sibling HLA-B alleles present. Relative quantitative scaling across the lane demonstrated by peak height/area with special reference to B*4501 indicates the prevalence of sibling donor fraction. Lanes 6289 and 6294: DNA extracted from PB non-differentiated leukocytes at +19 and +21 days show full CB chimerism. (b) HLA-B locus RSCA electropherograms of post-transplant PB and skin biopsy DNA. Lanes 6448A and 6448B show the full CB chimerism in DNA isolated from PB mononucleocytes and granulocytes, respectively, 123 days post transplant. Lane 6313 is DNA isolate of skin biopsy taken from patient when showing signs of GVHD. The B*51011 peak indicates the prevalence of patient cells but the mismatched B51 variant from the CB can be seen at lower levels. No DNA from the sibling was detected.

transplantable haematopoietic stem cells may reduce the need for a narrow HLA restriction. Indeed, available data suggest that CBTs result in a lower risk of severe GVHD and higher HLA disparity tolerance.1–12 However, CBT has problems related to risk of graft failure or relatively late engraftment, the median time of which is around 26 days, rarely occurring before day +20 in adult patients receiving Bone Marrow Transplantation

⬍3.7 × 107/kg nucleated cells.1–6 This late engraftment is most likely due to the relatively low number and early developmental stages of the haematopoietic progenitors present in the collected CB units.27–31 In patients Nos 1 and 2, who received transplants of CB without post-thaw culture plus cells obtained by positive selection and ex vivo expansion culture of the CD34+ cells from a HLA-different


Lanes

1

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3

4

5

6

7

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9

10

11

ers well before full engraftment of the uncultured CB unit and of any manifestation of GVHD. The lack of adverse effects that could be related to the simultaneous transplantation of cultured cells allows the dual transplant model to be regarded as a potentially useful tool to evaluate the engraftment potential of CB cells grown in ex vivo expansion cultures in human recipients, as it has been recently described in a xenogeneic model.21 This could allow progress in searching for culture conditions that could be more efficient than those we have used for our patients. Our data also suggest that the reported results of CB transplants in which an aliquot of the transplanted unit has been cultured prior to its delayed infusion should be interpreted with caution.15–18 Results obtained in the three patients (Nos 3, 4 and 5) who received the CB transplants together with the infusion of purified uncultured haploidentical CD34+ cells highly depleted of contaminating lymphocytes suggest a promising alternative for reducing the risks inherent to the post-transplant period of neutropenia. Their different post-transplant dynamics, compared to cultured CD34+ CB cells, could be related to the larger number of infused cells or to a higher capacity for proliferation, as they are native and not precultured. Their otherwise limited capacity for lasting engraftment, reflected in the transient generation of granulocytes, may be related to the relatively low number of progenitors that we deliberately infused to keep the number of contaminating lymphocytes low. This may have resulted in a rapid exhaustion of the most primitive cells under highly proliferative stimuli, as grafts of a much larger number of lymphocyte-depleted HLA haploidentical CD34+ cells have been reported to result in long-lasting engraftment.32 As previously discussed, the possibilities of immune rejection seems unlikely. Simultaneous infusion of a limited number of haploidentical CD34+ cells highly devoid of contaminating lymphocytes in conjunction with CB may be beneficial not only in reducing risks of bacterial and fungal infections, but also in allowing early initiation of prophylaxis or pre-emptive treatment for CMV with gancyclovir. Further study thus

12

Figure 5 Patient 3: double transplant of unexpanded CB cells and purified peripheral blood haploidentical CD34+ cells. Microsatellite analysis of DNA chimerism (MCT 118 marker). Predominant engraftment of haploidentical cells observed in days +11 and +13 samples. Progressive CB engraftment was detected from day +11. 1: haploidentical cells; 2: cord blood cells; 3: patient’s cells pre-transplant; 4: day +11 bone marrow mononuclear cells; 5: day +11 peripheral blood mononuclear cells; 6: day +13 bone marrow mononuclear cells; 7: day +13 bone marrow mononuclear cells; 8: day +19 peripheral total cells; 9: day +21 peripheral total cells; 10: day +23 bone marrow total cells; 11: negative control.

second CB unit, our results do not show contribution by the cultured cells to the recovery of circulating granulocytes. These reached the 500/␮l level after intervals (27 and 17 days) that are similar to what is usually observed in plain CBT both in children and adults,1–6 and DNA of the cultured cells was detected only in BM and PB sampled at earlier times (days +10 to +12). Similar results were observed in three other patients transplanted in another hospital using our protocol.19 The lack of effectiveness of culture-expanded CB cells in producing a significant number of circulating granulocytes may reflect a lack of capacity for sustained proliferation. Alternatively, it could be a consequence of rejection, either by the residual immune system of the patient or by immune cells derived from the co-transplanted unmanipulated CB unit. The lack of contaminating lymphocytes in the cells of the cultured CB units might play a role in this regard. Although immune rejection could be a possibility, we believe it is unlikely, not only because of the intensive immune suppressive regimen the patients were receiving, but because of the disappearance of the cultured cell mark-

361

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% DNA

WBC and ANC/ml

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Day WBC

ANC

% UCB DNA in neutrophils

% Haplo DNA in neutrophils

Figure 6 Patient 4: double transplant of unexpanded CB and purified PB haploidentical CD34+ cells. Bars representing DNA % in neutrophils show data derived from the RSCA analysis and of HLA markers. Transient engraftment of haploidentical cells. Long-lasting engraftment of CB cells. Bone Marrow Transplantation


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PB granulocytes

cells; and (4) the culture conditions we used to expand CB cells have not resulted in early recovery of ANC. It can be theorized that the double transplant approach could be combined with methods of pre-transplant coculturing in order to induce selective immunotolerance of the CB cells to the recipient antigens.33 This might allow us to reduce the intensity of immunosuppressive regimens, thus reducing risks of opportunistic infections or even tumour relapse, since the possible anti-tumour immunotherapeutic effect of CBT might be enhanced.

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We wish to thank the ‘Fundacio´ n Ramo´ n Areces’ and the ‘Fundacio´ n Josep Carreras’ for financial support of this work. Mrs P Dodi is acknowledged for her skilled editorial help.

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References

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Days after Tx Figure 7 Patient 5: double transplant of unexpanded CB and purified PB haploidentical CD34+ cells. Bars indicate % of DNA from haploidentical and CB origin in peripheral blood granulocytes (upper panel) and mononuclear cells (lower panel) as evaluated on the basis of HLA markers determined by the RSCA method. Transient and initially predominant engraftment of haploidentical cells. Later progressive predominance of CB-derived cells.

seems justified and desirable and this could benefit from a co-operative, multi-institutional approach. It can be speculated that the remarkably mild manifestations of GVHD observed in our patients might suggest that co-transplanted cultured CB cells or purified haploidentical PB CD34+ cells could affect the reactivity of the engrafted CB cells against the host. In conclusion, we believe our data shows that: (1) the simultaneous infusion of uncultured CB cells and purified HLA mismatched haematopoietic progenitor cells has not resulted in unfavourable effects (prohibitive GVHD or CBT engraftment failure); (2) the double transplant model we have used is suitable for evaluating the engraftment potential of ex vivo cultured CB cells in the clinical setting; (3) the model may be an alternative strategy to improve engraftment of umbilical cord blood by co-infusion of either ex vivo expanded cells of a second umbilical CB unit or a limited number of uncultured sibling haploidentical Bone Marrow Transplantation

1 Wagner JE, Kernan NA, Steinbuch R et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995; 346: 214–219. 2 Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996; 88: 795–802. 3 Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New Engl J Med 1996; 335: 157–166. 4 Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. New Engl J Med 1997; 337: 373–381. 5 Rubinstein P, Carrier C, Scaradavou A et al. Outcome among 562 recipients of placental blood transplants from unrelated donors. New Engl J Med 1998; 339: 1565–1577. 6 Rocha V, Wagner JE, Sobocinski KA et al for Eurocord. Graft-versus-host disease in children who have received a cord blood or bone marrow transplant from an HLA–identical sibling. New Engl J Med 2000; 342: 1846–1854. 7 Rocha V, Cornish J, Sievers E et al for Eurocord. Comparison of the outcome of unrelated bone marrow or cord blood transplant in children with acute leukemia. Blood 1999; 94 (Suppl. 1): (Abstr. 3143). 8 Wagner JE, DeFor T, Barker J et al. Superior survival in recipients of umbilical cord blood (UCB): results of a case controlled analysis comparing UCB and bone marrow from unrelated donors. Blood 1999; 94 (Suppl. 1): (Abstr. 3142). 9 Long G, Madan J, Kurtzberg J et al. Unrelated umbilical cord blood transplants in adult patients with hematologic malignancies or genetic disorders. Blood 1999; 94 (Suppl. 1): (Abstr. 2544). 10 Barker NJ, DeFor T, Verfaille CM et al. Transplantation of mismatched unrelated donor umbilical cord blood in adult patients with high risk disease: outcome in recipients of 1– 2 × 107 nucleated cells/kilogram. Blood 1999; 94 (Suppl. 1) (Abstr. 2562). 11 Thomson BG, Robertson KA, Gowan D et al. Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation. Blood 2000; 96: 2703–2711. 12 Rocha V, Arcese W, Sanz G et al for Eurocord. Prognostic factors of outcome after unrelated cord blood transplant in


13 14

15

16

17

18

19 20

21

22

23

adults with hematologic malignancies. Blood 2000; 96: 587a (Abstr. 2521). Fernandez MN, Millan I, Gluckman E. Cord blood transplants (letter). New Engl J Med 1999; 340: 1287. Migliaccio, AR, Adamason JW, Stevens CE et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 2000; 96: 2717–2722. Ko¨ gler G, Nu¨ rberger W, Fisher J et al. Simultaneous cord blood transplantation of ex vivo cells expanded together with non-expanded cells for high risk leukaemia. Bone Marrow Transplant 1999; 24: 397–403. Schpall EJ, Quinones R, Jones R et al. Transplantation of adult and paediatric cancer patients with cord blood progenitors expanded ex vivo. Blood 1999; 94 (Suppl. 1): (Abstr. 3145). Kurtzberg J, Martin PL, Driscol T et al. Augmentation of umbilical cord blood transplantation with ex vivo expanded cells, a phase I trial using the replicell system. Blood 1999; 94 (Suppl. 1): (Abstr. 2545). Pecora AL, Stiff P, Jennis A et al. Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood. Bone Marrow Transplant 2000; 25: 797–799. Fernandez MN, Gran˜ ena A, Milla´ n I et al. Evaluation of engraftment of ex vivo expanded cord blood cells in humans. Bone Marrow Transplant 2000; 25: (Suppl. 2): S61–S67. Gu¨ enechea G, Segovia JC, Albella B et al. Delayed engraftment of non obese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34+ cord blood cells. Blood 1999; 93: 1097–1105. Rosler ES, Brandt JE, Chute J et al. Competitive engraftment potential of primary ex vivo expanded cord blood CD34+ cells in a triple HLA-mismatched SCID-hu bone model. Blood 1999; 94 (Suppl. 1): (Abstr. 3120). Piacibello W, Sanavio F, Severino A et al. Engraftment in non-obese diabetic severe combined immunodeficient mice of human CD34+ cord blood cells after ex-vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999; 93: 3736–3749. Weinreb S, Yunis EJ, Cohn G et al. Transplantation of unrelated cord blood cells. Bone Marrow Transplant 1998; 22: 193–196.

24 Rachamin N, Gan J, Segall H et al. Tolerance induction by ‘megadose’ hematopoietic transplants: donor type human CD34 stem cells induce potent specific reduction of host antidonor cytotoxic T lymphocyte precursors in mixed lymphocyte culture. Transplantation 1998; 65: 1386–1393. 25 Querol S, Capmany G, Azqueta C et al. Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex-vivo expansion protocol. Transfusion 2000; 40: 625–631. 26 Arguello JR, Little AM, Pay AL et al. Mutation detection and typing of polymorphic loci through double-strand conformation analysis. Nat Genet 1998; 18: 192–194. 27 Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992; 89: 4109–4113. 28 Payne TA, Traycoff CM, Laver J et al. Phenotypic analysis of early hematopoietic progenitors in cord blood and determination of their correlation with clonogenic progenitors: relevance to cord blood stem cell transplantation. Bone Marrow Transplant 1995; 15: 187–192. 29 de Wynter EA, Nadali G, Coutinho LH, Testa NG. Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identifications of long term culture initiating cells present in two subsets. Stem Cells 1996; 4: 566–576. 30 Wang JCY, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997; 89: 3919–3924. 31 Noort WA, Rad M, van der Marel A et al. Qualitative differences in CD34+ cells of umbilical cord blood, bone marrow and mobilized blood are responsible for differential engraftment in NOD/SCID mice. Blood 1999; 94 (Suppl. 1): (Abstr. 2546). 32 Aversa F, Tabilio A, Velario A et al. Treatment of high-risk leukemia with T-cell depleted stem cells from related donors with one fully mismatched HLA haplotype. New Engl J Med 1998; 339: 1186–1193. 33 Guinan EC, Boussiotis VA, Neuberg D et al. Transplantation of anergic histoincompatible bone marrow allografts. New Engl J Med 1999; 340: 1704–1714.

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