Progenitor cells trapped in marrow filters can reduce GvHD ... - Nature

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ORIGINAL ARTICLE

Progenitor cells trapped in marrow filters can reduce GvHD and transplant mortality D Vicente1, M Podesta`1, A Pitto1, S Pozzi1, S Lucchetti1, T Lamparelli1, E Tedone1, A Ibatici1, O Figari1, F Frassoni1, MT Van Lint1, G Piaggio1, N Sacchi2 and A Bacigalupo1 1 Divisione Ematologia 2, Ospedale San Martino, Genova, Italy and 2The Italian Bone Marrow Donor Registry, Ospedale Galliera, Genova, Italy

A bone marrow harvest is filtered either in the operating room, in the laboratory or during infusion to the patient. Filters are usually discarded. Little is known of haemopoietic progenitor cells (HPCs) trapped in the filters. The aim of the study was to evaluate HPC content in the filters and to assess the outcome of transplants with filterdiscarded or filter-recovered cells. Haemopoietic progenitors were grown from filters of 19 marrow transplants. We then compared the outcome of 39 filter-recovered transplants from HLA-identical siblings (years 2001– 2004) with a matched cohort of 43 filter-discarded marrow grafts (years 1997–2000). Filters contained on average 21% long-term culture-initiating cells (LTC-IC) and 15% fibroblasts colony-forming units (CFU-F) of the total progenitor cell content. Filter-discarded transplants had significantly more grade II–IV graft-versus-host disease (GvHD) (42 vs 15%, P ¼ 0.008) as compared to filter-recovered transplants, and more transplant-related mortality (TRM) (20 vs 3%, P ¼ 0.04). The actuarial survival at 5 years is 69 vs 87%, respectively (P ¼ 0.15). This study suggests that a significant proportion of LTCIC is lost in the filters together with CFU-F. Recovery and add back of progenitors trapped in the filters may reduce GvHD and TRM. Bone Marrow Transplantation (2006) 38, 111–117. doi:10.1038/sj.bmt.1705413; published online 5 June 2006 Keywords: mesenchymal stem cells; haemopoietic progenitor cells; bone marrow filter; GvHD

Introduction Cell dose has been shown to play a crucial role in bone marrow transplantation (BMT): this was known in the early 1970s when Storb et al.1 showed the impact of cell dose in transplantation on severe aplastic anaemia.

It was thereafter confirmed in other studies of allogeneic BMT in leukaemia patients.2,3 The study from the European Group for Blood and Marrow Transplantation (EBMT) in patients with acute myeloid leukaemia (AML) in first remission showed that so-called ‘rich marrow’ (42.7 108 cells/kg) had significantly better outcome when compared to ‘poor marrow’.4 It is unclear which cells contribute to improved outcome: is it the haemopoietic stem cells (HSCs) or is it accessory cells? Within the bone marrow (BM), the nonhaemopoietic cells contribute greatly to the fate of HSC supporting long-term haemopoiesis.5 It has been postulated that critical stromal cells and haemopoietic progenitor cells (HPCs) form solid complexes termed haematons, identified in the very low-density floating layer of freshly aspirated human BM. Endothelial cells, fibroblasts, lipid-laden cells and macrophages are found within the haematon complex, tightly packed with haemopoietic cells. Because the majority of haematon particles are over 50 mm in diameter, they are trapped and removed by the BM filters commonly used.6,7 Because cell dose is crucial in allogeneic BMT, care should be taken as to how marrow is harvested from the posterior iliac crests: in a randomized study, we have shown that small volume aspirates (2 ml) produce significantly more cells and progenitors as compared to large volume (20 ml) aspirates.8 Therefore, harvesting can be optimized. What is done to marrow cells after harvest is left to each single centre: some centres filter the marrow in the operating room; some centres filter the marrow at the bedside. Filters used are usually 180–263 mm. In our unit, after marrow cells harvesting, we have always used filters at the bedside, and then discarded the filters after infusion. As of January 2001, we have started to wash the marrow filters after infusion. We have counted progenitor cell colonies from cells trapped in the filters, and then added back the cells recovered. We now report the results of this study with a comparison in BMTs performed in the years 1997–2000 and 2001–2004.

Patients and methods Correspondence: Dr D Vicente, Divisione Ematologia 2, Ospedale San Martino, Largo Rosanna Benzi 10, Genova 16132, Italy. E-mail: [email protected] Received 9 March 2006; revised 28 April 2006; accepted 2 May 2006; published online 5 June 2006

BM harvest Marrow blood is harvested with 20 ml syringes and injected directly into a plastic 500 ml container. The marrow from

Progenitor from marrow filters reduce GvHD and TRM D Vicente et al

112

two or more containers is transferred to a single 2000 ml bag through a filter and then infused to the patient via an infusion pump.

Filters We have always used filters (180–263 mm) during infusion of the marrow to the patients. The filters have always been discarded at the end of the marrow infusion. As of 1 January 2001, we have changed this procedure: we are now using the 180 mm filter as we transfer the marrow from single containers to the 2000 ml bag. At this point, the filter has a yellowish appearance and is clearly loaded with cells, fat and some fragments of bone. We then wash the filter with an additional 500 ml of saline, manually manipulating the filter until it becomes clear. The saline and recovered cells are added to the 2000 ml transfer bag. The marrow is now ready to be infused into the patients with no additional filter. An aliquot of the recovered cells was taken for cell count, in vitro progenitor cell growth and flow cytometry. Twelve samples were from BM harvested from related donors in our unit and seven were from unrelated donors harvested in other centres (Table 1). Flow cytometry The HPC were identified by flow cytometry using the Stem-kit Reagents (Immunotech, Marseille, France), for the simultaneous identification and enumeration in a single platform of CD45 þ and dual positive CD45 þ CD34 þ cell population percentages and absolute counts in BM specimens. One hundred microlitres of BM samples were incubated for 20 min in the dark and room temperature with 20 ml of CD45FITC/CD34PE, 581 clone (Immunotech, Marseille, France), or with 20 ml of control and 7-AAD viability dye (Immunotech). Then, the cells were lysed with NH4Cl and incubated for 10 min in the dark at room temperature. One hundred microlitres of fluorospheres (the absolute count reagent) were added. The committed progenitor cells were identified with the 10 ml CD45PC5/CD34PE/CD33FITC (Immunotech) per tube on 100 ml of the BM sample. The CD3 þ T lymphocytes were identified by 10 ml CD3 FITC (Caltag, Burlingame, CA, USA) per tube on 100 ml of the BM sample.

Table 1

All the tubes were analysed by COULTER EPICS XLMCL flow cytometer, equipped with System II Software/ Stem-one System (Beckman-Coulter, Miami, FL, USA).

Progenitor cell growth CFCs were tested using standard techniques. Briefly, 2 104 BM mononuclear cells (MNCs) were plated in 0.9% methylcellulose supplemented with 30% fetal calf serum, L-glutamine 200 mM, 2-mercaptoethanol 104 in the presence of 50 ng/ml of interleukin-3, GM-CSF, G-CSF and 2 U/ml of erythropoietin. Colonies were scored after 12–14 days of incubation at 371C and 5% CO2 in air. Long-term culture-initiating cell. BM MNCs were plated onto a preirradiated (1500 cGy) murine stromal cell line M210-B4 and maintained for 3 days at 371C, then switched to 331C and fed weekly by replacement of half of the growth medium (Myelocult; Stem Cell Techologies Inc., Vancouver, BC, Canada) þ 106 mol/l hydrocortisone (Sigma Chemical Co, St Louis, MO, USA) containing half of the non-adherent cells with fresh growth medium. After 5 weeks, adherent cells were trypsinized and combined with non-adherent fraction. These harvested cells were washed and aliquots were assayed for clonogenic precursors in standard methylcellulose culture as described above. Limiting dilution assay. The absolute number of longterm culture-initiating cells (LTC-IC) was determined using a limiting dilution technique to calculate their proliferative potential. Briefly, the cells were seeded into 96-well flatbottomed trays; the following day, four dilutions of cells were added to each of 96 wells (total volume 200 mm). A minimum of 12 replicates for each dilution were performed. Cultures were fed at weekly intervals for 5 weeks, then they were overlaid with semisolid culture medium and growth factors as described above. After 18–20 days of incubation, wells were examined for the presence of colonies. The frequency of LTC-IC and the mean number of colonies per positive well were calculated by determining the cell dilution that resulted in p37% negative wells, equivalent to single hit kinetics (1 LTC-IC/well) according to the Poisson distribution. The clonogenic capacity of a single LTC-IC was also calculated by dividing the number of colonies derived from bulk cultures by the frequencies of LTC-IC.9

Characterization of cells recovered from marrow filters BM harvested in Genoa (n ¼ 12)a

Nucleated cells CD34+ CD3+ CFU-GM CFU-F LTC-IC

3.5% 4.6% 2.5% 5.0% 19.3% 30.4%

(1.08–9.14) (0.52–15.16) (0.79–4.60) (0.34–45) (2.73–75.6) (1.06–90.4)

BM harvested in other centres (n ¼ 7)a 0.9% 1.08% 0.5% 1.3% 5.4% 2.4%

(0.04–1.76) (0.23–1.80) (0.02–1.15) (0.005–5.2) (2.0–12) (0–8.13)

P 0.006 0.07 0.02 0.18 0.05 0.03

Abbreviations: BM ¼ bone marrow; CFU-F ¼ colony-forming units-fibroblast; CFU-GM ¼ colony-forming units-granulocyte macrophage; LTC-IC ¼ longterm culture-initiating cells. Average values (and range) expressed as percentage (%) of the total cell (or progenitor) content of the harvest: 3.5 means 3.5% of the total cell content. Bold indicate significant P-values. a Number of donors.

Bone Marrow Transplantation


113 Table 2

Clinical characteristics of patients selected for outcome analysisa P

Year of transplant 1997–2000 filter-discarded (n ¼ 43)b

2001–2004 filter-recovered (n ¼ 39)b

Patients age (years)c

34 (16–50)

31 (17–47)

0.28

Patients sex M F Donor age (years)c

24 19 35 (7–52)

27 12 29 (9–57)

0.21

26 17

20 19

0.40

14 8 21 197 (88–800) 5.2 (3.3–12.2) 65 (43–102) 17 (11–28)

16 12 11 147 (102–1094) 4.6 (1.56–9.3) 71 (43–96) 19 (12–27)

Donor sex M F Diagnosis AML ALL CML Interval diagnosis–transplant (days)c Cells infused ( 108/kg)c Patient body weight (kg) Engraftment (days)c

0.05

0.27

0.06 0.008 0.1 0.05

Abbreviations: ALL ¼ acute lymphoblastic leukaemia; AML ¼ acute myeloid leukaemia; CML ¼ chronic myelogenous leukaemia. Bold indicate significant P-values. a These patients include only primary or secondary acute or chronic leukaemias, in first remission/first chronic phase, allografted with marrow from an HLAidentical sibling, in the periods 1997–2000 (filter-discarded) and 2001–2004 (filter-recovered). b Number of patients. c Median values.

Fibroblast colony-forming units (CFU-F) were tested by culturing 5 105 light density BM MNCs in Mesencult Medium (Stem Cell Technologies Inc., Vancouver, BC, Canada). This cell suspension was placed in 35 mm culture dishes and was incubated at 371C and 5% CO2. After 2 days, the medium was completely changed. Cultures were refed twice a week. After 14 days, medium was removed and the well washed with phosphate-buffered saline. The adherent CFU-F colonies were fixed with methanol and stained with May Grunwald Giemsa. Colonies were scored using an Olympus IM.

Patients Patients chosen for the matched pair analysis were primary or secondary acute leukaemias (30 patients with AML and 20 with ALL) and chronic myeloid leukaemia (32 patients) in first remission/first chronic phase, allografted with marrow from an HLA-identical sibling, in the periods 1997–2000 (filter-discarded) and 2001–2004 (filter-recovered). Median ages of patients were 34 (16–50) and 31 (17– 47) years, respectively (P ¼ 0.28). Patient’s and donor’s characteristics were comparable in the two groups, except for younger donor age in the filter-recovered group (35 vs 29 years, P ¼ 0.05) (Table 2). Median days from diagnosis to transplant was 197 (88–800) in the filter-discarded and 147 (102–1094) in the filter-recovered group (P ¼ 0.06). Conditioning regimen and graft-versus-host disease prophylaxis The conditioning regimen consisted of cyclophosphamide (120 mg/kg) and total body irradiation (TBI) (9.9–12 Gy).

Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporin (CyA) 1–2 mg/kg starting on day 1 and methotrexate (MTX) at the dose of 10–15 mg/kg on day þ 1 and 8–10 mg/kg on days þ 3, þ 6 and þ 11.

Laboratory parameters and GvHD The two groups of patients were compared for the following parameters on day þ 50 after transplant: platelet counts, haemoglobin level, white blood cell counts, lymphocyte counts, cholinesterase serum level, total serum protein, serum albumin and dose of oral CyA and oral cortisone (Table 3). GvHD was graded using standard criteria.10,11 Statistical analysis The w2 and Mann–Whitney rank sum tests were used to test for differences between groups. Kaplan–Meier plots were used for survival analysis (event ¼ death due to any cause). Cumulative incidence (CI) was used for competing risks: relapse (failure ¼ relapse), transplant-related mortality (TRM) (failure ¼ death in remission).

Results Flow cytometry and progenitor cells grown from filters The proportion of cells recovered from filters and expressed as average percentage of the total cell content of the transplant, including minimum and maximum, was as follows: total nucleated cells: 2.67 (0.04–9.1); CD34 þ cells: 3.4 (0.2–15); CD3 þ cells: 1.8 (0.02–4.6); CFU-GM: 3.8 Bone Marrow Transplantation


114 Table 3

Clinical outcome of patients receiving or not cells recovered from marrow filter

Day+50 parameters

P

Year of transplants 1998–2000 Filter-discarded (n ¼ 43)a

Platelets ( 109/l)b Haemoglobin (g/dl)b WBC ( 106/l)b Lymphocyte ( 106/l)b Total serum protein (g/dl)b Serum albumin (g/dl)b Cholinesterase (IU/ml)b CyA dose (mg/kg)b Oral cortisone dose (mg/kg)b Acute GvHD II–IV Acute GvHD III–IV

113 9.8 6.1 0.9 6.3 3.8 3388 5.6 0.15 18 3

Chronic GvHD Limited Extensive Overall TRM Relapse Relapse-related death Overall survival Follow-up (days)

26 (65%) 14 (35%) 20% 36% 13 % 69% 2647 (2090–3287)

2001–2004 Filter-recovered (n ¼ 39)a

(19–235) (7.6–12.3) (1.9–14) (2–47) (3.8–7.8) (2.3–4.4) (770–5296) (0–9.8) (0–0.8) (42%) (7%)

142 10.4 4.9 1.7 6.7 3.9 4037 3.3 0 6 0

(28–321) (7.7–13.6) (2.6–11.9) (4–51) (4.6–8.1) (3.0–4.7) (1545–6590) (0.7–9.2) (0–0.6) (15%) (0%)

0.02 0.07 0.56 0.02 0.007 0.02 0.004 0.0001 0.04 0.008 0.09

27 (69%) 8 (20%) 3% 23% 11% 87% 1353 (454–1847)

0.06 0.04 0.42 0.98 0.15 o0.001

Abbreviations: CyA ¼ cyclosporin; GvHD ¼ graft-versus-host disease; TRM ¼ transplant-related mortality; WBC ¼ white blood cells. Bold indicate significant P-values. a Number of patients. b Median values. Chronic GvHD for patients at risk at day +100.

Clinical outcome of patients grafted with filter-discarded or filter-recovered cells The median time to neutrophil engraftment was slightly shorter in the filter-discarded patients (17 days) as compared to 19 days in the filter-recovered group (P ¼ 0.05) (Table 2). We then analysed the laboratory values on day þ 50 after transplant for the two groups (Table 3). Patients receiving filter-recovered grafts had significantly higher platelet counts (113 vs 142 109/l, P ¼ 0.02), lymphocyte counts (0.9 vs 1.7 106/l, P ¼ 0.02), total serum protein (6.3 vs 6.7 g/dl, P ¼ 0.007) and serum albumin levels (3.8 vs 3.9 g/dl, P ¼ 0.02). They also had significantly higher cholinesterase serum levels (3388 vs 4037 IU/ml; P ¼ 0.004) and less acute GvHD grade II–IV (42 vs 15%, P ¼ 0.008) and grade III–IV (7 vs 0%) (P ¼ 0.09) (Table 3). Limited chronic GvHD was observed in 65% of patients in the filter-discarded group and in 69% of those in the filter-recovered group, whereas extensive chronic GVHD occurred in 35 and 20%, respectively (P ¼ 0.06). Patients in the filter-discarded group were also receiving on day þ 50 a significantly higher dose of oral cyclosporin (5.6 vs 3.3 mg/kg, Pp0.0001) and oral prednisolone (0.15 vs 0 mg/kg, P ¼ 0.04) (Table 3). Bone Marrow Transplantation

100

75

% of TRM

(0.005–45); CFU-F: 15 (2.0–75); LTC-IC: 21 (0–90). Table 1 outlines the average percentage of cells recovered from filters of marrow harvested in Genova or in other transplant centres. Filters from marrows harvested in Genova contained significantly more total nucleated cells, CD3 þ cells, CFU-F and LTC-IC as compared with filters harvested in other centres.

50

P = 0.03

25 A

B

0 0.0

1000.0 2000.0 3000.0 Days from BMT

4000.0

Figure 1 Overall CI of TRM for patients in filter-discarded (A) and filterrecovered (B) groups.

TRM and survival Overall CI of TRM was 20 vs 3% (P ¼ 0.04) (Figure 1) in patients receiving filter-discarded or filter-recovered marrow, respectively (Table 3). Causes of TRM were as follows: in the filter-discarded group: acute GvHD in two patients (5%), infection in one patient (2%) and other transplant-related causes (hepatitis, congestive heart failure, multiorgan failure) in five patients (12%); and in the filter-recovered group, one patient (3%) died of infection. Relapse and relapse-related death were comparable in both


115

% of relapse related death

100

75

50 P = 0.98

25 A B 0 0.0

1000.0 2000.0 3000.0 Days from BMT

4000.0

Figure 2 Relapse-related death for patients in filter-discarded (A) and filter-recovered (B) groups.

100 B

% of overall survival

75 A P = 0.15 50

25

0 0.0

1000.0

2000.0 3000.0 Days from BMT

4000.0

Figure 3 Actuarial overall survival for patients in filter-discarded (A) and filter-recovered (B) groups.

groups (Figure 2) and overall actuarial 5-year survival was 69 vs 87% (P ¼ 0.15) (Figure 3) for filter-discarded or filterrecovered groups, respectively. Median follow-up of living patients is 2647 and 1353 days, respectively.

Discussion We have shown in this study that a significant proportion of LTC-IC are lost in the filters together with CFU-F: recovery and add back of progenitors, trapped in the filters, reduces GvHD and transplant mortality. As to the first point, we studied 19 filters from marrow harvested in our unit (Genova) or coming from marrow

donor centres (unrelated): we found no significant trapping of total nucleated cells and of CD3 þ cells. On the contrary, we could show trapping of progenitors in the filters, most evident for LTC-ICs: in some marrow samples, the proportion of LTC-IC in the filters exceeded 70% of the total LTC-IC content of the transplant. This indicates that when filters are used in the operating room and at the bedside, the transplant may be depleted of a significant proportion of stem cells. In this regard, we could show that filters recovered from marrow harvested in our unit contained significantly more progenitors as compared to filters recovered from marrow harvested in other centres. This is probably owing to the fact that in other centres, it is customary to use filters in the operating room, and the marrow shipped to destination, has already been filtered. Our findings are in keeping with studies showing that stromal cells and haemopoietic progenitors form solid complexes (haematons) that are trapped in BM filters.6,7 In a study performed by Blasek,12 there were 3.5 times more LTC-ICs in the haematon particles extracted from filters than in the buffy coats of filtered human BM isolates. The authors also studied the association of HPP-CFC and GMCFU with stromal cells by determining the frequencies of CFU-F in buffy coat and haematon fractions: only in the latter, HPP-CFC generated CFU-F in long-term culture. Having confirmed that filters contain a significant proportion of marrow progenitors, we then asked whether adding back these cells would change the clinical outcome of the transplant. We therefore compared two groups of patients grafted from filter-discarded or filter-recovered grafts. Patients had leukaemia in first remission/first chronic phase, and were grafted from HLA-identical siblings with cyclophosphamide TBI conditioning, and CyA – MTX for GvHD prophylaxis: filter-recovered grafts were comparable to the filter-discarded grafts, except for younger donor age (favourable), lower cell dose (unfavourable) and borderline shorter interval diagnosis transplant. The lower nucleated cell dose may be explained by heavier patients in the filter-recovered group (65 vs 71 kg), although this was not statistically significant. Of course, the two groups are consecutive and this should also be kept in mind when looking at the results. Recovery of platelet and lymphocyte counts was significantly enhanced in filterrecovered patients and this may be explained by the greater number of early haemopoietic progenitors in filter-recovered grafts; we also saw less grade II–IV acute GVHD, higher cholinesterase levels,13 and higher total protein and serum albumin levels. Why should filter-recovered patients have less acute GvHD? One explanation could come from the additional dose of mesenchymal stem cells (MSCs) infused in filter-recovered patients: indeed, MSCs have been shown to have potent immunosuppressive effects in vitro and in vivo, including suppression of T/B lymphocyte activation/proliferation and modulation of cytokine production.14–20 Preclinical and clinical models have confirmed an immunoregulatory role of MSC in autoimmunity and transplantation.16,21,22 Finally, MSC support long-term haematopoiesis by providing an ideal microenvironment for the division, proliferation and differentiation of HSC.5 The final question is whether reduced GvHD translates into reduced mortality: this is a difficult issue, as Bone Marrow Transplantation


116

manoeuvres reducing GvHD often increase the likelihood of infections or reduce engraftment, and do not change TRM. In this study, on the contrary, we found a significantly lower TRM in the filter-recovered group, suggesting a clinically relevant effect. The main cause of TRM in the filter-discarded group was related to organ failure (liver, heart or multiorgan failure). It could well be that MSC recovered from filters and co-infused with haemopoietic cells could migrate to other sites and provide help for regeneration.23–25 In a recent paper, the co-infusion of MSC and HSCs has been reported to be safe, but evidence on a protective role of this combination on GvHD is still lacking.26 On the other hand, some indication of an effect of MSC in the management of acute and chronic GvHD has recently been presented.27 Interestingly, relapse and relapse-related death were similar in both groups. This could be owing to the comparable rate of chronic GvHD, shown to have a more pronounced protective effect on relapse. Although not statically significant, the overall survival tended to be higher in the filter-recovered group (87 vs 69%) as compared to the filter-discarded group (P ¼ 0.15). The better outcome of the filter-recovered group is possibly a combined effect of better haematologic reconstitution (as indicated by platelet counts), better immune reconstitution (as indicated by lymphocyte counts), and lower GvHD (as indicated by GvHD grading, cholinesterase and protein serum levels). It is quite possible that several cell subtypes trapped in filters may contribute to this outcome, mimicking the results of the EBMT study, which showed that rich marrow is the best stem cell source for acute leukaemia 1st CR.4 In conclusion, filters used in the operating room or at the bedside for marrow harvests should not be discarded, but rather washed and the recovered cells should be added back to the marrow infusion: in our hands, this has resulted in reduced GvHD and TRM. This may be particularly relevant in the unrelated donor setting, which we were unable to assess, owing to the wide-spread use of filtering procedures in marrow donor centres.

Acknowledgements This work was partly supported by Fondazione CARIGE Genova, Associazione Italiana Ricerca contro il Cancro (AIRC) Milano and Associazione Ricerca Trapianto Midollo Osseo (ARITMO) Genova.

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