Background: We previously reported a pattern of lymphocyte expansion in transplant recipients prior to neutrophil engraftment following cyclophosphamide-fludarabine (Cy/Flu) nonmyeloablative allotrans-plantation (NMAT) in humans. T-cells and NK with approximately 85-90% donor chimerism were the predominant leukocyte population detectable in peripheral circulation in patients with acute myeloid leukemia (AML)/myelodysplasia (MDS) between days +6 and +8. This observation appears unique and reproducible and the mechanism is unknown. In an exploratory study, we analyzed TH1, TH2 and homeostatic cytokines present on day +7 in the serum of engrafting patients by Multiplex.

Methods: Sixteen patients with a variety of hematological malignancies who underwent Cy/Flu NMAT according to a phase II protocol (NCT00975975) at Indiana University between 2009 and 2011 were included. Frozen serum samples collected from patients on day +7 were thawed and cytokine levels measured using a commercially available multiplex platform (MPXHCYTO-60K, Millipore Corp, Billerica, MA, USA). Samples were run in triplicate and compared against sample matrix alone; sample matrix alone was spiked with known levels of the specific cytokines and standardized quality controls revealed low and high ranges for each cytokine. Clinical parameters included age, diagnosis, donor source, stem cell dose, daily temperature, serum albumin and days to engraftment. Data were analyzed using SAS.

Results: Median age was 60 years. Hematological diseases included acute myeloid leukemia/myelodysplasia (n=8), concomitant myelodysplasia and multiple myeloma (n=1), chronic lymphocytic leukemia (n=3), non-Hodgkin’s lymphoma (n=1), cutaneous T-cell lymphoma (n=1), acute lymphoblastic leukemia (n=1) and primary myelofibrosis (n=1). Patients received peripheral blood mononuclear cell grafts from matched related (n=8) or unrelated (n=8) donors. Median CD34+ dose was 4.7x108 cells/kg (range 2.0-8.1) and median CD3+ dose was 1.7x108 cells/kg (range 0.8 to 4.3). Median time to engraftment was 13.5 days (range 10 to 18). Table-1 summarizes clinical features and levels of various cytokine at day +7. Ten patients had a fever (>38.2oC) by day +7. Median baseline (day -7) albumin was 3.5 mg/dl (range 3.1 to 4.1). We observed a median decrease in albumin by 1.0 mg/dl (range 0.1 to 1.6; P<0.001) comparing baseline to day +7. Median day +7 cytokine levels were as follows: IFNγ, 5pg/dl; TNFα, 20pg/dl; IL-4, undetectable; IL-6, 68pg/dl; FLT3 ligand, 624pg/dl; IL-10, 59pg/dl; IL-15, 51pg/dl; IL-2, undetectable; IL-7, undetectable; sIL2-Rα, 1121pg/dl and IL-8, 73pg/dl.

Conclusion: Although confirmatory studies with larger numbers of patients are needed, this exploratory analysis of day +7 cytokine levels suggests that pre-engraftment lymphoid expansion following Cy/Flu NMAT is mediated predominantly by homeostatic proliferative mechanisms.

Abstract 1148. Table
DonorCD34+ CD3+Fever >38.2C Alb <2.8mg/dL Days to ANC>500IFNγTNFαIL4IL6FLT3LIL2IL7IL10IL15sIL2RαIL8
       TH1 TH2 Growth Factors Other* 
  Cell dose ( X108/kg) by Day +7  pg/dL 
MUD 3.0 3.4 Yes Yes 13 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 
MRD 2.4 2.2 No Yes 14 27 1263 59 23 375 32 
MUD 4.7 0.8 No No 15 532 29 225 20 
MUD 6.1 1.7 Yes Yes 12 22 34 1129 70 26 1230 28 
MRD 5.9 1.2 No No 17 14 15 115 991 863 35 1533 73 
MRD 4.4 1.5 Yes No 15 20 210 624 46 137 3275 154 
MRD 5.2 1.4 Yes Yes 11 233 13 481 21 
MRD 8.1 2.3 Yes Yes 11 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 
MRD 3.5 2.6 Yes Yes 17 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 
10 MUD 4.7 0.9 Yes Yes 15 519 23 174 152 328 106 41 60 93 1443 142 
11 MRD 5.0 1.9 No No 16 22 68 893 27 29 645 24 
12 MUD 2.0 4.3 Yes Yes 13 30 90 59 506 18 33 17 51 125 39 
13 MUD 5.0 2.0 No Yes 18 42 34 721 293 902 111 1121 236 
14 MUD 4.2 1.6 Yes Yes 11 106 106 48 552 62 3802 80 7735 707 
15 MUD 6.0 1.0 Yes Yes 10 25 39 95 1412 17 489 58 2653 99 
16 MRD 4.7 1.7 No No 11 11 15 1036 15 63 172 316 
DonorCD34+ CD3+Fever >38.2C Alb <2.8mg/dL Days to ANC>500IFNγTNFαIL4IL6FLT3LIL2IL7IL10IL15sIL2RαIL8
       TH1 TH2 Growth Factors Other* 
  Cell dose ( X108/kg) by Day +7  pg/dL 
MUD 3.0 3.4 Yes Yes 13 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 
MRD 2.4 2.2 No Yes 14 27 1263 59 23 375 32 
MUD 4.7 0.8 No No 15 532 29 225 20 
MUD 6.1 1.7 Yes Yes 12 22 34 1129 70 26 1230 28 
MRD 5.9 1.2 No No 17 14 15 115 991 863 35 1533 73 
MRD 4.4 1.5 Yes No 15 20 210 624 46 137 3275 154 
MRD 5.2 1.4 Yes Yes 11 233 13 481 21 
MRD 8.1 2.3 Yes Yes 11 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 
MRD 3.5 2.6 Yes Yes 17 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 
10 MUD 4.7 0.9 Yes Yes 15 519 23 174 152 328 106 41 60 93 1443 142 
11 MRD 5.0 1.9 No No 16 22 68 893 27 29 645 24 
12 MUD 2.0 4.3 Yes Yes 13 30 90 59 506 18 33 17 51 125 39 
13 MUD 5.0 2.0 No Yes 18 42 34 721 293 902 111 1121 236 
14 MUD 4.2 1.6 Yes Yes 11 106 106 48 552 62 3802 80 7735 707 
15 MUD 6.0 1.0 Yes Yes 10 25 39 95 1412 17 489 58 2653 99 
16 MRD 4.7 1.7 No No 11 11 15 1036 15 63 172 316 

MUD: matched unrelated donor, MRD: matched related donor, Alb: Albumin, ANC: absolute neutrophil count, N/A: not available

*other activation/inflammatory cytokines

Disclosures

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.

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