Lineage Specific Chimerism Analysis Reveals That Mixed Donor T-Cell Chimerism Is Common in the Early Post-Transplant Period Following Myeloablative Allotransplants from HLA-Matched but Not HLA-Haploidentical Donors: A Multivariable Analysis of Allografted Patients from a Single Center

Failure to achieve  complete donor T-cell chimerism in the early post-transplant period  may impair the graft-versus malignancy effect and contribute to a higher risk of disease relapse.   Lineage specific chimerism  analysis (LSCA) of T-lymphocytes versus myeloid cells  is routinely performed following reduced-intensity or non-myeloablative  hematopoietic cell allografts. However, many centers do not perform LSCA following myeloablative conditioning.  We performed  LSCA in all consecutive  patients receiving a first T-replete allotransplant using myeloablative conditioning as defined by the CIBMTR (Giralt 2009 BBMT 15;367) between 2/2006 and 2/2015 (n=232; patient characteristics: median age 49 (18-73); Female 50%, Black 19%, Asian 3% white 78%; donor MRD 41%, MUD 39%, Haploidentical 20%; graft PBSC 88%, BM 11.5%, both 0.5%; diagnosis AML  52%, ALL 18%, MDS 11%, CML 11%, NHL 4% MPS1%, CLL1%, HL 1%; DRI low 10%, intermediate 51%, high 29%, very high 10%. Our objectives were to determine the rate of full-donor and mixed T-cell chimerism  in the early post-transplant period (d 30 and d 90) , to determine the association of T-cell chimerism with patient, disease and regimen specific factors, and  to determine whether early  mixed chimerism impacts post-transplant outcomes following myeloablative conditioning. LSCA was performed on peripheral blood using a  RoboSep instrument for automated sorting of CD3-positive T-cells and CD33 positive myeloid cells  to 96.5-100% and  98.3-100% purity respectively, and short tandem repeat analysis by PCR.   Full donor chimerism was defined as > 90% donor derived cells. Probability of achieving full donor T-cell chimerism in evaluable patients on d 30 and d 90 post transplant were 55% and 71%. In contrast the probabilities of achieving full-donor myeloid chimerism were 99.5% and 93.5% respectively. On univariate analysis the following factors were significantly associated with achievement of full-donor T-cell chimerism on d 30: donor type (haploidentical 100%, MRD 39%, MUD 47%, p<0.001), diagnosis (AML 58%, ALL 60%, MDS/MPS/CML 35%, NHL/HL/CLL 90%, p=0.003), conditioning regimen (busulfan based 37%, TBI based 56%, post-transplant Cy 100%, p<0.001), and on d 90: donor type (haploidentical 100%, MRD 56%, MUD 70%, p<0.001), diagnosis (AML 76%, ALL 77%, MDS/MPS/CML 49%%, NHL/HL/CLL 100%, p=0.002), conditioning regimen (busulfan based 56%, TBI based 77%, post-transplant Cy 100%, p<0.001). For multivariate analysis  (Table 1) the exact logistic regression method was used to accommodate the nature of the data.  The odds ratios (OR) were estimated by exponentiating median unbiased estimates of regression coefficients. Donor type (haploidentical vs other) and diagnosis (MDS/MPS vs AML) were significant factors. Our institutional approach to patients failing to achieve full-donor chimerism by d 90 is to withdraw immnosuppressive therapy in the absence of  active GVHD if T-cell chimerism is  > 50% or to administer DLI  (starting at 1 x 10e6 CD3+ cells/kg) for patients with CD3 chimerism < 50% . Using this approach long term outcomes for patients  who failed to achieve full-donor CD3 chimerism by d 30  were not significantly different from those achieving this threshold (2 yr estimated  survival -81% vs 67%; disease-free survival  62%% vs 57%; 6month CI  of grade 2-4  acute GVHD  33% vs 41%; 2 yr CI of non-relapse mortality 9% vs 15%, relapse 29% vs 28%; p=NS for all). These data demonstrate that failure to induce early full-donor T-cell chimerism is relatively common following myeloablative allotransplantation using  HLA-matched  but not haploidentical donors. Although spontaneous improvement can occur, routine LSCA followed by immunologic manipulation of patients with suboptimal chimerism may assist in preventing  adverse long term outcomes.

Table 1a - D 30 - OR = odds ratio of achieving full-donor CD3 chimerism

Factor	Effect	OR	95% CI	P value
Diagnosis	ALL vs AML	0.98	0.36 – 2.65	1.000
	MDS/MPS/CML vs AML	0.37	0.13 – 0.94	0.035
	NHL/HD/CLL vs AML	5.22	0.48 – 270	0.255
DonorType	MRD vs MUD	0.63	0.29 – 1.35	0.266
	Haploidentical vs MUD	57.3	12.0 – ∞	<0.001

Table 1b D 90 OR = odds ratio of achieving full-donor CD3 chimerism

Factor	Effect	OR	95% CI	P value
Diagnosis	ALL vs AML	0.70	0.20 – 2.49	0.719
	MDS/MPS/CML vs AML	0.15	0.04 – 0.48	<0.001
	NHL/HD/CLL vs AML	3.87	0.68 – ∞	0.216
Donor Type	MRD vs MUD	0.35	0.12 – 0.98	0.045
	Haplo vs MUD	19.0	3.64 – ∞	<0.001