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American Society for Blood and Marrow Transplantation
Peripheral Blood Eosinophilia Has a Favorable Prognostic Impact on Transplant Outcomes after Allogeneic Peripheral Blood Stem Cell Transplantation
Dong Hwan Kim,1,3 Gizelle Popradi,1 Wei Xu,2 Vikas Gupta,1 John Kuruvilla,1 Janice Wright,1
Hans A. Messner,1 Jeffrey H. Lipton1
Peripheral eosinophilia after allogeneic stem cell transplant (ASCT) may reflect the activation of the Th2 cytokine pathway. A retrospective analysis was performed to evaluate the impact of early- (before day 100: EEo) or late-onset (beyond day 100: LEo) eosinophilia ($0.5 x 109/L in peripheral blood) on transplant outcomes after peripheral blood SCT (PBSCT) in 237 patients. The incidence of EEo and LEo was 43% at day 100 and 62% at 2 years, respectively. Compared with patients without LEo, improved transplant outcomes were observed in patients with LEo: better overall survival (OS; 86% versus 41%,P = 5 x 102'1), lower non-relapse mortality (NRM; 10% versus 37%, P = 3 x 10~6), lower relapse incidence (11% versus 31%, P = 3 x 10~5), and higher GVHD-specific survival (GSS; 90% versus 64%, P = 1 x 10~6) were observed. In addition, similar finding was observed when transplant outcomes were analyzed according to the occurrence of eosinophilia at the onset of cGVHD. The multivariate analyses confirmed a favorable implication of LEo on OS, NRM, and GSS. LEo was associated with: (1) less severe chronic GVHD (cGVHD), (2) higher prevalence of autoantibodies, and (3) rapid lymphocyte count recovery after ASCT. In summary, the development of eo-sinophila after allogeneic PBSCT seemed to be a prognostic marker for improving transplant outcome. Biol Blood Marrow Transplant 15: 471-482 (2009) © 2009 American Society for Blood and Marrow Transplantation
KEY WORDS: Peripheral eosinophilia, GVHD, Allogeneic peripheral blood stem cell transplantation, Th2 cytokine pathway, Humoral immunity
INTRODUCTION
The implication of eosinophilia after allogeneic transplantation is presently to be fully elucidated. Previous reports suggested that blood eosinophilia precedes or coincides with the occurrence of acute or chronic graft-versus-host disease (aGVHD, cGVHD)
From the 'Allogeneic Blood and Marrow Transplant Program, Department of Medical Oncology and Hematology, Princess Margaret Hospital, University Health Network, University of Toronto, Toronto, Canada; Department of Biostatistics, Princess Margaret Hospital, University Health Network, University of Toronto, Toronto, Canada; and ^Department of Hematology and Medical Oncology, Samsung Medical Center, Sung-kyunkwan University School of Medicine, Seoul, Korea. Financial disclosure: See Acknowledgments on page 481. Correspondence and reprint requests: Dong Hwan Kim, MD, PhD, Department of Hematology/Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine Irwon-Dong, 50, Kangnam-gu, Seoul, 700-721 Republic of Korea (e-mail: drkim@medimail.co.kr). Received September 16, 2008; accepted January 6, 2009 ©2009 American Society for Blood and Marrow Transplantation 1083-8791/09/154-0001$36.00/0 doi:10.1016/j.bbmt.2009.01.003
[1,2]. In the first report describing the disease entity of cGVHD after allogeneic marrow transplant, elevated eosinophil counts had been reported, especially in patients with cGVHD [3]. Przepiorka et al. [4] also observed eosinophilia in as many as 34% of patients with cGVHD after allogeneic peripheral blood stem cell transplantation (PBSCT), and noted that eosinophilia occurred more frequently in patients with low-risk cGVHD compared with those with high-risk cGVHD (47% versus 12%, respectively). In addition, Daneshpouy et al. [5] suggested the inflammation-mediating role of eosinophils in the pathogenesis of GVHD based on the findings that activated eosino-phils were observed in gastrointestinal tract tissue of patients with GVHD, and that eosinophil density correlated with GVHD severity.
Generally, peripheral blood eosinophilia has been associated with underlying disease. Eosinophil efflux from the bone marrow and in situ tissue accumulation in response to local chemokine and cytokine release are complex processes. In peripheral tissues such as lung, skin, or gut, activated eosinophils may induce damage through degranulation and release of stored
inflammatory mediators including cytokines such as interkeukin (IL)-3, IL-4, IL-5, IL-6, and granulocyte macrophage-colony stimulating factor (GM-CSF).
The pathogenesis of cGVHD remains to be fully understood. Different from aGVHD, which is mediated by alloreactive cytotoxic T cell-lymphocyte activation and subsequent target organ inflammation through the concerted actions of type 1 cytokines (ie, tumor necrosis factor alpha [TNF-a] or interferon-g), the pathogenesis of cGVHD is associated with activation of Th2 cytokines such as IL-10, IL-4, IL-5, or IL-6 [6,7], which are the eosinophilopoietic factors [8,9]. Thus, eosinophilia after allogeneic stem cell transplantation (ASCT) could be a surrogate marker of activation of the Th2 pathway, which is located in the center of the pathogenesis of cGVHD.
Previous investigations reported that the development of peripheral blood eosinophilia within 100 days (early-onset eosinophilia [EEo]) was a sign of favorable transplant outcomes after (ASCT) [10,11]. However, the occurrence of eosinophila is not limited to the first 100 days after PBSCT. It has also been observed beyond day 100, and was potentially in association with active cGVHD. Accordingly, the current study investigated the clinical impact of either EEo (before day 100) or late-onset (beyond day 100) eosinophilia (LEo) on PBSCT outcomes in terms of overall survival (OS), non-relapse mortality (NRM), recurrence of primary disease, and GVHD-specific survival (GSS) in 237 allogeneic PBSCT recipients. Also, to evaluate the significance of eosinophilia during the course of cGVHD, the prognostic role of eosinophilia at the onset time of limited or extensive cGVHD has been also examined.
METHODS AND MATERIALS
Transplant Procedures
A total of 237 consecutive patients who received allogeneic PBSCTs from related donors between June 2001 and December 2006, at the Princess Margaret Hospital, Toronto, Canada, were included in this retrospective study. Institutional research board review and approval of the retrospective data collection and analysis was obtained. Patient characteristics and transplant details are summarized in Table 1. All patients received granulocyte-colony stimulating factor (G-CSF) mobilized PBSC grafts collected as previously described [12]. The transplantation procedures and posttransplantation management adhered to previously described institutional policies [12-17]. Posttransplant G-CSF support was not routinely administered.
GVHD Prophylaxis and Treatment
The majority of GVHD prophylactic regimens included cyclosporine (CsA; n = 233). It was used either
alone (n = 6) or in combination with methotrexate (MTX; n = 152), mycophenolate mofetil (MMF; n = 71), or antithymocyte globulin (ATG; n = 4). Four patients received FK506 plus MTX. CsA taper was initiated at 3 months posttransplantation in patients receiving from HLA-matched sibling or related donor, and at 6 months in recipients of HLA-mismatched grafts unless ongoing therapy of GVHD necessitated continuation of systemic immunosuppressive treatment.
First-line treatment for aGVHD treatment was oral prednisone, 1 mg/kg/day (n = 151) or i.v. methylpred-nisolone, 2 mg/kg/day (n = 15). Steroid refractory aGVHD was defined by any of the following modified criteria [18,19]: (1) progression of GVHD within 3 days of instituting treatment, (2) no change in GVHD grade within 10 days, (3) incomplete resolution of GVHD within 4 weeks, or (4) recurrence of GVHD when the steroid dose reached <0.5 mg/kg/day. Steroid refractory aGVHD was treated with second-line therapies including high-dose i.v. methylprednisolone (2 mg/ kg/day), a switch to FK506 from CsA, or institution of ATG, pentostatin, rapamycin, and MMF according to physician discretion.
Therapy of extensive cGVHD was decided after considering an individual patient's clinical status and current immunosuppression. Steroid therapy was used as the first-line regimen in patients not on immu-nosuppression, whereas either steroids, CsA, or both agents in combination were used in patients on immu-nosuppression. When additional treatment was needed to control cGVHD activity, a salvage regimen was introduced according to each patient's clinical manifestations or toxicity profiles. Salvage regimens included azathioprine, hydrochloroquine, MMF, FK506, or combination therapy.
Definitions
The day of the stem cell infusion was defined as day 0. Engraftment was defined as consecutive achievement of sustaining absolute neutrophil counts (ANC) above 0.5 x 109/L for 2 days and peripheral platelet counts exceeded 20 x 109/L for at least 3 consecutive days without transfusion.
Eosinophilia was defined as an eosinophil count of $0.5 x 109/L. EEo was defined as the peripheral eosinophilia occurring before day 100 after PBSCT, whereas late eosinophila (LEo) was defined as the eosinophilia occurring beyond day 100 after PBSCT.
Disease risk was defined as described previously [20]. aGVHD and cGVHD were diagnosed and graded using established criteria [3,21]. The severity of cGVHD was graded at the time of first manifestation of cGVHD using the Hopkins' risk category [22,23]. The Hopkins' risk category consists of 3 risk factors including progressive type onset, extensive skin involvement (>50% of body surface area), and
Table 1. Properties of Patients and Transplant Procedures According to the Development of Late-Onset or Early-Onset Eosinophilia after Allogeneic PBSCT
Overall Patients Early Eosinophilia No Early Eosinophilia Late eosinophilia No Late Eosinophilia
(n 5 237, 100%) (n 5 76, 32%) (n 5 161,68%) P-Value (n 5 97, 50%) (n 5 98, 50%) P-Valu
Recipients
Sex (F/M) 100/137 (42/58) 28/48 (37:63) 72/89 (45:55) NS 41/56 (42:58) 38/60 (39:61) NS
Age (years, median) 51.0 (18-71) 51.5 (18-69) 50.5 (18-71) NS 45.5 (18-71) 48.5 (19-67) NS
Diseases
AML/ALL 94/25 (40/10) 27/7 (36/9) 67/18 (42/11) NS 38/10 (39/10) 38/11 (39/11) NS
MDS/MF 18/12 (8/5) 10/1 (13/1) 8/11 (5/7) 8/3 (8/3) 9/6 (9/6)
CML/CLL 26/19 (11/8) 5/9 (7/12) 21/10(13/6) 14/6 (14/6) 10/10 (10/10)
NHL/HD 37/3 (16/1) 13/3 (17/4) 24/0(15/0) 15/2 (16/2) 12/1 (12/1)
MM/solid 1/2(0/1) 0/1 (0/1) 1/1 (1/1) 1/0 (1/0) 0/1 (0/1)
Disease risk
Low/intermediate/high 23/191/23 6/63/7 17/128/16 NS 13/76/8 9/80/9 NS
(10/80/10) (8/83/9) (11/80/10) (13/78/8) (9/82/9)
Conditioning regimen
Myeloablative 162 (68) 43 (57) 119 (74) .002 71 (73) 68 (69) NS
TBI-/non-TBI based 120/42 (50/18) 26/17 (34/22) 94/25 (58/16) 56/15 (58/15) 49/19 (50/19)
Reduced intensity 75 (32) 33 (43) 42 (26) 26 (27) 30 (31)
GVHD prophylaxis
CsA + MTX/CsA + MMF 152/71 (64/30) 42/30 (55/40) 110/41 (68/26) NS 67/25 (69/26) 64/27 (65/28) NS
Others* 14(6) 4(5) 10(6) 5 (5) 5 (5)
Cell dose
CD34+ cells (x 106/kg) 6.0(2.2-18.7) 6.3 (2.7-16.6) 5.9 (2.2-18.7) NS 5.8(2.3-18.7) 6.6 (2.3-16.9) NS
Donors
Sibling/family 224/13 (95/5) 72/4 (95/5) 152/9 (94/6) NS 89/8 (92/8) 95/3 (97/3) NS
HLA-disparity
Matched/1-Ag mm 214/23 (90)10 71/5 (93/7) 143/18 (89/11) NS 88/9 (92/8) 89/9 (91/9) NS
Engraftment
Neutrophil/platelet 232/228 (98/96) 76/76 (100/100) 156/152 (99/97) NS 97/97(100/100) 98/94 (100/98) NS
Acute GVHD
Overall/Gr 2-4/Gr 3,4* 196/168/67 64/51/19 132/117/48 NS 87/73/20 80/74/35 NS
(83/71/28) (84/67/25) (84/75/31) (90/75/21) (82/76/36)
Steroid refractory 78/161 (48) 23/50 (46) 55/111 (50) NS 29 (42) 36 (51) NS
No/limited/extensive 31/29/145 6/11/90 25/18/55 <.001 10/11/52 34/20/88 NS
(15/14/71) (6/11/83) (26/18/56) (14/15/71) (24/14/62)
PBSCT indicates peripheral blood stem cell transplantation; F, female; M, male; S.E., standard error; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; MF, myelofibrosis; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; NHL, non-Hodgkin' lymphoma; HD, Hodgkin' disease; MM, multiple myeloma; CMV, cytomegalovirus; PBSC, peripheral blood stem cells; TBI, total body irradiation; CsA, cyclosporine A; MTX, methotrexate; MMF, mycophenolate mofetil; HLA, human leukocyte antigen; Ag, antigen; mm, mismatched. *P = .02 between patients with and without late-onset eosinophilia.
thrombocytopenia (<100 x 109/L), then stratifying the patients into 3 risk groups according to their survival.
The incidence of relapse was defined according to the time from transplantation until disease progression. NRM was defined as death not related to disease recurrence or progression. OS was defined as the time from transplantation until death from any cause. GSS was defined as the time from diagnosis of aGVHD or cGVHD until death from GVHD-specific causes including GVHD, infection associated with immuno-suppressive treatment, or both.
Clinical Data
Clinical data was collected until the end of July 2007. Information about the following events was recorded: peripheral cell engraftment (neutrophils, platelets, lymphocytes, and eosinophils), aGVHD and cGVHD, disease relapse, NRM, and deaths.
Absolute eosinophil and lymphocyte counts were recorded monthly during the first 3 months after transplantation, then every 3 months until the end of year 1, then every 6 months until year 3, and then at 3 years after PBSCT.
Statistics
Patients' characteristics, transplantation procedures, and outcomes were compared according to the development of EEo or LEo by chi-square, Fisher's exact, or Mann-Whitney's U-tests. The cumulative incidence of posttransplant eosinophilia was estimated considering death as competing risk. The probabilities of OS and GSS were estimated by Kaplan-Meier methods, whereas the cumulative incidence of GVHD was estimated considering death as a competing risk. The cumulative incidence of NRM was estimated using relapse as a competing risk, and relapse incidence using NRM as a competing risk. Transplantation
outcomes were compared by the log-rank test or Gray test as appropriate.
The development of autoantibody and the severity of cGVHD were compared using the chi-square test according to the development of LEo. Serial changes in absolute lymphocyte counts were compared using the Student t-test.
The prognostic impact of clinical factors on OS, NRM, relapse incidence, and GSS was examined by uni- and multivariate analyses using the nontime-dependent Cox proportional hazard model. Multivar-iate survival analyses using the nontime-dependent Cox proportional hazard model was performed with forward conditional selection procedures. The univar-iate analyses included the following variables: grouping by EEo (no EEo versus EEo), LEo (no LEo versus LEo), conditioning regimen (myeloablative versus reduced intensity [RIC]), HLA matching (matched versus mismatched), donor type (sibling versus alternative), disease risk (low or intermediate versus high risk), age of recipients (#50 years versus >50 years), aGVHD (grade 0-ii versus grade iii-iv), steroid refractory aGVHD, cGVHD (absent versus present), and extensive cGVHD (absent versus present).
To illustrate the effect of eosinophilia on survival, "semilandmark plots'' were constructed for OS, NRM, and relapse [24,25]. In patients who developed peripheral blood eosinophilia, the posttransplant day of development of eosinophilia was defined as the landmark day; in patients who did not develop eosinophilia, posttransplant day 196, which was the median day of onset of eosinophilia, was defined as the landmark day. OS, NRM, and relapse was calculated from the landmark day to the corresponding event or date of last follow-up. In the same way, semilandmark analysis also compared the GSS rates between the cases with versus without eosinophila accounting the onset day of overall or extensive cGVHD as landmark. In patients who did not develop eosinophilia, posttransplant day 146 and day 271, which was the respective median onset day of overall or extensive cGVHD, was defined as the landmark day for analysis.
To demonstrate a strong association between the development of LEo and OS, we also performed multivariate analyses using the time-dependent Cox proportional hazard model. The occurrence of LEo, the onset of any grade cGVHD, or the onset of extensive cGVHD were treated as time-dependent covari-ates in the model, whereas other prognostic factors were treated as nontime categoric covariates. P < .05 was considered significant. The hazard ratio (HR) and 95% confidence interval (CI) were estimated with a predetermined reference risk of 1.0. SPSS software package (SPSS 13.0 Inc., Chicago, IL) and SAS version 9.0 (SAS Institute, Cary, NC) were used for the statistical analyses.
RESULTS
The Serial Change of Peripheral Eosinophil Counts after Allogeneic PBSCT
A biphasic pattern of eosinophilia was noted after PBSCT. The first peak occurred prior to day 100 then the second one, beyond day 100. The incidence of EEo at day 100 was 33.4% 6 3.1% (Figure 1A), whereas that of LEo at 2 years after PBSCT was 61.7% 6 4.4% (Figure 1B). The median time for the eosinophils counts over 0.5 x 109/L beyond day 100 after PBSCT was 196 days (95% CI, 277-453 days). In most cases with LEo, a trend of rising eosinophil counts were observed for 2 weeks to 4 months prior to reach the level of 0.5 x 109/L of eosinophils.
GVHD after Allogeneic PBSCT
With a median follow-up duration of 31 months among survivors (range: 2-71 months), the cumulative incidence of aGVHD grades ii to iv and grades iii to iv was 76.7% 6 3.0% and 47.6% 6 5.0%, respectively. Of 166 patients who received steroid therapy for grades ii to iv aGVHD, 161 patients were evaluated for their responses. Good response to steroids was noted in 83 patients (51.6%) and the remainders (48.4%) were subsequently diagnosed with steroid refractory GVHD.
The cumulative incidence of overall and extensive cGVHD at 2 years was 91.6% 6 2.7% and 78.3% 6 3.4%, respectively. The median time to onset of overall and extensive cGVHD was 146 days (95% CI, 132160 days) and 271 days (95% CI, 223-319 days), respectively. The development of limited cGVHD was followed by the occurrence of LEo within a median interval of 80 days, then by extensive cGVHD within a median of 35 days following LEo occurrence. The 2-year GSS rate was estimated as 77.3% 6 3.1% after the diagnosis of either aGVHD or cGVHD.
Early-Onset Eosinophilia and Its Contribution to Transplantation Outcomes
The development of EEo was not associated with the occurrence of grades iii-iv aGVHD (P = .1), but there was a trend toward having more patients developing EEo in the group with grade ii-iv aGVHD compared with those with grade 0 to i GVHD (P = .053). When analyzed according to the organ-specific manifestations of aGVHD, EEo did not affect the developments of cutaneous (P = .07), gastrointestinal (P = .15), or hepatic GVHD (P = .13). Steroid refractory aGVHD was not associated with the development of EEo (P = .7).
In univariate analyses, the group with EEo showed a better OS (72.0% 6 5.9% versus 52.5 6 4.2% at 3 years, P = .002; Figure 2A), a lower NRM (20.8% 6 5.5% versus 32.0% 6 4.0% at 3 years, P = .05;
0 20 40 60 80 100 0 1 2 3 4 5 6
Days after transplantation Years after transplantation
Figure 1. The probability of development of early-onset eosinophlia ($0.5 x I09/L) after allogeneic PBSC transplantation before day 100 (A) or the probability of development of late-onset eosinophlia occuring beyond day 100 (B).
Figure 2B), and a lower relapse (15.5% 6 4.7% versus 29.7% 6 4.4% at 3 years, P = .05; Figure 2C) compared with the group without EEo, although no difference was observed in GSS (83.1% 6 4.7% versus 73.0% 6 4.1%, P = .2; Figure 2D).
Late-Onset Eosinophilia Is Associated with Improved Transplantation Outcomes after Allogeneic PBSCT
Patients who had experienced previous episodes of EEo showed a higher episode of LEo than those with-outprior experienceofEEo (73% versus 38%; P = 4 x 10"6 by chi-square test, P = 7 x 10"5 by McNemar
test).
With a median duration of follow-up of 773 days and 679 days in the groups with and without LEo, significant differences were observed between the 2 groups in terms of 3 year OS (86.3% 6 3.9% versus 46.2% 6 5.6%, P = 5 x 10"11; Figure 2E), NRM at 3 years (9.5% 6 3.3% versus 36.5% 6 5.6%, P = 3 x 10"6; Figure 2F), 3 years relapse incidence (10.8% 6 3.7% versus 30.5% 6 5.6%, P = 3 x 10"5; Figure 2G) and 3 years GSS (90.4% 6 3.3% versus 64.5% 6 6.0%, P = 1 x 10 5; Figure 2H).
With respect to cGVHD, no association of LEo was observed with the occurrence of overall (P = .3) or extensive cGVHD (P = .5). When analyzed according to the organ-specific manifestations of cGVHD, LEo was significantly associated with more frequent involvement of cutaneous (P = .001), oral (P = .04),
Figure 2. The plots of the overall survival, nonrelapse mortality, relapse incidence, and GVHD-specific survival of patients with and without the development of early-onset eosinophilia before day 100 (A-D) and of late-onset eosinophilia beyond day 100 (E-H).
ocular (P = .02), and pulmonary GVHD (P = .03), but not of gastrointestinal (P = .2), hepatic (P = .7), or musculoskeletal involvement (P = .6).
Peripheral Blood Eosinophilia at the Time of Onset of Overall or Extensive cGVHD after Allogeneic PBSCT, and Its Prognostic Role
The peripheral blood eosinophilia at the onset of first manifestation of cGVHD was documented in 47 of 171 patients having any grade of cGVHD (28%), whereas it was noted in 66 of 137 patients having extensive cGVHD (48%) at the onset time of extensive cGVHD.
Significant differences of OS or NRM were observed in favor of the patients presenting eosino-philia when diagnosed initially with either any grade of cGVHD (limited and extensive) or extensive cGVHD. The group with eosinophilia at the onset time of cGVHD showed a better OS (89.7% 6 5.0% versus 66.6% 6 4.5% at 2 years, = .003; Figure 3A), a lower NRM (6.8% 6 3.8% versus 24.5% 6 4.2% at 2 years, P = .02; Figure 3B), but similar incidence of relapse (9.2% 6 5.2% versus 11.4% 6 3.3% at 2 years, P = .4; Figure 3C) compared with the group without eosinophilia.
These differences were more profound when analyzed according to the occurrence of eosinophilia at the onset of extensive cGVHD. The group with eosinophilia
at the onset time of extensive cGVHD showed a better OS (94.8% 6 3.0% versus 49.7% 6 6.5% at2 years, P = 4 x 10"8; Figure 3D), a lower NRM (3.3% 6 2.3% versus 41.5% 6 6.6% at 2 years, P = 6 x 10"7; Figure 3E), but similar incidence of relapse (5.8% 6 5.2% versus 12.2% 6 4.8% at 2 years, P = .2; Figure 3C) compared with the group without eosinophilia.
Late-Onset Eosinophilia Was Associated with Less Severe Grade cGVHD at Diagnosis, Faster Lymphocyte Count Recovery after Transplantation, and the Development of Autoantibodies during the Course of cGVHD
To explain why the patients developing late-onset eosinophilia together with cGVHD, especially with extensive cGVHD, had better transplant outcome compared with those without eosinophilia, further analyses were performed.
Forty-seven of 170 patients (28%) had eosinophilia at the time of onset of cGVHD, whereas 66 of 137 (48%) had eosinophilia at the time of onset of extensive cGVHD. The group developing LEo had less severe cGVHD compared with those without LEo according to the Hopkins' grading system (P = .0004; Table 2). In addition, when we compared the severity of cGVHD according to the presence of eosinophilia, especially at the time point of the onset of cGVHD,
Figure 3. The plots of the overall survival, nonrelapse mortality, and relapse in patients with versus without the development of eosinophilia at the time of onset of overall chronic GVHD (A-C) and of extensive chronic GVHD (D-F).
the patients both presenting eosinophilia and cGVHD had milder degree of cGVHD compared with those presenting cGVHD without evidence of eosinophilia (P = .004; Table 3).
Lymphocyte reconstitution after PBSCT was compared between the patients with and without, LEo. As shown in Figure 4, the patients with LEo showed a faster recovery of absolute lymphocyte counts than those without LEo, which were significantly different up to 9 months posttransplantation. Thereafter, the lymphocyte counts became similar between the 2 groups.
We collected data on the development ofautoanti-bodies during the course of cGVHD. Sixty-three patients were evaluated for the presence or titer of antinuclear antibody (ANA) measured during the course of cGVHD. A positive ANA was noted in 38 patients (60%), and a titer of ANA over 1:160 was observed in 21 patients (33%). Those who developed a positive ANA had better OS and GSS compared with those with a negative ANA (P = .009 for OS and P = .1 for GSS). Interestingly, a significant correlation was noted between the development of LEo and a positive ANA. A positive ANA was noted in 35 of 51 patients with LEo (69%), whereas it was only seen in 3 of 9 patients without LEo (33%, P = .04). Moreover,
Table 2. The Association of the Late-Onset Eosinophilia with the Severity of cGVHD and Organ Involvement
Late eosinophilia No late eosinophilia
(n 5 97, 50%) (n 5 98, 50%) P-Value
Seattle's classification of cGVHD
No cGVHD 6(6) 25 (26) <.001
Limited cGVHD 11 (11) 18(18)
Extensive cGVHD 80 (83) 55 (56)
Hopkins' risk category
Components of risk category
Progressive type onset 10/92 (11) 25/72 (35) .0002
Thrombocytopenia 18/92 (20) 32/73(44) .001
Extensive skin involvement 16/92(17) 21/72(29) .07
Severity of cGVHD by risk category
Mild grade 59/92 (64) 25/72 (35) .0004
Moderate grade 29/92 (32) 36/72 (50)
Severe grade 4/92 (4) 11/72 (15)
Organ involvements (n 5 91, 55%) (n 5 73, 44%)
Skin 73 (80) 40 (55) .0004
Oral 55 (60) 27 (37) .003
Ocular 26 (29) 6 (8) .001
Pulmonary 27 (30) 7(10) .002
Gastrointestinal 31 (34) 34(47) .1
Hepatic 71 (78) 54 (74) .5
Musculoskeletal 22 (24) 9(12) .05
Deaths (n 5 97, 50%) (n 5 98, 50%)
Death 12(12) 49 (50) <.001
Because of progression 4 (4) 20 (20) .6
Because of nonrelapse 8 (8) 29 (30)
mortality
GVHD 3(3) 10(10)
GVHD + infection 3 (3) 8 (8)
Infection 1 (1) 10(10)
Others 1 (1) 1 (1)
GVHD indicates graft-versus-host disease; cGVHD, chronic GVHD.
an ANA titer over 1:160 was observed in 21 of 51 patients with LEo (42%), but 0 of 9 patients without LEo (0%, P = .02).
Multivariate Survival Analysis Using Nontime-Dependent and Time-Dependent Cox's Proportional Hazard Model
A uni- and multivariate analysis using a Cox's proportional hazard model was performed to evaluate the contribution of LEo to OS, NRM, relapse incidence, and GSS in the context of other potentially relevant clinical parameters (Table 4). The LEo, rather than EEo, was identified as a significant favorable prognostic factor for OS (P = 5 x 10"6, HR 0.19, 95% CI [0.09-0.39]), NRM (P = .00005, HR 0.17 [0.07-0.41]), relapse incidence (P = .003, HR 0.21 [0.08-0.59]), and GSS (P = .00006, HR 0.17 [0.07-0.41]).
The beneficial effect of LEo on OS was proved consistently (P = .0009, HR 0.32 [0.17-0.63]; Table 5) in the time-dependent Cox's proportional hazard model that was adopted to confirm the powerful independent effect of LEo on transplant outcomes. Other variables such as overall or extensive cGVHD, and aGVHD grades iii to iv, were also identified as independent prognostic factors for survival after PBSCT.
The Semilandmark Analysis for OS, NRM, and Relapse
The group with eosinophilia showed a better OS (86.7% 6 3.8% versus 61.4% 6 6.6% at 2 years, P = .00003; Figure 5A), a lower NRM (9.4% 6 3.2% versus 24.3% 6 5.8% at 2 years, P = .02; Figure 5B), but similar incidence of relapse (8.7% 6 3.2% versus 13.5% 6 5.3% at 2 years, P = .4; Figure 5C) compared with the group without eosinophilia in the semilandmark analysis
Table 3. The Association of the Development of Peripheral Blood Eosinophilia at the Time of First Manifestation of chroinc GVHD with the Severity of cGVHD
At the Onset of Overall cGVHD
Eosinophilia No Eosinophilia
(n 5 47, 28%) (n 5 123, 72%) P-Value
Seattle's classification of cGVHD
Limited cGVHD 12(37) 20 (25) .2
Extensive cGVHD 35 (63) 103 (75)
Hopkins' risk category
Components of risk category
Progressive type onset 3 (6) 33 (27) .004
Thrombocytopenia 6(13) 44 (36) .004
Extensive skin involvement 7(15) 32 (123) .1
Severity of cGVHD by risk category
Mild grade 34 (72) 54 (44) .004
Moderate grade 11 (23) 55 (45)
Severe grade 2(4) 14(11)
GVHD indicates graft-versus-host disease; cGVHD, chronic GVHD.
2.0 ■
X, 1.5 ■
3 o 1.0 ■
3 0.5 ■
* p<0.05 * p<0.001
~I-I-I-I-I-I-I-I-I-I-I
0 2 4 6 8 10 12 14 16 18 20 22 24 months after transplant
Figure 4. Serial change of absolute lymphocyte counts according to the development of late eosinophila after allogeneic transplantation.
accounting for the days from the onset of eosinophilia to the corresponding events. Also, the group with LEo showed better GSS rates compared with those without LEo when semilandmark analyses were applied accounting the onset day of overall (P = .00003; Figure 5D) or extensive cGVHD as landmark (P = .0001; Figure 5E).
DISCUSSION
The current study revealed the following findings: (1) LEo after allogeneic PBSCT is a favorable prognostic indicator for OS and GSS; (2) the occurrence of eosinophilia together with cGVHD, especially with extensive cGVHD, seemed to be a favorable prognostic factor for OS and NRM; and (3) the favorable effect of LEo on transplant outcomes might be from the association of LEo with a milder severity of cGVHD, a higher precvelance of autoantibodies, and faster lymphocyte count recovery after PBSCT.
Based on the finding that the development of eosinophilia together with cGVHD, especially with extensive cGVHD, can be a risk factor for OS and NRM (Figures 3 and 5), it is possible to stratify the patients with cGVHD into 2 subgroups having distinct prognoses: cGVHD with eosinophilia and cGVHD without eosinophilia. Herein, because eosinophilia can be a simple marker of Th2 cytokine activation, we propose that the biologic correlate of this stratification is differences in levels of activation of the Th1 and Th2 cytokine pathways: cGVHD with eosinophilia may represent a predominance of Th2 activation compared with Th1 activation, and the opposite may be true when cGVHD is not associated with eosinophilia. Further investigation is needed to validate the current hypothesis to stratify the patients with cGVHD according to the presence of eosinophila and to understand their biologic basis at the level of cytokine or gene expression profiles.
The beneficial effect of eosinophilia during the course of cGVHD or in long-term outcomes of
PBSCT is supported by 3 key findings: (1) the group that developed LEo presented with a milder severity of cGVHD as scored by the Hopkins' grading system. When the severity of cGVHD was compared among patients with and without LEo (Table 4), and according to the presence of eosinophilia at the time that cGVHD was diagnosed, we found that the group with LEo presented with a milder grade of cGVHD. We hypothesized that the group with the milder grade of cGVHD at diagnosis in association with LEo had a predominantly Th2 cytokine profile. (2) The group with LEo demonstrated a higher prevalence of ANA positivity compared with those without LEo. This finding may reflect deregulated immunity or activity of the humoral immune pathway. (3) Last, lymphocyte count recovery was faster in the group with LEo compared with those without LEo up to 9 to 12 months after PBSCT (Figure 4). Alloreactive T cells stimulated by the Th1 activation pathway attack the thymus and delay the immune reconstitution of T cell-lymphocytes [26,27]. Thus, lymphocyte recovery could be expected to be faster in patients with LEo reflecting a state of lower Th1 cytokine activation. A cy-tokine profile that is shifted from a Th1 to a Th2 state may be associated with thymic sparing from damage because of alloreactive T cells (Figure 6).
Several investigations have reported that autoanti-body such as ANA or rheumatoid factor appear frequently (20%-46%) after ASCT [28,29]. Patriarca et al. [29] reported that the development of autoanti-body after ASCT is related, not only to the development of cGVHD, but also to faster B cell immune recovery. These findings suggest a role for humoral immunity in the pathogenesis of cGVHD [30,31]. However, the prognostic significance of auto-antibody on transplantation outcomes has yet to be clarified.
Alloreactive donor T cells are believed to be key mediators in the pathogenesis of GVHD. Th1 pathway activation stimulates these lymphocytes, and plays a central role in the deleterious effects of aGVHD in humans. Conversely,Th2 pathway activation may have a protective role against the effects of GVHD [32-34]. There are evidences that activated tissue eosinophils are linked to the pathogenesis of GVHD [5,35]. The activation of eosinophils is mediated by cytokines involved in the Th2 pathway including IL-4, IL-5, and IL-6 [32-34]. A predominant Th2 peripheral blood cytokine profile may be associated with a reciprocal decline in the activity of Th1 cytokines. In addition, the B cell-mediated mechanism is also involved in the pathogenesis of cGVHD given that the rituximab is effective for the treatment of cGVHD [36]. Furthermore, previous studies in a murine GVHD model support the hypothesis that blockade of the Th1 activation pathway during the course of GVHD may result in the activation of the Th2
Table 4. Uni-and Multivariate Analyses to Evaluate the Contribution of Late Eosinophilia in the Context of Clinical Risk Factors on Overall Survival, Nonrelapse Mortality, Relapse Incidence, and GVHD-Specific Survival
Univariate Multivariate
Prognostic Factor
P-Value HR (95% CI) P-Value HR (95% CI))
Overall survival
Late eosinophilia .000000009 0.15 (0.08-0.29) 0.000005 0.I9 (0.09-0.39)
Early eosinophilia .003 0.45 (0.27-0.76) NS —
RIC .08 1.46 (0.95-2.25) NS —
Mismatched transplant .4 1.28 (0.68-2.40) NS —
Sibling transplant .2 0.48 (0.I5-I.51) NS —
High risk disease .008 2.17 (1.13-3.84) NS —
Old age (50 years or over) .004 1.87 (1.22-2.87) NS —
Steroid refractory aGVHD .002 2.16 (I.32-3.55) NS —
aGVHD, grade iii-iv .00005 2.40 (I.57-3.65) 0.0I 2.05 (I.I6-3.64)
Overall GVHD .000000000003 0.17(0.10-0.27) 0.00003 0.23 (0.II-0.46)
Extensive GVHD .001 0.47 (0.29-0.75) NS —
Nonrelapse mortality
Late eosinophilia .00003 0.18 (0.08-0.40) 0.00005 0.I7 (0.07-0.4I)
Early eosinophilia .05 0.54 (0.29-I.00) NS —
RIC .9 0.96 (0.54-I.70) NS —
Mismatched transplant .1 I.76 (0.86-3.58) NS —
Sibling transplant .8 0.83 (0.26-2.65) NS —
High risk disease .002 2.78 (I.44-5.36) NS —
Old age (50 years or over) .008 2.I0 (I.2I-3.63) NS —
Steroid refractory aGVHD .00007 2.00 (0.00-7.82) 0.00006 3.87 (I.73-8.62)
Acute GVHD, grade 3-4 .00000003 4.73 (2.73-8.I8) NS —
Overall GVHD .002 0.32 (0.I6-0.66) NS —
Extensive GVHD .6 0.82 (0.42-I.59) NS —
Relapse incidence
Late eosinophilia .0001 0.2I (0.09-0.47) 0.003 0.2I (0.08-0.59)
Early eosinophilia .05 0.5I (0.25-I.00) NS —
RIC .0003 2.93 (I.64-5.24) NS —
Mismatched transplant .5 0.66 (0.20-2.I2) NS —
Sibling transplant .3 0.3I (0.04-2.27) NS —
High risk disease .01 2.6I (I.22-5.6I) NS —
Old age (50 years or over) .2 I.47 (0.82-2.65) NS —
Steroid refractory aGVHD .9 0.94 (0.46-I.95) NS —
Acute GVHD, grade 3-4 .3 0.68 (0.33-I.42) NS —
Overall GVHD .0000000000003 0.08 (0.04-0.I6) 0.0000004 0.09 (0.04-0.23)
Extensive GVHD .0000004 0.I9 (0.09-0.36) NS —
GVHD specific survival
Late eosinophilia .00007 0.20 (0.09-0.44) 0.00006 0.I7 (0.07-0.4I)
Early eosinophilia .2 0.66 (0.34-I.27) NS —
RIC .8 I.09 (0.57-2.I2) NS —
Mismatched transplant .2 I.65 (0.74-3.7I) NS —
Sibling transplant .6 0.65 (0.I6-2.67) NS —
High risk disease .04 2.37 (I.06-5.32) NS —
Old age (50 years or over) .02 2.05 (I.II-3.77) NS —
Steroid refractory aGVHD .0001 3.87 (I.95-7.66) 0.0008 5.68 (2.43-I3.25)
Acute GVHD, grade 3-4 .000000001 7.84 (4.04-I5.2I) NS —
Overall GVHD .08 0.44 (0.I8-I.09) NS —
Extensive GVHD .7 I.I7 (0.53-2.57) NS —
RIC, reduced-intensity conditioning; GVHD, graft-versus-host disease; HR, hazard ratio; 95% CI, 95% confidence interval; NS, not significant.
pathways, and, consequently, alter the clinical presentation of GVHD [37-39].
Because patients were ascertained and categorized prior to establishment of the NIH consensus criteria for diagnosis and stating of cGVHD in the current study [40], our cohort may include some patients with late-onset aGVHD, a powerful adverse prognostic factor for GSS [41,42]. Therefore, this limitation needs to be addressed in future prospective trials.
Previous studies have suggested that the development of EEo might have a favorable implications on
transplantation outcomes [1,2]. However, based on our findings, stronger association of LEo with transplant outcomes was revealed in comparison with those of EEo. The reason why significant association was noted between EEo and transplant outcomes is from strong correlation of the development of LEo with prior episode of EEo.
The finding that LEo did not predict the risk of cGVHD implies that the eosinophils-activating mechanism is not the only pathway to develop cGVHD. The other pathway would include a Th1 activated, alloreactive cytotoxic T cell-mediated mechanism. The
Table 5. Multivariate Analysis for Overall Survival According to the Time-Dependent Cox's Proportional Hazard Model
Prognostic Factor Parameter Estimate Chi-Square P-Value HR (95% CI)
Overall survival
Late eosinophilia -1.13414 11.0223 .0009 0.32 (0.17-0.63)
Overall GVHD -2.39024 18.9166 <.0001 0.09 (0.03-0.27)
Extensive GVHD -2.05429 14.9408 .0001 7.80(2.75-22.11)
High risk disease -0.57434 2.7657 .1 1.78 (0.90-3.45)
Old age (50 years or over) -0.53244 4.7533 .03 1.69 (1.05-2.78)
Acute GVHD, grade 3-4 -0.85984 11.6401 .0006 2.38 (1.45-3.85)
GVHD indicates graft-versus-host disease; HR, hazard ratio; 95% CI, 95% confidence interval.
better prognosis in the patients presenting eosinophilia together with cGVHD suggest that GVHD predominantly induced by Th2 activation pathway, may have a less severe course of cGVHD compared with other subtype of Th1 predominating GVHD.
Herein, we suggest that (1) there seemed to be a similarity between cGVHD associated with the Th2 activation pathway and those associated with humoral immunity, (2) the LEo could be a surrogate for the cGVHD with the Th2 activation pathway, and (3) the prognosis of cGVHD with the Th2 activation pathway, presenting as eosinophilia, may be better than those with cGVHD with predominantly a Th1 activation pathway.
In conclusion, peripheral blood eosinophilia (especially LEo) has favorable prognostic implications on
outcomes after allogeneic PBSCT. The benefits of late-onset eosinophilia may be from a shift in the balance between the Th2 and Th1 cytokine activation pathways during the course of cGVHD. Further studies are warranted to elucidate the causative mecha-nism(s) behind the clinical benefits of LEo and to validate its prognostic impact on ASCT outcomes.
CONTRIBUTION OF THE AUTHORS
D.K. contributed to the design of the study, the supervision of data collection, and of data interpretation, data analysis, and writing the manuscript. J.L. contributed to the design of the study, the supervision of data collection, and of data interpretation, data analysis,
Figure 5. Semilandmark analysis for the overall survival, nonrelapse mortality, relapse, and GVHD-specific survival in patients with versus without the development of eosinophilia (A-E).
Figure 6. Proposed mechanism of pathogenesis of cGVHD presenting with peripheral blood eosinophilia. This subtype of cGVHD is induced by the activation of Th2 cytokine pathway, which is shifted from Th1 activation pathway.
Acute GVHD
Tissue Damage by Conditioning
Shift of cytokine profiles
Chronic GVHD
Host tissue
Th1 activation*
Thymic damage —► Immunity Severity of GVHD —► Better OS
r IL-4, IL-5, IL-6 —► Eosinophilia
Th2 activation h
CD8CTL
Oy Cellular & ^^ inflammatory CD8 CTL phase
Humoral immunity
Autoimmune Autoantibody
and critical revision of the manuscript. G.P. was involved in the data collection and data interpretation, data analysis, and critical revision of the manuscript. W.X. was involved in the design of the study and statistical analysis of the data. J.W., V.G., J.K., and H.M. were involved in the supervision of the data collection, interpretation of the data, and critical revision of the manuscript.
ACKNOWLEDGMENTS
Financia/ disclosure: The authors have nothing to disclose.
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