Complications after bone marrow transplantation are ... - Nature

Bone Marrow Transplantation (2000) 26, 419–426  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt

Complications after bone marrow transplantation are manifestations of systemic inflammatory response syndrome H Takatsuka1, Y Takemoto1, S Yamada1, H Wada1, S Tamura1, Y Fujimori1, T Okamoto1, A Suehiro1, A Kanamaru2 and E Kakishita1 1

Second Department of Internal Medicine, Hyogo College of Medicine, Hyogo; and 2Third Department of Internal Medicine, Kinki University School of Medicine, Osaka, Japan

Summary: Bone marrow transplantation has been established as a useful treatment for various hematological disorders and is now performed widely, but the mortality rate is still high due to various complications. A clear therapeutic policy for such complications has not yet been established because of their complex nature. We investigated whether the major complications occurring after bone marrow transplantation could be classified as aspects of the systemic inflammatory response syndrome. Subjects were 10 patients who developed severe complications after bone marrow transplantation (graft-versus-host disease, thrombotic microangiopathy, respiratory disorders, and cytomegalovirus interstitial pneumonitis) and 16 patients without complications. Their symptoms, serum cytokines, and factors related to vascular endothelial damage were compared before and after transplantation. Whereas all 10 patients who developed complications had fever in the aplastic phase after transplantation, 15 of the 16 patients without complications remained afebrile (P ⬍ 0.001, t-test). When compared with the patients who did not develop complications, the patients with complications also showed significantly higher cytokine levels during the recovery phase after transplantation (P ⬍ 0.0001, t-test). Thus, the patients with complications developed fever in the aplastic phase and showed an increase of cytokines during the recovery phase, which triggered the occurrence of vascular endothelial damage shown by factors such as the thrombomodulin and plasminogen activator inhibitor type 1. This sequence of events corresponds with that occurring during systemic inflammatory response syndrome, so many of the complications of bone marrow transplantation can be considered as manifestations of this syndrome. Bone Marrow Transplantation (2000) 26, 419–426. Keywords: allogeneic bone marrow transplantation; systemic inflammatory response syndrome; cytokines; endothelial damage

Correspondence: H Takatsuka, Second Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinimiya, Hyogo 663-8501, Japan Received 8 February 2000; accepted 6 May 2000

It is well known that multiple complications can occur after bone marrow transplantation (BMT) as a result of various factors.1–5 Control of these complications is complex and difficult, and it is not unusual for there to be an unfavorable outcome. We have previously demonstrated that vascular endothelial damage and hypercytokinemia are involved in central nervous system disorders,6 adult respiratory distress syndrome (ARDS)-like respiratory disorders,7 thrombotic microangiopathy (TMA),8 acute graft-versus-host disease (GVHD),9 and cytomegalovirus (CMV) infection10 among the complications occurring after BMT. In this study, we attempted to unify the various complications occurring after BMT by considering them as manifestations of systemic inflammatory response syndrome (SIRS). SIRS is a systemic inflammatory response caused by hypercytokinemia with excess production of proinflammatory cytokines.11 Organ impairment in SIRS is considered to occur in SIRS secondary to vascular endothelial damage.12

Materials and methods Patients The subjects were 26 consecutive patients who underwent allogeneic BMT at our institution from October 1996 to June 1998. Table 1 shows age, gender, underlying disease, disease stage, donor type, and human leukocyte antigen (HLA) status of the subjects, as well as conditioning regimen and GVHD prophylaxis. The basic conditioning regimen was busulfan (BU), cyclophosphamide (CY), and total body irradiation (TBI) (BU at a dose of 4 mg/kg/day ⫻ 2 days, CY at 30 mg/kg/day ⫻ 2 days, and TBI at 3 Gy ⫻ 4) or BU and CY (BU at 4 mg/kg/day ⫻ 4 days and CY at 60 mg/kg/day ⫻ 2 days). GVHD prophylaxis The basic treatment was cyclosporin A (CsA) plus methotrexate (MTX) plus methylprednisolone (mPSL)13 or CsA plus MTX. CsA was infused from day ⫺1 for 24 h, and was kept at a target blood level of 400–600 ng/ml until marrow engraftment, after which the dosage was varied depending on the symptoms of GVHD. If GVHD became worse, the doses of CsA and mPSL were increased or CsA was replaced with FK506.

Complication after BMT are SIRS H Takatsuka et al

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Table 1

Clinical characteristics of the patients with or without complications

Age, range (mean) Sex, M:F Underlying disease ALL AML CML MDS SAA ML Disease stage CR1 CR2 CR3 NR CP1 Donor type R UR HLA match Full 1-locus mismatch Conditioning regimen BU⫹CY⫹TBI BU⫹CY CY⫹TLI GVHD prophylaxis CsA⫹MTX⫹mPSL CsA⫹MTX

With complications (n = 10)

Without complication (n = 16)

P value

20–38 (28.3) 4:6

21–50 (31.1) 7:9

NS NS

3 3 2

4 3 7 1

NS

1 1

1

3 2 1 1 2

5 1 2 2 7

4 6

12 4

NS

8 2

16

NS

8 1 1

9 7

NS

8 2

5 11

⬍0.05

NS

ALL = acute lymphoblastic leukemia; AML = acute myelogenous leukemia; CML = chronic myelogenous leukemia; MDS = myelodysplastic syndrome; SAA = severe aplastic anemia; ML = mixed leukemia; LBL = lymphoblastic lymphoma; CR1/2 = complete remission 1st/2nd; NR = no remission; CP = chronic phase; AP = accelerated phase; R = related; UR = unrelated; BU = busulphan; CY = cyclophosphamide; TBI = total body irradiation; TLI = total lymphoid irradiation; CsA = cyclosporin A, MTX = methotrexate; mPSL = methylprednisolone.

CMV prophylaxis

Veno-occlusive disease (VOD) prophylaxis

We administered acyclovir (250 mg three times daily) from day ⫺7 to approximately day 30. All patients received 12.5 g of immunoglobulin containing a high titer of antiCMV antibodies biweekly until 3 months after transplantation. To detect CMV infection, both the bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) were checked for CMV-DNA on approximately day 35 after BMT by the polymerase chain reaction (PCR) method. The direct immunoperoxidase method was also used for CMV antigen screening, employing a peroxidase-labelled monoclonal antibody for matrix protein p65 of CMV (HRPC7).14 The HRP-C7 method was used for CMV antigen screening of BALF on day 35 and screening of peripheral blood once weekly until day 50, and then biweekly to monthly depending on the clinical manifestations until day 100. If either the peripheral blood or BALF was CMVpositive by at least one of these methods, ganciclovir was administered according to the schedule proposed by Singhal et al15 (5 mg/kg twice a day in week 1 and once a day in weeks 2 and 3). Then the dose was adjusted depending on the CMV-DNA level and the C7-HRP antigen in peripheral blood and the extent of myelosuppression in weeks 2 and 3.

We administered heparin (100 IU/kg/day) for 24 h.

Bone Marrow Transplantation

Diagnosis of acute GVHD The classification of acute GVHD was based on the Seattle diagnostic criteria, and the highest grade until day 100 after transplantation was determined. Diagnosis of CMV interstitial pneumonitis (CMV-IP) CMV-IP was diagnosed from the following five criteria: (1) presence of fever, nonproductive cough, tachypnea; (2) hypoxia; (3) radiological findings suggestive of interstitial pneumonitis; (4) detection of CMV-DNA or CMV antigen; and (5) no evidence for another cause of pneumonitis. Diagnosis of VOD The diagnosis of VOD was made according to the Seattle clinical criteria, which required at least two of the following three features: hyperbilirubinemia (total serum bilirubin ⬎2 mg/dl or 34.2 ␮ mol/l), hepatomegaly or right upper quadrant pain of hepatic origin, and sudden weight gain


(⬎2% of baseline body weight) because of fluid accumulation, along with no other explanation for these signs and symptoms.

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Assay of plasminogen activator inhibitor type 1 (PAI-1) and thrombomodulin (TM)17

Diagnosis of thrombotic microangiopathy (TMA) We made a diagnosis of TMA when the reticulocyte count increased and the platelet count decreased in addition to criteria corresponding to grade 2 or higher of the classification system of Zeigler et al16 (fragmented erythrocytes comprising at least 1.3% of 2000 counted peripheral blood cells and elevation of LDH). Assay of cytokines Levels of the following cytokines were investigated: TNF␣, IFN-␥, IL-1␤, IL-2, IL-6, IL-8, IL-10 and IL-12. These cytokines were determined on five occasions: (1) before conditioning; (2) day 0 of BMT (before bone marrow infusion); (3) during the aplastic phase after BMT (days 5–10); (4) during the rapid WBC recovery phase (days 11–20); and (5) at the time of WBC stabilization after discontinuation of granulocyte colony-stimulating factor (days 21–28). Blood samples were collected into tubes containing 3.8% citric acid at a ratio of 1:9. After separation by centrifugation at 1500 r.p.m. for 10 min at 4°C, plasma was stored at ⫺80°C. All parameters were measured in duplicate at each specified time using ELISA kits from Table 2

Endogen (Woburn, MA, USA) and the mean values were determined.

PAI-1 activity was determined using a commercially available chromogenic assay (Biopool, Ventura, CA, USA) (normal; 4–43 ng/ml). Soluble TM was measured by a commercially available sandwich enzyme immunoassay based on monoclonal antibodies (Fuji-rebio, Tokyo, Japan) (normal ⭐4.5 FU/ml). These parameters were determined three times: (1) during the aplastic phase; (2) during the rapid WBC recovery phase; and (3) at the time of WBC stabilization after discontinuation of granulocyte colonystimulating factor. This study design was prospective, and the data were analyzed blindly and independently. We made the diagnosis of complications without knowledge of the laboratory results and measured the various cytokines and other factors without knowledge of which patients had complications. Statistical analysis Statistical analysis was done using Student’s t-test or twoway layout analysis of variance as appropriate, and P ⬍ 0.05 was considered to indicate a significant difference.

Outcome

Case No.

Complications

Febrile days

CRP (max)

Days from BMT to ANC ⬎500

Days from BMT to platelets ⬎50 000

1 2 3 4 5 6 7 8 9 10 11 12 13 14

TMA, GVHD, CMV-IP GVHD TMA, GVHD GVHD ARDS TMA ARDS ARDS CMV-IP GVHD, CMV-IP — — — —

4 2 6 23 12 8 3 20 6 15 0 0 0 0

8.6 1.5 0.6 26.1 13.6 10.4 9.6 24.9 3.0 29.2 ⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3

14 15 14 15 14 17 18 12 15 12 13 17 17 13

25 22 23 31 25 ⬎100 — 33 22 26 22 31 24 17

15 16 17 18 19 20 21 22 23 24 25 26

— — — — — — — — — — — —

0 0 0 0 0 0 0 0 0 3 0 0

⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3 ⬍0.3 1.3 ⬍0.3 ⬍0.3

14 19 15 15 20 12 14 14 17 15 14 16

19 21 24 22 36 18 23 52 24 24 17 27

Outcome (source)

D D A D A A D D A D D A A A

(TTP/HUS) (GVHD) (infection) (MOF) (infection) (infection) (relapse)

D (relapse) A A A A A A A A A D (relapse) D (relapse)

TMA = thrombotic microangiopathy; GVHD = graft-versus-host disease; CMV-IP = interstitial pneumonitis due to cytomegalovirus; ARDS = adult respiratory distress syndrome; CRP = C-reactive protein; ANC = absolute neutrophil count; A = alive; D = dead; TTP/HUS = thrombotic thrombocytopenic purpura/hemolytic uremic syndrome; MOF = multiple organ failure. Bone Marrow Transplantation


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100 a

b

80 60

100 P < 0.01* 0

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180

40 20

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0 20

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IL-8 (pg/ml)

TNF- a (pg/ml)

120

50 P < 0.01*

10 0 10 0

Preconditioning

Day 0

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Figure 1 Levels of various cytokines before and after BMT. Black circles of the upper parts show patients with complications and white circles of the lower parts show patients without complications. *Two-way layout analysis of variance. (a) IL-10: Patients with severe acute GVHD (grade III or IV) showed a marked rise of IL-10 during the WBC recovery phase (P ⬍ 0.001). (b) IL-8: Patients with CMV-IP showed a marked rise of IL-8 during the WBC recovery phase (P ⬍ 0.001). (c) IFN-␥: Patients with CMV-IP showed a marked rise of IFN-␥ during the WBC recovery phase (P ⬍ 0.001). (d) IL-12: Patients with TMA showed a marked rise of IL-12 during the WBC recovery phase (P ⬍ 0.001). (e) TNF-␣: Patients with ARDS-like respiratory disorders showed a marked rise of TNF-␣ during the WBC recovery phase (P ⬍ 0.01). (f) IL-8: Patients with ARDS-like respiratory disorders showed a marked rise of IL-8 during the WBC recovery phase (P ⬍ 0.01).

G-CSF (lenograstim (5 ␮g/kg), filgrastim (5 ␮g/kg), or natrograstim (8 ␮g/kg)) was administered to all patients from day 5 until the WBC exceeded 10 ⫻ 109/l. Results Among the 26 patients, five developed severe GVHD (grade III or IV), three had CMV-IP, three had TMA, three had ARDS-like respiratory disturbance, and 16 had no complications (Table 2). Clinical characteristics were compared between the 10 patients with complications and the 16 patients without complications. There were no significant differences in age, sex, underlying disease, conditioning Bone Marrow Transplantation

regimen, GVHD prophylaxis, time (days) from BMT to an ANC ⬎500/␮l and to a platelet count ⬎50 000/␮l, or donor type, but the febrile period during the aplastic phase was significantly longer and the C-reactive protein (CRP) level was significantly higher in patients with complications compared with those without complications (P ⬍ 0.01; t-test). The changes of various cytokines were studied from before conditioning until the time of WBC stabilization after discontinuation of G-CSF and these parameters were compared between patients with and without each complication. The patients with severe GVHD (grade III or IV) showed a marked rise of IL-10 (Figure 1a), those with CMV-IP showed a marked rise of IL-8 (Figure 1b) and


423

a

a

50

180

IL-10 (pg/ml)

100 P < 0.05* 0 60 40 20 0 Preconditioning

IFN-g (pg/ml)

40 30 20 P < 0.01*

10 0 20 10 0

Day 0

Aplastic phase

Recovery phase

Preconditioning

Stable phase

Day 0


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b

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100 P < 0.01 * 0 20 0

Preconditioning

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50 40

P < 0.05 * 0 40 20 0

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IL-10 (pg/ml)

100

30 20 10

P < 0.01*

0 40 20

Day 0

Aplastic phase

Recovery phase

Stable phase

Figure 2 IL-10 level before and after BMT. Black circles of the upper parts show with severe GVHD and white circles of the lower parts show patients with CMV-IP (a), TMA (b) and ARDS (c). There are significant differences between the two groups, respectively (a, P ⬍ 0.05; b, P ⬍ 0.01; c, P ⬍ 0.05). *Two-way layout analysis of variance.

IFN-␥ (Figure 1c), those with TMA showed a marked rise of IL-12 (Figure 1d), and those with ARDS-like respiratory disturbance showed a marked rise of TNF-␣ (Figure 1e) and IL-8 (Figure 1f) during the WBC recovery phase (twoway layout analysis of variance: P ⬍ 0.01) (Figure 1). There were no significant differences between the patients with and without complications with regard to the other parameters studied, including IL-1␤, IL-2, and IL-6 (data not shown). In addition, the changes of various cytokines were compared between the two groups with different complications. The levels of IL-10 (Figure 2 a–c), IFN-␥

0

Day 0


Stable phase

Preconditioning

Figure 3 IFN-␥ level before and after BMT. Black circles of the upper parts show with CMV-IP and white circles of the lower parts show patients with severe GVHD (a), TMA (b) and ARDS (c). There are significant differences between the two groups, respectively (a, P ⬍ 0.01; b, P ⬍ 0.01; c, P ⬍ 0.01). *Two-way layout analysis of variance.

(Figure 3a–c), IL-12 (Figure 4a–c), and TNF-␣ (Figure 5a– c) were significantly higher during the WBC recovery phase in the patients with severe GVHD, TMA, CMV-IP, ARDS compared with those with other complications, respectively. IL-8 level was significantly higher at the same time in the patients with CMV-IP or ARDS compared with those with severe GVHD or TMA (Figures 6 and 7a and b). There were no significant differences between other two groups with different complications with regard to the other parameters studied. Changes in TM and PAI-1 were also studied from the Bone Marrow Transplantation


424

a

150

80 IL-12 (pg/ml)

60 40 20

P < 0.05*

0 20 0

Preconditioning

TNF- a (pg/ml)

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P < 0.05

0 30

Day 0

Aplastic phase

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0

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Preconditioning

Day 0

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b

IL-12 (pg/ml)

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P < 0.01*

TNF- a (pg/ml)

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0 20 0


120 90 60

P < 0.05

30 0 30


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Stable phase

Preconditioning

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c 150 120

60 40 20

P < 0.05*

0 20

TNF- a (pg/ml)

IL-12 (pg/ml)

80

90 P < 0.05

60 30 0 30

0 PreDay 0 conditioning


Stable phase

Figure 4 IL-12 level before and after BMT. Black circles of the upper parts show with TMA and white circles of the lower parts show patients with severe GVHD (a), CMV-IP (b) and ARDS (c). There are significant differences between the two groups, respectively (a, P ⬍ 0.05; b, P ⬍ 0.01; c, P ⬍ 0.05). *Two-way layout analysis of variance.

aplastic phase until the time of WBC stabilization and these parameters were compared between patients with and without complications. Both TM and PAI-1 levels were significantly higher at the time of WBC stabilization in the patients with complications compared with the patients without complications (P ⬍ 0.0001; t-test) (Figure 8). Discussion Various mechanisms have been proposed to explain the complications of BMT,1–5 but these are diverse and comBone Marrow Transplantation

0 Preconditioning

Day 0

Aplastic phase

Recovery phase

Stable phase

Figure 5 TNF-␣ level before and after BMT. Black circles of the upper parts show with ARDS and white circles of the lower parts show patients with severe GVHD (a), CMV-IP (b) and TMA (c). There are significant differences between the two groups, respectively (a, P ⬍ 0.05; b, P ⬍ 0.05; c, P ⬍ 0.05). *Two-way layout analysis of variance.

plex. By applying the concept of SIRS11,12 as the basic state that occurs after BMT, complications such as multiple organ failure can be considered to be derived from SIRS. All of our patients who developed complications had fever during the aplastic phase (Table 2) and an increase in cytokines during the recovery phase (different cytokines increased when different complications occurred) (Figure 1). After the increase of cytokines, the serum TM and PAI1 levels always increased (Figure 8). Taking these events into consideration, it seems that the patients were in a similar state to that of SIRS, in which proinflammatory


425

a

a 100

1000

IL-8 (pg/ml)

60 40 20

P < 0.05*

0

IL-8 (pg/ml)

80

20

Day 0

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Recovery phase

P < 0.05*

50 10 0 50 10 0

0 Preconditioning

100

Stable phase

Preconditioning

Day 0


Stable phase

b

b

1000

100 80 IL-8 (pg/ml)

40 20

P < 0.05*

0 20

50

P < 0.05*

10 0 50 10 0

0 Preconditioning

IL-8 (pg/ml)

100

60

Day 0

Aplastic phase

Recovery phase

Stable phase

Figure 6 IL-8 level before and after BMT. Black circles of the upper parts show with CMV-IP and white circles of the lower parts show patients with severe GVHD (a) and TMA (b). There are significant differences between the two groups, respectively (a, P ⬍ 0.05; b, P ⬍ 0.05).

cytokines are overproduced in response to infection (inflammation) and organ impairment or failure is precipitated by these cytokines. Vascular endothelial damage caused by neutrophils has been suggested to be involved in the progression of SIRS to organ impairment,12 rather than disseminated intravascular coagulation and microthrombus formation as is proposed conventionally. A role for neutrophils would be more consistent with our data. Based on the present findings, patients with fever during the aplastic phase and an increase of pro-inflammatory cytokines (such as TNF-␣, IFN-␥, or IL-12) during the recovery phase who thereafter show an increase in serum TM and PAI-1 (suggesting vascular endothelial damage) seem to be likely to develop complications after BMT. If conditioning with TBI or chemotherapy and the occurrence of fever during the aplastic phase are regarded as the ‘first attack’ and multiple organ disease syndrome (MODS) arising from vascular endothelial damage primed by cytokines during the recovery phase and triggered by some stimulation (eg CsA or FK506 at high concentrations, or methylprednisolone, GVHD, and CMV infection), is regarded as the ‘second attack’, then complications such as acute GVHD, VOD, TMA, and CMV-IP after BMT are manifestations of SIRS and the mechanism of onset corresponds to the ‘second attack’ theory proposed by Ogawa et al.18 Accordingly, the following preventive measures can



Stable phase

Figure 7 IL-8 level before and after BMT. Black circles of the upper parts show with ARDS and white circles of the lower parts show patients with severe GVHD (a) and TMA (b). There are significant differences between the two groups, respectively (a, P ⬍ 0.05; b, P ⬍ 0.05).

be taken to decrease complications after BMT: (1) investigation of new conditioning regimens; (2) prevention of fever during the aplastic phase; (3) adjustment of immunosuppressive therapy after successful graft take; and (4) avoidance of vascular endothelial damage. In this study, we determined the levels of cytokines and indications of vascular endothelial damage throughout the period before and after BMT. Our results suggest that complications after BMT can be regarded as manifestations of SIRS. This concept simplifies the mechanism of complications after BMT, and suggests that countermeasures against infection and vascular endothelial damage are more important than has been realized. Acknowledgements We wish to thank Ms A Utsumi and Ms Y Shikita for their expert technical assistance.

References 1 Krenger W, Hill GR, Ferrara JML. Cytokine cascades in acute graft-versus-host disease. Transplantation 1997; 64: 553–558. 2 Shulman HM, McDonald GB, Matthews D et al. An analysis of hepatic venocclusive disease and centrilobular hepatic Bone Marrow Transplantation


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a

P < 0.0001*

6

8

TM (FU/ml)

6.71 ± 1.78 6

P < 0.0001*

7

4

2

4.02 ± 0.80 NS*

8

1.73 ± 0.43 1.18 ± 0.30 Aplastic phase

1.73 ± 0.43

1.38 ± 0.47

Recovery phase

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10

b P < 0.0001* 8

PAI-1 (ng/ml)

72.77 ± 13.32 6

P < 0.05*

4

2

11

12 30.32 ± 15.64

NS*

18.49 ± 9.44 10.95 ± 4.54 9.00 ± 5.98 Aplastic phase

15.61 ± 9.30

Recovery phase

13 Stable phase

Figure 8 TM and PAI-1 levels during the aplastic, recovery, and stable phases are shown for patients with complications (solid squares) and patients without complication (solid circles). *Student’s t-test. (a) TM levels were significantly increased in the recovery phase and stable phase for the patients with than without complications. (b) PAI-1 levels were significantly increased during the recovery phase in the patients with complications compared to those without, and PAI-1 became even higher during the stable phase in the patients with complications.

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