We monitored levels of C-reactive protein (CRP) in 96 consecutive adult allogeneic BMT patients (age 15–50 years) transplanted in our unit. Major transplant-related complications (MTC) occurred in 32% of cases and included: hepatic veno-occlusive disease, pneumonitis, severe endothelial leakage syndrome and >II acute GVHD. Transplant-related mortality (TRM) before day 100 post-BMT was 13.5%. Variables included in a stepwise logistic regression model were: gender, age, disease category, donor type, T cell depletion, TBI, use of growth factors, bacteremia, mean CRP-levels >50 mg/l between days 0 and 5 (CRP day 0–5) and >100 mg/l between days 6 and 10 (CRP day 6–10) post-BMT. Only high CRP-levels (for MTC and TRM) (P < 0.001) and donor-type (for TRM) (P = 0.02) were independent risk factors. The estimated probability for MTC was 73% (CRP day 6–10 >100 mg/l) vs 17% (CRP day 6–10 <100 mg/l). Using the same cut-off levels, the probabilities for TRM were 36.5% vs 1% in the identical sibling donor situation and 88% vs 12.5% in other donor-type transplants. We conclude that the degree of systemic inflammation, as reflected by CRP-levels, during the first 5–10 days after BMT identifies patients at risk of MTC and TRM. Our data may be useful in selecting patients for clinical trials involving pre-emptive anti-inflammatory treatment.
Despite improvements in supportive care, the practice of allogeneic BMT remains limited by its toxicity. Overall transplant-related mortality (TRM) varies between 10 and 50% and is mainly due to the occurrence of major transplant-related complications (MTC) during the first months after BMT, including: infections, hepatic veno-occlusive disease (VOD), acute graft-versus-host disease (AGVHD), pneumonitis and severe endothelial leakage syndrome (ELS). Several risk factors for MTC and TRM have been identified: older age and more advanced disease status of the patient, a more intensive conditioning regimen, use of alternative donor sources and less efficient methods of graft-versus-host prophylaxis.1,2,3 However, even in homogeneous cohorts of adult BMT patients, MTC/TRM occur in some but not all cases. To a certain extent, this may be explained by differences in individual sensitivity to the tissue-damaging effects of the conditioning regimen. Methods of assessing this differential sensitivity may lead to early identification of patients at risk who might benefit from intensified prophylactic measures. Some biological markers in the early transplant phase have been associated with an increased risk of TRM, such as low capacity of IL-10 production by blood mononuclear cells4 and high levels of TNF-alpha during the conditioning regimen.5 Also, an early increase in serum bilirubin and/or blood urea nitrogen (BUN) after BMT has been associated with TRM.6
Tissue damage can lead to a systemic inflammatory response. C-reactive protein (CRP) is an acute phase protein produced by hepatocytes and is a reliable marker of systemic inflammation.7 We have previously shown that high peak levels of CRP were associated with the occurrence of MTC.8 In this study, we demonstrate that an early increase of CRP-levels, before day 10 after BMT, is highly predictive of MTC and TRM before day 100 post-BMT.
Patients and methods
Patients and transplant conditions
From a total of 103 consecutive allogeneic marrow transplants performed on adult patients (age ⩾15 years) in our unit between 1989 and 1999, seven were excluded from this analysis: five second transplants and two patients who died before day 10 post-BMT. The median age of the 96 patients included was 37 years (range: 15–50) and 68 (71%) were male. Diagnosis at time of BMT was: chronic myeloid leukemia (n = 37), acute leukemia (n = 33), aplastic anemia (n = 8), myelodysplasia (n = 7), lymphoma (n = 6), myeloma (n = 4), and solid tumor (n = 1). Patients with acute leukemia or myelodysplasia in first complete remission and chronic myeloid leukemia in first chronic phase were considered to be in the good risk category, representing 65% of cases. Marrow was the source of stem cells in all cases and donors were HLA-identical siblings (n = 82), volunteer-unrelated donors (n = 13) or a haploidentical brother (n = 1). From the start of the conditioning regimen until the end of neutropenia and stabilization of their clinical condition, patients were hospitalized in an isolation unit with laminar air flow protection. At the same time they received norfloxacin (400 mg three times a day p.o.), itraconazole (200 mg twice a day p.o.) or fluconazole (200–400 mg every day p.o. or i.v., adapted to renal function) and started mouth washes with povidon-iodine solution (four times a day) and liquid nystatin (100 000 IU four times a day). The conditioning regimen was always myeloablative and included total body irradiation in 82% of cases. GVHD prevention consisted of either cyclosporin in combination with methotrexate (53%) or partial T cell depletion by E-rosetting (47%). Granulocyte colony-stimulating factor was administered in 45% of cases after marrow infusion until stable neutrophil recovery, defined as the achievement of neutrophil levels above 0.5 × 109/l for 2–3 consecutive days.
Definition of major complications
Fluid balance and body weight as well as blood levels of bilirubin were prospectively monitored in all patients. Fluid retention was rigorously treated with fluid and salt restriction and diuretics, with addition of dopamine in severe cases. All patients received a continuous infusion of heparin 100 units/kg. VOD was defined according to the modified Seattle criteria9 as the occurrence, within the first 20 days after BMT, of fluid retention of at least 2% body weight in combination with a direct bilirubinemia above 2 mg/100 ml. Hepatomegaly, right upper quadrant pain and ascites were considered additional criteria. No other explanation for these signs and symptoms could be present at the time of diagnosis. As major transplant-related complications (MTC), other than VOD, were considered: >grade II AGVHD, severe endothelial leakage syndrome and pneumonitis. AGVHD was diagnosed and staged according to the Seattle criteria.10 Severe endothelial leak syndrome (ELS) was defined as the occurrence of fever, fluid retention and weight gain of >3% without hyperbilirubinemia and in the absence of cardiac failure with insufficient response to diuretics, requiring fluid restriction and dopamine therapy. Pneumonitis was defined as the presence of fever and respiratory symptoms that could not be related to cardiac failure or generalized fluid retention and with clear demonstration of pulmonary infiltrates on chest X-ray. TRM was defined as death due to any reason in a patient without evidence of relapse and occurring before day 100 after BMT. As reason for TRM, the primary cause was taken into account, eg AGVHD, when a patient died of infection in the context of AGVHD.
Serum samples of CRP were taken during all 96 post-BMT episodes, every 2 days or daily at times of fever, from the day of BMT until stable neutrophil recovery and discharge from the BMT unit. Levels of CRP were measured on a Behring nephelometer analyzer (normal = <5 mg/l). A mean of 19 CRP measurements for each post-BMT episode, including the day of transplant and the following 25 days, were performed. For all patients, we calculated the average CRP value for each 5-day period post BMT, the first also including the day of transplant. This was done to reduce the influence of very low or high levels of short duration and to more accurately reflect the degree of inflammation over several consecutive days in a particular patient. In addition, and by considering one average value per 5-day post-BMT period and per patient, each patient equally contributed to the statistical analysis, again to reduce the influence of extreme values. Not all patients were analyzed during each 5-day episode because follow-up stopped before day 25 after transplant. This could be due to the end of neutropenia and full clinical recovery, or to early death. However, the minimum number of mean values per 5-day period which was used for subgroup comparison was 16, 20 and 17 for uncomplicated, minor complications and MTC subgroups, respectively.
All statistical tests were carried out two-tailed, at the 5% level of significance, using the SPSS (version 10) software for statistics. For comparison of continuous variables the Mann–Whitney test was used. The association between categorical variables was investigated by the Chi-square or Fisher's exact test. Time to TRM was estimated by the Kaplan–Meier method and for comparison between groups the logrank test was used. Stepwise logistic regression was used as a multivariate model to predict MTC and TRM.
Based on a study of the clinical records, all 96 post-BMT episodes were divided into three distinct categories: absence of any clinical complication except for fever without documented infection (uncomplicated) (n = 29), fever in association with documented bacteremia or some degree of fluid retention, but no criteria for MTC (minorTC) (n = 36) and one or more MTC (n = 31). The uncomplicated and minorTC groups taken together constitute the MTC− (68%) as opposed to the MTC+ (32%) group. None of the patients who developed no, or only minor complications, died of toxicity within the first 100 days after BMT. In contrast, 42% of patients with MTC died early (P < 0.001). The early BMT-related toxicity is summarized in Table 1. Bacteremia before day 30 post transplant occurred in 28 (29%) patients and with comparable frequency in patients with minor (39%) or major (45%) complications (P = 0.63). Bacteremia was due to coagulase-negative staphylococci in 61% of cases. VOD occurred in 14 of 93 evaluable patients, corresponding to an incidence of 15% in our series. The median times to reach maximal bilirubinemia (median 7 mg/100 ml, range 2.6–18 mg/100 ml) and fluid retention (median 4%, range 2.5–8%) were 13 and 14 days, respectively. Two patients who developed severe hyperbilirubinemia were not evaluable for VOD. They died as the result of hepatorenal failure and were also classified as MTC. ELS occurred in seven patients and the median time to maximal fluid retention (median 5%, range 3–11%) was 13 days. Eleven patients had pneumonitis and in nine of these, no infectious etiology could be documented or suspected as based on response to anti-infectious therapy. Median time to appearance of pulmonary infiltrates on X-ray was 15 days. Eleven patients had grade >II AGVHD, mostly as a secondary event and occurring later, at a median of 30 days after transplant. Death before day 100 post-BMT occurred in 13 (13.5%) patients and the primary causes were VOD (n = 2), hepatorenal failure (n = 2), pneumonitis (n = 5), AGVHD >II (n = 3) and infection (n = 1).
Figure 1 shows the comparison of CRP patterns obtained in the three clinical subgroups. Mean CRP levels were significantly higher in patients developing major, as opposed to minor, complications throughout the early post-BMT episode. This was also the case for mean CRP levels between days 0 and 5 (CRP d0–5) (P = 0.027) and between days 6 and 10 (CRP d6–10) (P < 0.001) after transplant. fifteen (16%) of the 96 patients had crp d0–5 >50 mg/l, whereas 26 (27%) had CRP d6–10 >100 mg/l. Of the 15 patients with CRP d0–5 >50 mg/l, 11 (73%) also developed CRP d6–10 >100 mg/l. In Figure 2, Kaplan–Meier analysis shows that the probability of TRM for patients with CRP d6–10 >100 mg/l was significantly higher (42.5% ± 9.5%) than for those with lower levels (3% ± 2%) (P < 0.001).
We further analyzed the relationship of early post-transplant CRP levels with MTC/TRM as we did with other pre- and post-transplant variables which might be relevant (Table 2). On univariate analysis, we found that the occurrence of MTC was associated with bad-risk disease category, no T cell depletion and bacteremia, whereas TRM was associated with bad-risk disease category and bacteremia. CRP d0–5 >50 mg/l, CRP d6–10 >100 mg/l and maximal levels reached within the first 25 days post transplant were all highly significantly associated with MTC and TRM. On multivariate analysis and including CRP d6–10 in addition to the same pre-transplant and clinical variables, we found that only CRP d6–10 >100 mg/l was significant for MTC (OR: 13 (4.4–38), P < 0.001) and that crp d6–10 >100 mg/l (OR: 52 (6–456), P < 0.001) and sibling donor (OR: 0.08 (0.01–0.8), P = 0.03) were significantly associated with TRM. Based on the stepwise logistic regression model, the risks of MTC and TRM could be estimated, as shown in Table 3.
We studied the relationship between several pre- and post-transplant variables, including CRP levels, with the occurrence of major complications after BMT. Our definition of MTC was based on similar definitions to those used in other reports,11 as well as on our own observation that none of the patients without such complications died of transplant-related toxicity. Bacteremia occurred in 29% of cases and was mainly due to coagulase-negative staphylococci. Such infections usually have a relatively benign course and we therefore did not consider bacteremia as a major complication.
Our study shows that a sustained increase in CRP levels during the first 5–10 days after BMT is associated with the occurrence of MTC and TRM. In a stepwise logistic regression model, only CRP levels (for MTC and TRM) and, to a lesser degree, donor-type (for TRM) were independent risk factors. Early CRP release has more impact compared to some known factors associated with MTC/TRM such as advanced disease, no T cell depletion and early bacteremia. These factors were also identified in our study on univariate analysis. CRP is an acute phase protein which is generally considered to be a reliable marker of systemic inflammation.7 Our data thus suggest that the degree of systemic inflammation during the early post-BMT episode is the main determinant for the occurrence of MTC and TRM. Since CRP can amplify complement activation by T cells,12 it may even contribute itself to mechanisms involved in the pathogenesis of MTC.
CRP release has been documented in neutropenic patients with bacteremia.13 However, bacteremia could not be the main trigger for early CRP release in our patients since it occurred in roughly the same proportion of patients with either minor or major complications. On the other hand, our univariate analysis showed that bacteremia was associated with MTC and, to a lesser extent with TRM. We hypothesize that it may be a co-factor for the development of complications, as already suggested from our previous observations.14
Tissue damage due to the conditioning regimen leads to local inflammation and production of several pro-inflammatory cytokines by macrophages and monocytes. Cytokines induce a systemic inflammatory response and among them, IL-6 is the primary inducer of CRP in hepatocytes.15 We hypothesize that CRP levels closely correlate with cytokine-mediated systemic inflammation as the result of tissue damage produced by the conditioning regimen. High levels of TNF-alpha during or shortly after the conditioning regimen have been shown to precede MTC, in particular AGVHD.5,11,16,17 More recently, we showed that the release of IL-6, IL-8 and TNF-alpha contributes to the early pathophysiological events involved in VOD.18 Our hypothesis is consistent with these data. The sensitivity to tissue-damaging factors is different among individual patients. In our transplant setting, CRP levels proved to be useful for identifying patients who develop more systemic inflammation, thus increasing the risk of cytokine-triggered toxic events including VOD, capillary leakage syndrome, toxic pneumonitis and AGVHD. This may be less the case in situations where regimens used to prevent AGVHD contain agents such as corticosteroids which tend to block systemic inflammation. Finally, our data indicate that early CRP release is associated with the subsequent development of severe AGVHD which, in general, occurred at a later stage post BMT. It may be that alloreactive processes also contribute to the early CRP release without clinical signs of AGVHD.
The early identification of patients at risk of TRM is a relevant issue in allogeneic BMT. In a recent study including more than 300 patients, it was found that relatively small increases in bilirubin and blood urea nitrogen on day 7 after transplant increased the 100-day TRM from 10% to 28%.6 Patients with a high risk score died more often of AGVHD, infections and multi-organ failure. In our study, almost 50% of MTC were VOD. Many VOD patients already have small increases in bilirubin on day 7 after transplant and also develop some degree of renal dysfunction.19 Thus, it is reasonable to think that VOD may be predicted early by both scoring systems. On the other hand, some small increases in indirect bilirubin can quite often be seen post transplant as a result of, for instance, some degree of post tranfusion hemolysis. Also, the BUN can be increased by cyslosporine toxicity. Pneumonitis patients who develop infiltrates at a median of 2 weeks after BMT are unlikely to develop increases in BUN or bilirubin during the first week after BMT. Therefore, we argue that high levels of CRP are likely to be more reliable as a risk factor for MTC/TRM as compared to early bilirubin and BUN increases.
Since early post-transplant systemic inflammation appears to be highly predictive of MTC/TRM, a logical consequence of our study would be to treat patients with high levels of CRP d6–10 by anti-inflammatory therapy as a pre-emptive approach. Several treatment options may be considered. Early treatment with high-dose methylprednisolone has been shown to be beneficial to the majority of patients with regimen-related hepatotoxicity, of which many had VOD.20 Defibrotide has anti-thrombotic, but also anti-inflammatory properties and leads to a complete response in around 50% of VOD cases. Results were better when treatment was started earlier.21 The toxicity observed during the first weeks after BMT shows some similarities to sepsis and septic shock. Both conditions are characterized by a pro-inflammatory host response. In a recent randomized trial performed in patients with severe sepsis, treatment with recombinant human activated protein C (drotecogin alfa) reduced mortality very significantly by 6.1%.22 The use of anti-cytokines in AGVHD has thus far been disappointing, mainly because of initiating therapy at advanced stages and recurrence of AGVHD after stopping the treatment.23,24 Combinations of several anti-cytokines25 or of anti-cytokines with other forms of immunotherapy may be more effective. A novel candidate therapeutic agent may be anti-CD14 which has led to a reduction of lipopolysaccharide-induced symptoms and inflammatory reactions in humans.26 In murine models, lipopolysaccharide has been shown to trigger TNF-alpha release by macrophages which in turn primed the graft-versus-host reaction.27
In summary, our data lead us to advocate close monitoring of CRP levels within the first weeks after BMT. High levels within the first 5–10 days after transplant identify patients at risk of MTC and TRM. These patients are most likely to benefit from timely anti-inflammatory therapies and should be included in clinical trials that aim to demonstrate the efficacy of such therapies.
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We thank Mrs A Willekens and her nursing staff from the BMT-Unit for the excellent care of our patients. This work was supported by grant from the scientific Fund W Gepts AZ-VUB.
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Schots, R., Van Riet, I., Ben Othman, T. et al. An early increase in serum levels of C-reactive protein is an independent risk factor for the occurrence of major complications and 100-day transplant-related mortality after allogeneic bone marrow transplantation. Bone Marrow Transplant 30, 441–446 (2002). https://doi.org/10.1038/sj.bmt.1703672
- C-reactive protein
- severe complications
- transplant-related mortality
- allogeneic BMT
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