Association between pretransplant iron overload determined by bone marrow pathological analysis and bacterial infection

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Iron overload (IO) before allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is regarded as a poor prognostic factor after allo-HSCT, although there is controversy regarding the clinical relevance.1 Previous studies identified pretransplant IO as a poor prognostic factor after allo-HSCT, whereas other studies including a meta-analysis using liver magnetic resonance imaging showed no association between pretransplant IO and poor overall survival (OS).2, 3 Although serum ferritin (SF) is the most widely used indicator of total body iron burden, its level is affected by factors causing inflammation such as infection or active disease, and before red blood cell transfusions.4 The development of assays to easily and accurately determine IO is warranted from a clinical standpoint because the oral iron chelating agent deferasirox is commercially available. With only a few studies directly assessing IO by pathological approaches, the impact of pathologically determined IO especially on clinical outcomes has not been fully clarified.5, 6, 7 Here, we compared clinical relevance of IO estimated by Prussian blue staining on post-transplant outcomes with that by SF.

Pretransplant serum ferritin (SF) was measured for 193 adult patients with acute leukemia and myelodysplastic syndromes undergoing first allo-HSCT in our institution between 2006 and 2013. Except for two participating in a phase I study, and 22 whose bone marrow samples before allo-HSCT were unavailable or inappropriate for pathological analysis, 169 patients were included in further analyses. A pretransplant SF level of 800 ng/mL was defined as the cut-off value as the median level in this study, and this cut-off was used to divide patients into two groups: low-ferritin group, SF<800 ng/mL; and high-ferritin group, SF800 ng/mL. Prussian blue staining was newly performed for this study using stored bone marrow specimens, and cases with a few stained granules within normal range were assigned as negative, whereas those with focally or diffusely stained iron granules as positive by two pathologists (Supplementary Figure 1). The covariates included in the statistical analyses were sex, age, diagnosis, pretransplant SF level, conditioning regimen, disease status at allo-HSCT, stem cell sources, HLA disparity, GvHD prophylaxis and positivity of Prussian blue staining. Analysis of complications after allo-HSCT included the incidences of bacterial infection, bacteremia, fungal infection, aspergillus infection, acute GvHD, and veno-occlusive disease (VOD), where each complication was judged based on clinical decision from medical records or mycological and pathological data. Febrile neutropenia was included in bacterial infection because it is a clinically important adverse event, which is considered to be closely associated with bacterial infection. The end points included the P of OS and the cumulative incidences of relapse or progression, non-relapse mortality (NRM) and complications after HSCT including infectious diseases. Survival curves were estimated using the Kaplan–Meier method, and P-values were calculated using the log-rank test. Relapse or progression, NRM and complication incidences were calculated using cumulative incidence curves. Univariate and multivariate analyses of risk factors for OS were performed using Cox proportional hazards regression models, and the Fine and Gray regression model was used for analysis of relapse or progression, NRM, and complications. Categorical and continuous variables were analyzed using the Fisher’s exact test and Student’s t-test, respectively. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University).

Detailed patient characteristics are shown in Table 1. Patients in the high-ferritin group included more patients with active disease than those in the low-ferritin group (41 vs 23%, P=0.01). The median time to neutrophil engraftment in all patients was 17 days, and the period tended to be longer in patients with positive Prussian blue staining (P=0.06). The median pretransplant SF level was 807 ng/mL (range, 47–9130), and 85 patients (50%) had a high ferritin level (800 ng/mL), and 100 (59%) showed positive Prussian blue staining. There was a statistically significant association between SF level and positive Prussian blue staining (P<0.01). The cumulative incidence of bacterial infection at day 100 after allo-HSCT was 72%, and it was significantly higher in patients who showed positive Prussian blue staining (P<0.01, 82 vs 58% at day 100, Figure 1a). Although the incidence also tended to be higher in the high-ferritin group, it was not significant (P=0.10, 75 vs 69% at day 100, Figure 1b). There was no statistically significant difference in the cumulative incidence of bacteremia, invasive fungal infection, aspergillus infection, acute GvHD and VOD between the high-ferritin and low-ferritin groups, or between the positive and negative Prussian blue staining groups (Supplementary Table 1). The median follow-up time of survivors was 29.2 months (range, 1.7–105.1). The probabilities of 2-year OS in the high-ferritin and low-ferritin groups were 61% and 76%, respectively (Figure 1c). In a multivariate analysis, high-ferritin level was a poor prognostic factor for OS (hazard ratio (HR), 1.9; 95% confidence interval, 1.1–3.2; P=0.02) (Supplementary Table 2). In terms of immunohistochemical classification of IO, there was no significant difference in the P of OS between the Prussian blue negative and positive groups (P=0.57, 2-year OS rate 67 vs 70%; Figure 1d). The cumulative incidences of 2-year relapse in the high-ferritin and low-ferritin groups were 44 and 29% (Figure 1e), and a multivariate analysis showed that high-ferritin level was a marginal risk factor for relapse (hazard ratio, 1.6; 95% confidence interval, 1.0–2.6: P=0.05). On the contrary, there was no significant difference in the incidence of relapse between the negative and positive Prussian blue staining groups (P=0.98, 2-year relapse rate 35 vs 38%; Figure 1f). In terms of NRM, there was no significant difference according to ferritin level or Prussian blue staining positivity. The concurrent high-ferritin and positive Prussian blue staining group (high/pos group) was a significant risk factor for the cumulative incidence of bacterial infection and the P of OS (P<0.01, 85% in the high/pos group vs 64% in other groups at day 100; P=0.02, 2-year OS rate 61% in the high/pos group vs 73% in other groups).

Table 1 Patient and transplant characteristics
Figure 1
figure1

The cumulative incidence of bacterial infection in patients grouped according to Prussian blue staining positivity (a) and serum ferritin (SF) level (b). Kaplan–Meier curves for OS grouped according to SF level (c) and Prussian blue staining positivity (d). The cumulative incidence of relapse grouped according to SF level (e) and Prussian blue staining positivity (f).

We retrospectively analyzed the impact of pretransplant IO on post-transplant clinical outcomes, and showed that positive bone marrow staining for Prussian blue was a significant risk factor for bacterial infection after allo-HSCT, which could be partially affected by the delay of neutrophil engraftment. Young et al.8 reported that patients undergoing transplant from PBSC donors had fewer infection events than those transplanted from bone marrow donors due to quicker engraftment, which strengthens our hypothesis. Miceli et al.5 categorized patients into two groups according to the level of iron stored in bone marrow, and found that increased iron store was a risk factor for severe infection. Our findings were consistent with their report, as well as with those that described an increased incidence of bacterial infection in patients with IO as determined by ferritin levels.5, 9, 10 Moreover, we found that high ferritin level was a significant poor prognostic factor for OS, whereas positivity of Prussian blue staining was not. Contrary to several studies reporting the relevance of pretransplant IO for clinical outcomes using SF, only two studies by Sivgin et al.11, 12 used pathological approaches to identify the impact of IO on OS. They used a 5-point scale based on the degree of staining to classify liver biopsy and bone marrow specimens, and showed that high iron contents were associated with a poorer OS in both studies. They did not refer to complications after allo-HSCT, and our study is the first to evaluate both OS and complications associated with IO in bone marrow specimens. Patients with high ferritin levels before HSCT were previously found to have a higher rate of relapse.13, 14 Our results are consistent with these findings, as patients with high ferritin levels had inferior OS due to an increased incidence of relapse/progression. Our study showed that there was a clear association between high ferritin level and active disease status at allo-HSCT, which might have led to higher relapse rates in the high-ferritin group. There are several limitations to this study. First, although we found that patients in the Prussian blue-positive group were at high risk of bacterial infection, we did not find any significant difference when the analysis included only bacteremia as well as invasive fungal infection due to the relatively small sample size in this study. Second, there exist no standardized methods in assessing IO by pathological classification, and our approach is one attempt. Finally, SF before allo-HSCT and data on blood transfusion volumes were not available in about 20% and over 80% of cases, respectively.

In conclusion, we demonstrated that pathologically determined IO was associated with increased risk of bacterial infection after allo-HSCT. Our study suggests that pretransplant IO by bone marrow pathological analysis is relevant in the setting of allo-HSCT, and larger-scale, prospective studies are warranted to confirm for further confirmation.

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Acknowledgements

We thank C Kina and S Miura for their technical assistance with immunohistochemistry. This study was supported by grant from the National Cancer Research and Development Fund (26-A-26).

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Correspondence to S Fuji.

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The authors declare no conflict of interest.

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Supplementary Information accompanies this paper on Bone Marrow Transplantation website

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Ohmoto, A., Fuji, S., Miyagi-Maeshima, A. et al. Association between pretransplant iron overload determined by bone marrow pathological analysis and bacterial infection. Bone Marrow Transplant 52, 1201–1203 (2017) doi:10.1038/bmt.2017.93

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