Organ siderosis and hemophagocytosis during acute graft-versus-host disease.

Iron overload prior to allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to blood transfusions is associated with increased non-relapse mortality (NRM) and with low overall survival. After allo-HSCT, high iron content in liver biopsies and elevated ferritin concentrations in blood, used as a surrogate parameter for iron overload, are also related to NRM. The cellular and molecular mechanisms behind this association, however, are yet to be clarified. In particular, available data on the role of ferritin and iron metabolism during the pathophysiology of acute graft-versus-host disease (GvHD), a major contributor to NRM, are scarse. To shed light on the connection between GvHD, ferritin levels, and iron metabolism, we decided to analyse the clinical data of patients undergoing allo-HSCT as well as data from preclinical murine GvHD models. We generated our working hypothesis after observing concurrent presentations of acute GvHD (aGvHD) and hyperferritinemia in a series of patients undergoing alloHSCT. In a typical case, a 29 year old female underwent allo-HSCT for severe aplastic anemia. She received four erythrocyte concentrates in total: two prior to allo-HSCT and two during aplasia post allo-HSCT. Around day +200, she suffered from late aGvHD of the liver; stage 3, grade III. At the same time point, a high ferritin level and a high transferrin saturation was measured for the first time in this patient (Online Supplementary Figure S1A). Ferritin levels dropped rapidly after clinical improvement of aGvHD due to administration of high dose steroids. This repeated during a second exacerbation of GvHD. Notably, the transferrin saturation was not affected by the immunosuppression. We found siderosis in bone marrow in this patient (Online Supplementary Figure S1B). During the course of disease, no iron chelation was administered. No mutations were found on the HFE gene. After her second exacerbation, the patient recovered and is still alive. Based on this case, and others similar, we hypothesized that GvHD is associated with hyperferritinemia and organsiderosis, independent of blood transfusions. We retrospectively analysed allo-HSCT recipients at the Charité-CBF over a six-year period, with a survival of more than 60 days. Patient characteristics and their maximum ferritin levels after allo-HSCT (low/medium vs. high vs. very high) are given in Online Supplementary Table S1. Of 104 patients, 33 developed high ferritin levels (5000μg/l-10000μg/l), and 11 had very high ferritin levels (more than 10000μg/l) after allo-HSCT. There was no significant relationship between the ferritin levels prior to allo-HSCT and the maximum ferritin levels after alloHSCT. We analysed overall survival of patients with high/very high ferritin levels (5000μg/l or more) vs. patients with low/medium ferritin levels (<5000μg/l) after allo-HSCT. Median follow-up time was 661 days. We found that overall survival in patients with high ferritin levels was significantly lower than in the low/medium ferritin group (Fig. 1A). Of note, none of the patients with Ferritin levels >10.000μg/l had longterm survival. This was due to increased non-relapse mortality (Figure 1B). Next, we were interested to find out if high ferritin levels after allo-HSCT were associated with acute GvHD. For analyses we created three cohorts of allo-HSCT recipients according to their clinical aGvHD grade: 1) No aGvHD, 2) Grade I-II and 3) Grade III-IV. We then picked the maximum serum ferritin level of each patient after allo-HSCT. Figure 2 shows the distribution of ferritin levels between the cohorts. We found that maximum ferritin levels after allo-HSCT were significantly higher in patients during aGvHD than in patients without aGvHD (Figure 2A). Of note, very high ferritin levels (>10000μg/l) occurred exclusively in patients during aGvHD; they were not observed in the absence of aGvHD (Figure 2A). Interestingly, we did not observe an association between ferritin levels and chronic GvHD (Figure 2B). Importantly, mean ferritin levels taken prior to alloHSCT did not differ significantly between allo-HSCT recipients who later developed no aGvHD (1194 SEM ± 268.2), aGvHD Grade I-II (1295 SEM ± 174.7) or aGvHD Grade III+IV (1194 ± 268.2) (P=0.61). We were then interested to establish if we were able to detect hemophagocytes in patients with hyperferritninaemia after allo-HSCT. The rationale is that hyperferritinemic syndromes are usually characterized by macrophage activation and by hemophagocytosis. The standard technique to visualize hemophagocytosis is assessment of BM aspirates. BM aspirates were available in 9 out of 11 allo-HSCT recipients who developed ferritin levels above 10000μg/l during aGvHD. In one of these specimens, many siderophages were found; however, there was no clear evidence of hemophagocytosis. The remaining 8 specimens showed clear hemophagocytosis in the BM (Figure 2C). We conclude that very high ferritin levels after allo-HSCT are associated with hemophagocytosis. To investigate if aGvHD is associated with