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N. Luebbering et al.
men. We hypothesize that this finding may be due to the frequent use of myeloablative preparative regimens in our practice for children with leukemia who have received extensive prior chemotherapy while non-myeloablative regimens are used for children with non-malignant disor- ders who have not received chemotherapy. Children who have received prior chemotherapy are likely to have a reduced mass of hematopoietic cells to be lysed by the preparative regimen and hence have less release of F-actin into the circulation. Similar findings have been described in the settings of acute liver failure and massive trauma, and low VDBP levels predict poor outcome in both these clinical settings, in agreement with our findings in this study.15,16,28-30 Our findings differ from reports of acute liver failure in that HSCT recipients generally have healthy liv- ers and we were able to see rapid rebound of VDBP levels after initial depletion.
In this paper we show for the first time that VDBP levels are associated with outcomes of HSCT. Our data show both an association between day 30 VDBP level and TA- TMA, and a strong association of day 100 level with NRM. Our data show a differential effect of the VDBP genotype on the early outcome of TA-TMA, with lower TA-TMA in Gc2 homozygous transplant recipients. A large number of prior epidemiological studies have identi- fied associations between VDBP polymorphism and assorted chronic and frequent diseases, including chronic obstructive lung disease,31 diabetes mellitus32 and coro- nary artery disease,33,34 supporting some functional impor- tance to the variants, although reproducibility of associa- tion studies is mixed. Despite the many association stud- ies, there have been few functional studies that offer insight into potential mechanisms. Gc1 and Gc2 alleles dif- fer by a single amino acid and by differential post-transla- tional glycosylation. Gc1s and Gc1f alleles differ by a sin- gle amino acid but are similarly glycosylated. The func- tional consequences of these changes are poorly under- stood, although in general Gc2 homozygotes do have modestly reduced VDBP levels. We speculate that the dif- ference in TA-TMA risk by genotype might relate to dif- ferential binding of actin by genotype, or differential clear- ance of actin-VDBP complexes. This question is a subject of ongoing investigation.
We recognize that the frequency of TA-TMA reported from our center is notably higher than in other reports. We reported a prospective study of systematic screening for TA-TMA and identified an incidence of TA-TMA of 39%.21 It is important to stress that this incidence includes a majority of cases with biological evidence of TA-TMA that will never require clinical treatment due to absence of organ injury. Such cases would not be discerned in the absence of prospective detailed screening of markers such as lactate dehydrogenase and schistocytes, at our center or at others. In the same work we describe an incidence of TA-TMA with evidence of organ injury worthy of consid- eration of treatment of approximately 15%. The general- izability of these data is an important question. Our center treats a disproportionate number of children with immune deficiency disorders such as hemophagocytic lymphohistiocytosis and Fanconi anemia that are associat- ed with increased risk of TA-TMA, amplifying incidence at our site. We have addressed the question of incidence of TA-TMA at other centers directly with collaborators by performing a multi-center study of prospective screening for TA-TMA, using the same screening algorithms. This
PBMTC/PALISI sponsored study included 13 diverse (large and small, academic and community) pediatric transplant centers transplanting for largely malignant dis- ease (personal communication, CE Dandoy, February 2020). Six hundred and fourteen children were studied and 18% met criteria for TA-TMA, demonstrating the importance of prospective observations to identify the fre- quency of TMA. We believe that these data support the high frequency of TA-TMA consistently observed at our center, although a significant proportion of cases observed in this way do not merit changes in therapy. Clinical con- text must be carefully considered in initiating and therapy for TA-TMA.
VDBP functions as an actin scavenger, and in this role has been shown to be key in recovery from massive cell lysis occurring in settings such as acute liver failure and massive trauma. Higher levels of VDBP are associated with improved survival in those clinical settings, likely because VDBP serves to remove the angiopathic molecule F-actin, similar to the findings in our study. F-actin is clear- ly identified as directly angiopathic in previous literature. Surprisingly, however, direct infusion of monomeric actin into dbp null mice showed increased pulmonary toxicity in the dbp deficient compared to dbp sufficient animals. This study raises concern that the angiopathic molecule might be the actin-VDBP complex, and not unbound free actin. This distinction is important because if unbound free actin is the toxic molecule, one might speculate that infusion of additional VDBP around the time of transplant would be beneficial. VDBP is an abundant protein that could be prepared and infused, in a similar manner to albumin. Our human data show consistent advantage for a higher level of VDBP, in contrast to the mouse study, and no association of adverse outcomes with high levels of cir- culating actin-VDBP complex. These disparate findings may reflect important differences between the mouse model and clinical HSCT.
We found considerable variation in the amount of actin- VDBP complex present at day 0 (the day of stem cell infu- sion, after completion of conditioning therapy), between individuals. The amount of complex present at day 0 could be influenced by many variables, including the amount of hematopoiesis present to be lysed (high in active leukemia, low in marrow failure), the VDBP level, the efficiency of the reticulo-endothelial system in remov- ing complex and of the liver in replacing VDBP and the intensity and speed of delivery of the preparative regimen so variability is perhaps not surprising. It is important to recognize that studies in human disease are challenged by heterogeneity of cases, often small sample size, and need for assessment in multiple sample sets to validate findings and establish whether observations are generalizable to all populations.
The benefit of VDBP appears to last longer than the early weeks after transplant when endothelial injury occurs, and in this context appear independent of vitamin D transport. A higher day 100 VDBP level was associated with significantly improved NRM, a time long after lysis of the host hematopoietic system and clearance of F-actin, but a critical time for recovery of the donor immune sys- tem and for ongoing healing of epithelial injury. VDBP is known to act as a so-called “macrophage activating” fac- tor, so we hypothesized that VDBP levels might favorably influence immune recovery and tissue healing in the later weeks after HSCT. We found changes in cytokine secre-
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haematologica | 2021; 106(5)