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\begin{document}

\title{Thrombotic Microangiopathy: Multi-Institutional Review of Pediatric
Patients Who Underwent HSCT}



\author[1]{Archana Ramgopal}%
\author[2]{Shiva Sridhar}%
\author[2]{Jignesh Dalal}%
\author[1]{Ram Kalpatthi}%
\affil[1]{Children's Hospital of Pittsburgh of UPMC}%
\affil[2]{UH Rainbow Babies and Children's Hospital}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Thrombotic microangiopathy (TMA) is a rare but serious complication of
hematopoietic stem cell transplantation (HSCT). The purpose of our study
is to estimate the incidence and risk factors of TMA in 93 out of a
total of 12369 children (0.8\%) receiving HSCT. HHV6 infection was an
independent risk factor associated with increased mortality in patients
with TMA (Hazard Ratio: 2.86 {[}1.01, 8.39{]}, p=0.05), and our study
conducts a review of the literature with the association of HHV-6 and
complement activation. Studies exploring the pathophysiology of TMA and
its relationship to HSCT are needed to optimize the outcome of pediatric
patients.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Transplant-associated thrombotic microangiopathy (TA-TMA) is a rare but
serious complication of hematopoietic stem cell transplantation (HSCT).
As a part of the family of thrombotic endothelium disorders, the disease
causes microangiopathic hemolytic anemia leading to microvascular
thrombosis and fibrin deposition in the microvessels. Due to a lack of
clear diagnostic criteria, it is difficult to determine incidence and
mortality for the disease, but values are estimated to range from 3\% to
39\% for incidence and exceed 50\% for mortality
(\textsuperscript{1,2,3}).

Currently, research shows that TMA is induced by endothelial cell injury
which could be the result of either infection, graft-versus-host disease
(GVHD), chemotherapy or radiation.

Literature demonstrates increased incidence status post allogeneic HCST
when compared to its autologous counterpart
(\{\ldots{}\}\textsuperscript{4,5}). While a few case studies of
pediatric patients note that TA-TMA was preceded by a HHV6 infection,
there is an ultimate lack of data regarding HHV6 association with TA-TMA
(\textsuperscript{5}). The purpose of our study is to estimate the
incidence of TA-TMA in children receiving HSCT and reveal other
potential risk factors and its impact on healthcare outcomes in this
population.

\textbf{Methods}

We used the Pediatric Health Information System (PHIS), an electronic
database of children's hospitals in the US. The study was deemed exempt
by the local Institutional Review Board. De-identified patients under
the age of 21 who underwent HSCT at one of the 42 PHIS hospitals from
2000-2012 were analyzed using data abstracted with ICD-9 codes. All
statistical analyses were performed using SAS software, version 9.3 (SAS
Institute, Cary, NC) or the base R statistical package (R Foundation for
Statistical Computing, Vienna, Austria). All calculated \emph{P} values
were 2-sided, and \emph{P} \textless{} .05 were considered statistically
significant.

\textbf{Results}

From 2000 to 2012, a total of 12369 unique pediatric patients who
received HSCT were identified. Among these 93 (0.8\%) children were
identified to have the diagnosis of TMA. Overall, there was an
increasing trend of TMA diagnosis seen in our cohort over the years,
with the highest percent occurrence in 2012 (p = 0.0125).

In Table 1, we identified 12 cases of TMA (12.9\%) in patients who
received an autologous HSCT, 78 cases (83.9\%) in patients who received
an allogeneic transplant, and 3 cases (3.2\%) who were unspecified.
Furthermore, 25 cases of TMA (32.1\%) were from a bone marrow
transplant, 41 cases (52.6\%) were after a peripheral blood transplant,
and 12 cases (15.4\%) occurred after a cord blood transplant.

TMA was significantly associated with allogeneic HSCT
(p=\textless{}0.001), PBSCT (p=0.045), CMV (p=\textless{}0.001), HHV6
(p=\textless{}0.001), fungal infection (p=\textless{}0.001), GVHD
(p=\textless{}0.001) and VOD (p=0.01). Additionally, TMA was
significantly associated with hypertension (p=\textless{}0.001), and
renal failure (p=\textless{}0.001) (Table 1). Multivariate logistic
regression analysis of mortality using age, gender, HSCT type, CMV,
HHV6, fungal infection and plasmapheresis showed only HHV6 was an
independent risk factor associated with increased mortality in patients
with TMA (Hazard Ratio: 2.86 {[}1.01, 8.39{]}, p=0.05)

The mortality was significantly higher in patients with TMA compared to
those without (30.1\% vs. 12.2\%, p=\textless{}0.001, Table 1).
Additionally, median time (days) to mortality following HSCT was shorter
in patients with TMA than those without (754 {[}365, 1614{]} vs. 1439
{[}552, 2847{]}, p=\textless{}0.0001).

\textbf{Discussion:~}

Thrombotic microangiopathy is a severe complication seen following HSCT
in children, thought to be associated or preceded by graft-versus-host
disease (GVHD). We are currently working in the paradigm of a three-hit
hypothesis, where patients with underlying genetic factors (Hit 1) that
undergo conditioning regimens such as chemotherapy and/or radiation (Hit
2) and experience other potential sources of endothelial injury such as
infection, medication or GVHD symptoms (Hit 3) are more likely to
manifest the disease (\textsuperscript{6}).

As demonstrated by previous literature, our data show higher incidence
of transplant-associated thrombotic microangiopathy (TA-TMA) after
allogeneic HSCT when compared to its autologous counterpart
(\textsuperscript{7,8}). Prior literature has documented increased risk
of both acute and chronic GVHD associated with peripheral blood HSCT
(\textsuperscript{9,10}). Additionally, GVHD association with TA-TMA~is
both well-documented and in agreement with our data
(\textsuperscript{11,12}). We also discovered, in concordance with the
literature, that GVHD had a higher association with late TMA, defined as
TMA that presents 120 days after transplant, compared to early TMA (p =
0.03) (\textsuperscript{13}).

In chronic GVHD, the endothelium can be damaged as a result of cytotoxic
T-cell induced injury (\textsuperscript{14}). Other studies have also
shown that many of the immunosuppressive agents used to treat GVHD, such
as calcineurin inhibitors can cause damage to the endothelium
(\textsuperscript{15,16}). Treatments given following transplant such as
radiation and chemotherapy are also known to be associated with
development of TA-TMA (\textsuperscript{17--19}).

Our data shows a significant correlation between CMV, HHV6, and fungal
infections post-transplant and risk of TA-TMA (\textsuperscript{20}).
Interestingly, we also found that HHV6 infection increases both risk and
mortality in addition to risk of TA-TMA. HHV6 is known to directly
infect endothelial cells and inhibit angiogenesis
(\textsuperscript{21}). Several studies have noted elevated levels of
neutrophil extracellular traps (NETs) in patients with TA-TMA
(\textsuperscript{22}). Mechanistically, it is believed that IL-8 is
released by the damaged endothelial tissue, recruiting neutrophils that
will undergo netosis and form NETs. These NETs, known up-regulators of
complement, then induce complement activation on self-cells, leading to
TA-TMA. While normally NETs may be cleared, in patients with chronic
GVHD undergoing treatment, the endothelium is constantly being attacked
(\textsuperscript{23}). In patients additionally infected with HHV6,
angiogenesis is inhibited, affecting the body's ability to repair this
damage \textbf{(} \textsuperscript{24}\textbf{).}

HHV6 infection may simply play a role in TA-TMA via GVHD as many studies
identify HHV6 species B as a risk factor for acute GVHD after HSCT
(\textsuperscript{24}). It is also of specific interest that the
cellular receptor for HHV6B in humans is CD46 or Membrane Cofactor
Protein (MCP), a regulator of complement attack on self-cells. In
T-cells, it is documented that HHV6B infection leads to down-regulation
of MCP (\textsuperscript{25}). Although there is no data on MCP
expression on endothelial cells after HHV6B infection, it seems possible
that the same finding could hold true for both cell types, see proposed
mechanism in Figure 1 (\textsuperscript{26}). If MCP expression is
reduced, endothelial cells become more prone to damage via complement
attack. While more data is needed to corroborate this claim, HHV6
induced downregulation of MCP along with promotion of complement
signaling via NETs would serve to further expose endothelial cells to
damage complement attack and therefore promote development of TMA
(\textsuperscript{27}).

Limitations of the study relate to the age of the data collected between
2000 and 2012, however we believe that our discoveries remain relevant.
PHIS is an administrative database, therefore the patients studied here
were identified utilizing ICD-9 codes, which may not fully reflect all
complications.

\textbf{Conclusions}

Ours is a large cohort of TMA following HSCT in children. The prevalence
of TMA in our study is 0.8\% with an increasing trend in recent years.
The mortality in our pediatric TMA cohort is 30\% which is in contrast
to the higher mortality reported in previously published small case
series. HHV6 emerged as not only a risk factor for TMA but also
associated with increased mortality in these patients. Studies exploring
the pathophysiology of TMA and its relationship to other complications
of HSCT and to prove the effect of HHV6 on complement on a molecular
level are needed to optimize the outcome.

References

1. Jodele S, Davies SM, Lane A, et al. Diagnostic and risk criteria for
HSCT-associated thrombotic microangiopathy: a study in children and
young adults. \emph{Blood} . 2014;124(4):645-653.
doi:10.1182/blood-2014-03-564997

2. Schoettler M, Lehmann LE, Margossian S, et al. Risk factors for
transplant-associated thrombotic microangiopathy and mortality in a
pediatric cohort. \emph{Blood Adv} . 2020;4(11):2536-2547.
doi:10.1182/bloodadvances.2019001242

3. Epperla N, Li A, Logan B, et al. Incidence, Risk Factors for and
Outcomes of Transplant-Associated Thrombotic Microangiopathy. \emph{Br J
Haematol} . March 2020. doi:10.1111/bjh.16457

4. Davies JOJ, Hart AC, De La Fuente J, Bain BJ. Macrophage activation
syndrome and post-transplant microangiopathy following haploidentical
bone marrow transplantation for sickle cell anemia. \emph{Am J Hematol}
. 2018;93(4):588-589. doi:10.1002/ajh.24995

5. Matsuda Y, Hara J, Miyoshi H, et al. Thrombotic microangiopathy
associated with reactivation of human herpesvirus-6 following high-dose
chemotherapy with autologous bone marrow transplantation in young
children. \emph{Bone Marrow Transplant} . 1999;24(8):919-923.
doi:10.1038/sj.bmt.1702003

6. Dvorak CC, Higham C, Shimano KA. Transplant-Associated Thrombotic
Microangiopathy in Pediatric Hematopoietic Cell Transplant Recipients: A
Practical Approach to Diagnosis and Management. \emph{Front Pediatr} .
2019;7:133. doi:10.3389/fped.2019.00133

7. Ye Y, Zheng W, Wang J, et al. Risk and prognostic factors of
transplantation-associated thrombotic microangiopathy in allogeneic
haematopoietic stem cell transplantation: a nested case control
study.\emph{Hematol Oncol} . 2017;35(4):821-827. doi:10.1002/hon.2310

8. Sakellari I, Gavriilaki E, Boussiou Z, et al. Transplant-associated
thrombotic microangiopathy: an unresolved complication of unrelated
allogeneic transplant for hematologic diseases. \emph{Hematol Oncol} .
2017;35(4):932-934. doi:10.1002/hon.2346

9. Changsirikulchai S, Myerson D, Guthrie KA, McDonald GB, Alpers CE,
Hingorani SR. Renal thrombotic microangiopathy after hematopoietic cell
transplant: role of GVHD in pathogenesis. \emph{Clin J Am Soc Nephrol} .
2009;4(2):345-353. doi:10.2215/CJN.02070508

10. Stem Cell Trialists' Collaborative Group. Allogeneic peripheral
blood stem-cell compared with bone marrow transplantation in the
management of hematologic malignancies: an individual patient data
meta-analysis of nine randomized trials. \emph{J Clin Oncol} .
2005;23(22):5074-5087. doi:10.1200/JCO.2005.09.020

11. Keesler DA, St Martin A, Bonfim C, Seber A, Zhang M-J, Eapen M. Bone
Marrow versus Peripheral Blood from Unrelated Donors for Children and
Adolescents with Acute Leukemia. \emph{Biol Blood Marrow Transplant} .
2018;24(12):2487-2492. doi:10.1016/j.bbmt.2018.08.010

12. Gavriilaki E, Sakellari I, Anagnostopoulos A, Brodsky RA.
Transplant-associated thrombotic microangiopathy: opening Pandora's
box.\emph{Bone Marrow Transplant} . 2017;52(10):1355-1360.
doi:10.1038/bmt.2017.39

13. Heybeli C, Sridharan M, Alkhateeb HB, et al. Characteristics of late
transplant-associated thrombotic microangiopathy in patients who
underwent allogeneic hematopoietic stem cell transplantation. \emph{Am J
Hematol} . July 2020. doi:10.1002/ajh.25922

14. Biedermann BC, Sahner S, Gregor M, et al. Endothelial injury
mediated by cytotoxic T lymphocytes and loss of microvessels in chronic
graft versus host disease. \emph{Lancet} . 2002;359(9323):2078-2083.
doi:10.1016/S0140-6736(02)08907-9

15. Luft T, Dietrich S, Falk C, et al. Steroid-refractory GVHD: T-cell
attack within a vulnerable endothelial system. \emph{Blood} .
2011;118(6):1685-1692. doi:10.1182/blood-2011-02-334821

16. Garcia-Maldonado M, Kaufman CE, Comp PC. Decrease in endothelial
cell-dependent protein C activation induced by thrombomodulin by
treatment with cyclosporine. \emph{Transplantation} .
1991;51(3):701-705. doi:10.1097/00007890-199103000-00030

17. Uderzo C, Bonanomi S, Busca A, et al. Risk factors and severe
outcome in thrombotic microangiopathy after allogeneic hematopoietic
stem cell transplantation. \emph{Transplantation} . 2006;82(5):638-644.
doi:10.1097/01.tp.0000230373.82376.46

18. Hoorn CM, Wagner JG, Petry TW, Roth RA. Toxicity of mitomycin C
toward cultured pulmonary artery endothelium. \emph{Toxicol Appl
Pharmacol} . 1995;130(1):87-94. doi:10.1006/taap.1995.1012

19. Kohn S, Fradis M, Podoshin L, Ben-David J, Zidan J, Robinson E.
Endothelial injury of capillaries in the stria vascularis of guinea pigs
treated with cisplatin and gentamicin. \emph{Ultrastruct Pathol} .
1997;21(3):289-299. doi:10.3109/01913129709021925

20. Takatsuka H, Wakae T, Mori A, et al. Endothelial damage caused by
cytomegalovirus and human herpesvirus-6. \emph{Bone Marrow Transplant} .
2003;31(6):475-479. doi:10.1038/sj.bmt.1703879

21. Rizzo R, D'Accolti M, Bortolotti D, et al. Human Herpesvirus 6A and
6B inhibit in vitro angiogenesis by induction of Human Leukocyte Antigen
G. \emph{Sci Rep} . 2018;8(1):17683. doi:10.1038/s41598-018-36146-0

22. Arai Y, Yamashita K, Mizugishi K, et al. Serum neutrophil
extracellular trap levels predict thrombotic microangiopathy after
allogeneic stem cell transplantation. \emph{Biol Blood Marrow
Transplant} . 2013;19(12):1683-1689. doi:10.1016/j.bbmt.2013.09.005

23. Gloude NJ, Khandelwal P, Luebbering N, et al. Circulating dsDNA,
endothelial injury, and complement activation in thrombotic
microangiopathy and GVHD. \emph{Blood} . 2017;130(10):1259-1266.
doi:10.1182/blood-2017-05-782870

24. Phan TL, Carlin K, Ljungman P, et al. Human Herpesvirus-6B
Reactivation Is a Risk Factor for Grades II to IV Acute
Graft-versus-Host Disease after Hematopoietic Stem Cell Transplantation:
A Systematic Review and Meta-Analysis. \emph{Biol Blood Marrow
Transplant} . 2018;24(11):2324-2336. doi:10.1016/j.bbmt.2018.04.021

25. Santoro F, Kennedy PE, Locatelli G, Malnati MS, Berger EA, Lusso P.
CD46 is a cellular receptor for human herpesvirus 6. \emph{Cell} .
1999;99(7):817-827. doi:10.1016/S0092-8674(00)81678-5

26. Zipfel PF, Misselwitz J, Licht C, Skerka C. The role of defective
complement control in hemolytic uremic syndrome. \emph{Semin Thromb
Hemost} . 2006;32(2):146-154. doi:10.1055/s-2006-939770

27. Grivel J-C, Santoro F, Chen S, et al. Pathogenic effects of human
herpesvirus 6 in human lymphoid tissue ex vivo. \emph{J Virol} .
2003;77(15):8280-8289. doi:10.1128/jvi.77.15.8280-8289.2003\selectlanguage{english}
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