We have enclosed our article titled “Emerging Therapeutic and Preventative Approaches to Transplant-Associated Thrombotic Microangiopathy”.

Abstract

Purpose of Review:

Transplant associated thrombotic microangiopathy (TA-TMA) is a complication which can occur in both allogeneic and autologous hematopoietic cellular therapy (HCT) recipients and is associated with significant morbidity and mortality. While TA-TMA is a complex disease, there is emerging evidence that complement activation and endothelial dysfunction play a key role in the pathophysiology of the disease. The use of eculizumab has improved survival in patients with high risk and severe disease, but mortality rates in treated patients still exceed 30%, highlighting the need for novel approaches.

Recent Findings:

There are multiple ongoing and planned clinical trials investigating novel complement agents in TA-TMA and other TMAs. Drugs vary by targets of the complement system, mechanism, and form of administration. Clinical trial designs include single arm studies that span across multiple age groups including children, and double blind, randomized, placebo-controlled studies. These studies will provide robust data to inform the care of patients with TA-TMA in the future. In addition to multiple promising therapeutic agents, preventing TA-TMA is an emerging strategy. Agents known to protect the endothelium from damage and augment endothelial function by promoting anti-inflammatory and anti-thrombotic effects may have a role in preventing TA-TMA or ameliorating the severity, though additional studies are needed.

Summary:

Novel therapeutic agents for TA-TMA inhibition of the complement system are under investigation and prophylactic strategies of endothelial protection are emerging. Further understanding of the pathophysiology of the disease may identify additional therapeutic targets. Multi-institutional, collaborative clinical trials are needed to determine the safety and efficacy of these agents going forward.

Background

Transplant associated thrombotic microangiopathy (TA-TMA) is an increasingly recognized complication of allogeneic and autologous hematopoietic cellular therapy (HCT) associated with significant morbidity and mortality^1*,2*,3. TA-TMA shares the defining features of all TMAs: microangiopathic hemolytic anemia, thrombocytopenia, and organ dysfunction. Clinical manifestations of TA-TMA include hypertension, serositis, pulmonary hypertension, diffuse alveolar hemorrhage (DAH), renal insufficiency, posterior reversible encephalopathy syndrome (PRES) and gastrointestinal bleeding^3,4*. About 50% or more patients with TA-TMA develop a severe phenotype with multiorgan dysfunction that can progress to organ failure and death⁵. TA-TMA is primarily a clinical diagnosis as renal biopsies are often not obtained in suspected TA-TMA and there is no consensus on histologic criteria in other organs. While multiple diagnostic criteria have been proposed, the consensus definition requires that ≥4/7 features are met at 2 different time points: hemolytic anemia (or increased need for red blood cell transfusions), de novo thrombocytopenia (or increased need for platelet transfusions), elevated lactate dehydrogenase (LDH), schistocytes on the peripheral blood smear, proteinuria of ≥30 mg/dL in random urine, hypertension defined as ≥99^th percentile for age or greater than 140/90 in adults, and elevated soluble membrane attack complex (sC5b-9, ≥ 244 ng/mL)^6,7. A high degree of clinical suspicion is required to detect TA-TMA before the development of multiorgan dysfunction as these laboratory and clinical findings may be caused by other common HCT complications.

Given inherent limitations of the clinical diagnostic criteria and different screening practices, the incidence of TA-TMA varies. TA-TMA is thought to complicate 10–35% of allogeneic HCTs² and ~25% of pediatric autologous HCTs in children with neuroblastoma^8,9. Risk factors for TA-TMA can be grouped into patient characteristics, transplant characteristics, and complications post HCT. Patient characteristic risks for TA-TMA include: underlying diagnosis of HLH¹⁰, aplastic anemia¹¹, female sex¹², non-white race, and genetic variants in complement proteins¹³. Transplant characteristics include a second allogeneic HCT¹⁴, myeloablative conditioning regimen¹⁵, and HLA disparity¹⁶ and post-transplant complications of bacterial, viral and/or fungal infections², acute graft versus host disease (aGVHD)¹⁴, and the use of calcineurin inhibitors (CNI)¹⁷, particularly when combined with sirolimus¹⁸. This has led to the “three hit hypothesis” of TA-TMA genesis: hit 1) a predisposing endothelial injury and/or an underlying predisposition to complement activation, hit 2) receipt of chemotherapy which further damages the endothelium, and hit 3) additional insults like aGVHD or infection^4*.

TA-TMA confers an increased non-relapse mortality rate and decreased overall survival compared to patients without TA-TMA^7,19,20. In a prospective cohort, elevated sC5b-9 and proteinuria at the time of TA-TMA diagnosis was prognostic and associated with a 1-year OS rate of 16.7%, compared to 1-year survival rate of 100% in children with TA-TMA who did not have these features⁷. Thus, these markers are used most commonly for prognosis and treatment decisions. The “TMA index” (LDH IU/L divided by platelet count) is also prognostic, with patients scoring >100 having very poor outcomes²¹.

The pathophysiology of TA-TMA is not fully understood. It is thought to start with endothelial damage, leading to endothelial activation and a pro-coagulant, pro-inflammatory state with activation of complement and generation of microthrombi, leading to ongoing endothelial cell activation (Figure 2). Pathways in endothelial cell activation are largely derived from work in atherosclerosis, but there are some biomarker studies in TA-TMA patients that support similar processes²². The role of complement in the pathophysiology of the disease is suggested by an increased risk of TA-TMA in patients with complement genetic mutations¹³, perturbation in complement regulatory function, elevated levels of MASP-2^23**, significant increases in C3b and sC5b-9²⁴, and the therapeutic effect of eculizumab, a monoclonal antibody to C5. In a study of pediatric patients with high-risk TA-TMA (elevated sC5b-9 and proteinuria), the 1-year overall survival of patients treated with eculizumab was 66% compared to 16.7% in historical (untreated) controls^25**. Much of the literature on eculizumab use in TA-TMA is in pediatric cohorts. While responses to eculizumab have been reported in adults with TA-TMA, survival remains poor^26,27. Regardless of treatment, survivors of TA-TMA, have increased morbidity due to chronic kidney disease²⁰, persistent pulmonary hypertension²⁸, and neurologic sequelae²⁹.

Figure 2: Proposed pathophysiology of TA-TMA and potential therapeutic targets.

Proposed Pathophysiology of TA-TMA: Endothelial cells are damaged by chemotherapy given as part of the BMT, medications, and complications of BMT. Damaged ECs release ANG-2, leading to vascular destabilization, lead to decreased nitric oxide production (NO) and releases IL-8. Local recruitment of antigen presenting cells (APCs) and lymphocytes occurs due to the expression of increased adhesion molecules by activated APCs. APCs also express TNF-alpha, INF-gamma, IL-6, IL-8 and IL-12, further stimulating activation of T-cells and monocytes/macrophages. This stimulates neutrophils to release neutrophil extracellular traps (NETs). Decreased levels of NO leads to loss of suppression of complement and NETs activate complement. Activation of C3 leads to the formation of C3b, which binds to the endothelial surface leading to formation of the membrane attack complex (MAC) on the endothelial cells, causing additional endothelial cell damage. Tissue factor (TF), which is expressed on the cell surface binds factor VIIIA and von Willebrand factor (vWF) promoting activated platelets and subsequent thrombus formation. Microthrombi then lead to organ ischemia and end organ damage (e.g. renal failure). Potential prophylactic and therapeutic targets and agents are indicated. Figure was created using biorender.com.

Current Management Paradigm

The current management paradigm for TA-TMA first emphasizes preventing (when possible) and mitigating complications or medications that drive TA-TMA (Figure 1). Treating infections and aGVHD may decrease the severity of TMA by dampening the feedback loop of endothelial damage and inflammation. Removing calcineurin inhibitors (CNIs) may improve TA-TMA, by diminishing the direct toxic effects on the endothelium (studied in vitro)^{30,31 32}. However, this strategy is complicated in patients with concurrent aGVHD. Uncontrolled aGVHD is also a risk factor for the development of TA-TMA, and the period of decreased immunosuppression incurred while switching from a CNI to another agent may increase the severity of GVHD. In a small study, switching GVHD prophylaxis from a CNI to daclizumab resulted in improvement of TA-TMA in some patients; however mortality from aGVHD was unacceptable³³.

Figure 1: Current Management Paradigm.

TA-TMA is currently managed with supportive care of concurrent illnesses and complications, and in patients with high-risk TA-TMA or severe disease, eculizumab. Plasmapheresis and rituximab are generally not effective unless there is an autoantibody present.

Optimizing supportive care measures is recommended for associated complications of TA-TMA including: hypertension, acute kidney injury, pericardial or pleural effusion, PRES, DAH, and gastrointestinal bleeding. If patients have high titers of factor H autoantibodies, treatment with therapeutic plasma exchange (TPE) may be helpful³⁴, and treating with rituximab at the end of TPE can be considered³⁵. If there are no antibodies present, TPE and rituximab have limited efficacy³⁶. Eculizumab is recommended for patients with high risk features defined as elevated sC5b-9 (≥244 ng/mL) and proteinuria (>30 mg/dL)³⁷. Eculizumab may also be considered when patients have organ dysfunction and elevated sC5b-9 or proteinuria, or in those with progressive disease despite maximal supportive care. Eculizumab is dosed frequently early in the course of TA-TMA to achieve suppression of complement, with CH50 used as an indirect measure of complement blockade³⁸. After an early intense treatment period, eculizumab is spaced out over time. If the inciting factor has resolved, i.e., GVHD is quiescent or infection is treated, patients may be able to stop eculizumab without recurrent TA-TMA. More data is needed regarding the length of therapy, patients most likely to benefit, the pharmacokinetics of eculizumab in TA-TMA, and an ongoing phase 2 multi-institutional trial will likely provide important data to this effect (ClinicalTrials.gov: NCT03518203). Although eculizumab has shown success in some patients and generally been safe when prophylaxis against Neisserial bacterial infections are provided, it is expensive and nearly 40% do not respond and die^25**. Thus, new approaches are needed. Additional understanding of the pathophysiology, consensus on diagnostic criteria and response, and new diagnostic and prognostic biomarkers are critically important as additional therapeutic approaches emerge.

Emerging Therapeutic Approaches

There are multiple complement inhibitors under investigation that may have therapeutic implications in TA-TMA (Table 1). The agents currently FDA approved for paroxysmal nocturnal hemoglobinuria (PNH) or atypical hemolytic uremic syndrome (aHUS) include: eculizumab, ravulizumab, and pegcetacoplan. Medications with published data or current or planned trials specifically in TA-TMA include:

• Ravulizumab/Ultomiris® is a long acting humanized monoclonal C5 antibody, currently FDA approved for PNH and aHUS. There are two ongoing clinical trials in TA-TMA, one a phase 3 randomized, double blind, placebo-controlled trial (clinicaltrials.gov NCT04543591) and a phase 3 single arm study in pediatrics (clinicaltrials.gov NCT04557735). This is the first randomized trial in TA-TMA and is a critically important addition to the field of TA-TMA.

• OMS721/Narsoplimab® is a novel mannan-binding lectin-associated serine protease-2 (MASP-2) pathway inhibitor. A single arm phase 2 study of OMS721 demonstrated improved overall survival in adult patients with TA-TMA compared to a historic cohort and improvement in hematologic parameters^23**. The drug received FDA breakthrough approval, and the company has announced commitment to opening a pediatric trial in in the near future.

• Coversin/ Nomacopan® is a recombinant C5 inhibitor originally derived from the hematophagous tick with ongoing clinical trials in PNH and aHUS. Coversin overcomes variants in C5 that result in eculizumab resistance. In a case report, a child with eculizumab refractory TA-TMA responded to coversin³⁹. The company has announced a plan for clinical trials in both adults and pediatrics.

Table 1:

Complement agents currently with active clinical trials in TMAs or announced upcoming trials in TA-TMA

Agent	Target	Admin	Pharmaceutical Sponsor	TMA type	Current Active Studies	Age
Eculizumab/Soliris ®	mAb inhibits C5	IV	Alexion Pharmaceuticals	TA-TMA*	Phase 2 single arm	Any age
Ravulizumab/Ultomiris ®	mAb inhibits C5 (long acting, same epitope as eculizumab)	IV	Alexion Pharmaceuticals	TA-TMA*	Phase 3 Single arm Phase 3 DB RCT	≥1 month <18 years ≥12 years
Coversin/Nomacopan®	Recombinant protein, inhibits C5 & leukotriene B4	SQ	Akari Therapeutics	TA-TMA#	Announced in planning
OMS721/Narsoplimab®	Inhibits lectin pathway activation	IV	Omeros	TA-TMA^	Announced in planning
REGN3918	Inhibits C5	SQ	Regeneron	PNH	Phase 3 Single arm	Adults
LFG-316/Tesidolumab	Inhibits C5	IV	Novartis	PNH	Phase 2	Adults
ABP959	mAb inhibits C5 (biosimilar)	IV	Amgen	PNH	Phase 3 DB randomized crossover Study	Adults
SKY59/RO7112689/Crovalimab	mAb inhibits C5	SQ	Hoffmann-La Roche	aHUS, PNH	Phase 3, single arm	≥12 years
RA101495/Zilucoplan	Inhibit C5	SQ	Ra Pharmaceuticals	PNH	Phase 2	Adults
ALN-CC5/Cemdisiran	RNAi, silence hepatic C5 production	SQ	Alnylam	PNH	Phase 2	Adults
APL2/Pegcetacoplan	Pegylated peptide, inhibits C3	SQ	Apellis Pharmaceuticals, Inc.	PNH	Phase 3 DB RCT	Adults
LNP023/Iptacopan	Small molecule, inhibits factor B, preventing C3 convertase formation	oral	Novartis Pharmaceuticals	aHUS PNH	Phase 3 DB RCT	Adults
ALXN2040/Danicopan	Small molecule, inhibits factor D, preventing C3 convertase formation	Oral	Alexion Pharmaceuticals	PNH	Phase 2	Adults
Sutimlimab	Inhibits C1 protease activity	IV	Sanofi	CAD	Phase 2, Phase 3 DB RCT	Adults

^*completed studies in PNH and aHUS

^#completed study in PNH

^{^}completed study in aHUS

mAb- monoclonal antibody, IV- intravenous, SQ- sub cutaneous, DB RCT- double blind randomized control trial, PNH- paroxysomal nocturnal hemoglobinuria, aHUS- atypical hemolytic uremic syndrome, CAD- cold agglutinin disease

Agents currently investigated for other complement driven diseases, i.e., vasculitis or renal diseases, were not included in this review. Studies that were withdrawn on clinicaltrial.gov were also excluded.

Immune Effector Blockade:

Current understanding of the action of immune effector cells and subsequent inflammatory cytokines at the microvascular level in TA-TMA are derived from data generated primarily from atherosclerosis⁴⁰ and/or other TMAs⁴¹. There is emerging evidence from RNA sequencing data for an INF-gamma and activated complement loop in TA-TMA^42*. While elevated serum levels of inflammatory cytokines including TNF-alpha, INF-gamma, and IL-8 are reported in other HCT complications, to our knowledge elevation of different cytokines has not been described in TA-TMA. If these pathways are altered in TA-TMA and contribute to the disease pathogenesis, inhibitors including etanercept (TNF-alpha), infliximab (TNF-alpha), tocilizumab (IL-6), and emapalumab (INF-gamma) may be additional therapeutic options.

Defibrotide:

Defibrotide, an oligonucleotide derived from pig intestines and is currently FDA approved for the treatment of VOD with organ dysfunction. While its mechanism is not fully understood, defibrotide is thought to reduce endothelial activation and enhance fibrinolysis, exerting protective effects on the endothelium. There are retrospective reports using defibrotide to treat TA-TMA with overall survival rates ranging from 57%⁴³ to 77%⁴⁴. The duration of use of defibrotide varied considerably among the studies. Anecdotally, patients have developed TA-TMA while receiving defibrotide for VOD prophylaxis. Defibrotide is expensive but generally well tolerated and thus further studies evaluating the efficacy of defibrotide for TA-TMA may be warranted.

Recombinant thrombomodulin(rTM):

Thrombomodulin is a transmembrane glycoprotein that exerts antithrombotic, antifibrinolytic, anti-inflammatory, and cytoprotective properties⁴⁵. Activated neutrophils in endothelial activation cleave thrombomodulin, obviating the vasoprotective properties. rTM has mixed results in multiple clinical trials investigating the impact on sepsis and disseminated intravascular coagulation (DIC)^46,47 In small case series or reports, rTM has been used to treat TA-TMA with varying responses^48,49. While rTM has biologic features that make it a plausible therapeutic agent, there is currently limited data to support its use in TA-TMA.

Emerging Prophylactic Approaches

Preventing severe illness is easier than treating it, and thus prophylaxis is a keystone approach to multiple HCT complications including GVHD, infections and veno-occlusive disease (VOD). While some risk factors of TA-TMA are not modifiable, like race or donor match, there is evidence that general measures aimed at improving endothelial health are protective against other types of TMAs in animal models⁵⁰. Thus, there is biologic premise that endothelial protection may be an effective strategy to prevent TA-TMA in retrospective studies and series, in-vitro studies, and animal models (Figure 2).

Defibrotide:

Defibrotide is used for prevention of VOD in high risk HCT recipients^51–53. In vivo, defibrotide protects endothelial cells against damage from CNI induced apoptosis⁵⁴ and fludarabine⁵⁵. There are emerging studies that suggest the use of defibrotide may result in lower rates of acute GVHD^56,57 a known risk factor for the development of TA-TMA. Thus, defibrotide could be an effective agent for the prevention of TA-TMA via several mechanisms. The incidence of TA-TMA was not reported in previous clinical trials when defibrotide was administered for VOD prophylaxis. However, there is an ongoing single arm feasibility trial evaluating the use of defibrotide as prophylaxis for TA-TMA in children with high-risk disease (ClinicalTrials.gov NCT03384693). Further studies are warranted to investigate the role of defibrotide prophylaxis in TA-TMA and the effect if given only in the immediate post HCT period.

Vitamin D:

There is emerging data that Vitamin D plays a role in endothelial health by augmenting nitric oxide production and reducing IL-6 and vascular and intravascular cell adhesion molecule expression (VCAM and ICAM)⁵⁸. Vitamin D deficiency is linked to endothelial dysfunction as measured by flow mediated dilation⁵⁹ and linked to cardiovascular disease and atherosclerosis, also endothelial diseases. In-vitro vitamin D protects endothelial cells from radiation induced apoptosis⁶⁰. Some though not all prospective studies have shown vitamin D deficiency is associated with increased risk of death after HCT^61,62. To our knowledge, there are no published data to evaluate vitamin D deficiency prior to HCT as a risk factor for subsequent TA-TMA, though a phase 4 clinical trial is ongoing to test the safety and efficacy of a single high oral dose of vitamin D pre-HCT in children (Clinicaltrial.gov NCT03176849); secondary endpoints will capture the incidence of transplant complications. Given low cost, minimal side effects, and benefit to bone health, checking vitamin D levels prior to HCT and repleting deficiencies should likely be incorporated into practice, though the effect on TA-TMA risk is unknown.

Statins:

While the indication for statins is hypercholesteremia, they are also thought to improve endothelial cell function by increasing the bioavailability of nitric oxide, promoting reendothelialization, reducing oxidative stress, and inhibiting inflammatory responses⁶³. In an study in adult HCT, with retrospective diagnosis of TA-TMA, a multivariable analysis showed the use of pravastatin and ursodiol was associated with a decreased incidence of TA-TMA compared to an untreated historic cohort (HR 0.23, 95% CI 0.11–0.49)⁶⁴. Statins are relatively well tolerated with minimal side effects, and regularly used in the adult setting. Statins have limited dose formulations available, which could make their use in the pediatric setting challenging. Further, statins have multiple medication interactions which may complicate their use in the immediate peri-HCT period. Additional studies on the efficacy and safety of this approach are needed.

Eicosapentaenoic acid (EPA):

EPA is an omega-3 fatty acid thought to inhibit thrombosis, augment endothelial vasodilation via nitric oxide and suppress the production of inflammatory cytokines. In a small study, patients who were given EPA from day −21 to day +180 had a lower incidence of TA-TMA and improved survival compared to patients who did not receive EPA⁶⁵.

N-acetyl cysteine (NAC):

NAC is an antioxidant that removes reactive oxygen species by increasing glutathione biosynthesis. In a mouse model of shiga-toxin-induced hemolytic uremic syndrome, the administration of NAC was partially protective against the development of renal failure and death⁶⁶. In a retrospective study, a combination of EPA and NAC was administered to patients classified as high risk for developing TA-TMA (high risk if 3 of the following criteria are met: age>10 years, race ethnicity non-white, ABO blood group minor incompatibility, or haploidentical donor). Compared to historic high-risk controls, the incidence of TA-TMA among the EPA/NAC treated group decreased from 28.2% to 4.5%^11*. A phase 3, randomized, double blind placebo-controlled trial investigating NAC for prophylaxis of TA-TMA is ongoing (clinicaltrial.gov NCT03252925).

Complement blockade:

Agents that inhibit complement proteins are the leading area of growth in emerging therapeutic agents, but this strategy as a prophylactic approach has not been trialed. There is some evidence that elevation of sC5b-9 on day 28 compared to baseline levels prior to HCT are associated with the later development of TA-TMA⁶⁷. However, the effect (if any) of blocking complement pre-emptively is not known. This approach is expensive and currently lacks data. Additional investigation is needed to determine if activated complement early in the course of HCT is associated with later development of TA-TMA.

Conclusions

TA-TMA is associated with significant morbidity and mortality, and new approaches are needed. There are many promising novel complement blockade agents under investigation, and emerging approaches to prophylaxis. Further understanding of the pathophysiology and risk factors for the disease will inform additional prophylactic and therapeutic options. This field would benefit from specific consensus on diagnostic criteria and markers of prognosis to best define the population who would most benefit from novel therapies and pre-emptive approaches. Multi-institutional, collaborative clinical trials are then needed to develop these criteria, understand incidence and risk factors, and to critically evaluate the efficacy of therapies and side effects.

Key Points:

• Transplant associated thrombotic microangiopathy (TA-TMA) is a common, potentially severe complication of hematopoietic cellular therapy (HCT) thought to be driven by activated complement and endothelial dysfunction.

• Current standard of care in patients with high risk or severe disease includes supportive care and inhibition of the complement system with eculizumab, a monoclonal antibody to C5. While survival has improved with this approach, mortality rates still exceed 30%.

• Multiple clinical trials are announced or currently enrolling children and adults investigating novel complement inhibition agents in TA-TMA and other TMAs with complement activation.

• In addition to novel therapeutic agents, prophylactic approaches are emerging and merit further study.