Purpura Fulminans Triggered by the Formation of Anti-Endothelial Autoantibodies in a Patient with Chronic Lymphocytic Leukemia

    

    

    Figure 1: Purpura fulminans (PF) triggered by the formation of anti-endothelial IgG autoantibodies in a patient with chronic lymphocytic leukemia (CLL).
A) Representative images of the patient’s cutaneous lesions. On the left side, an image from the initial dermal lesion at the right upper leg is shown. The image on
the right side was taken postmortem. B) Histopathologic analysis of dermal lesions revealed cutaneous vasculopathy with thrombotic occlusions of microvessels
by hyaline thrombi (arrows), fibrinoid necrosis of small blood vessels, and hemorrhages (stars). Magnification 100×. C) IgG from the patient or a healthy control
was purified from serum and analyzed for binding to hybridoma EA.hy926 endothelial cells by indirect flow cytometry. D, E) Before and after initiation of plasma
exchange therapy (PET), patient IgG was analyzed for binding to EA.hy926 cells by flow cytometry (D), and thrombin generation was measured in patient plasma
in the presence (dashed lines) or absence (solid lines) of EA.hy926 cells using a modified fluorogenic assay (E).
    

Immunosuppressive therapy was continued with cyclophosphamide, and plasma exchange therapy (PET) was initiated based on the identification of anti-endothelial antibodies. Skin lesions did not further progress, and the patient’s clinical condition temporarily improved. Consistently, binding of patient IgG to EA.hy926 cells was markedly decreased following PET (Figure 1D). A few weeks later, however, the patient died from septic shock during cyclophosphamide-induced neutropenia.

We performed additional experiments to further investigate an autoimmune mechanism interfering with the endothelial protein C-protein S-thrombomodulin (PC-PS-TM) system. To this end, we used a modified fluorogenic thrombin generation assay, which assesses thrombin generation in the presence of EA.hy926 cells, as previously described [4]. In this assay, thrombin generation is highly regulated by the EA.hy926 PC-PS-TM system. In patient plasma obtained after PET, addition of EA.hy926 cells almost completely abolished tissue factor (TF)-triggered thrombin generation (Figure 1E). In patient plasma obtained before PET, however, this effect was significantly less pronounced (Figure 1E), suggesting that patient IgG attenuated the inhibitory effect of EA.hy926 cells on TF-triggered thrombin generation. Interestingly, patient IgG neither bound to immobilized soluble TM (sTM) in an in-house solid-phase ELISA (Supplementary Figure 2A) nor affected the anticoagulant activity of sTM in the thrombin generation assay in the absence of EA.hy926 cells (Supplementary Figure 2B).

Discussion/Conclusion

PF is a rare, but life-threatening manifestation of DIC and characterized by thrombotic occlusion of cutaneous microvessels with subsequent hemorrhagic tissue necrosis [5,6]. In most cases, PF is triggered by severe sepsis, but it is also associated with functional defects of the PC-PS-TM system [5,6].

Our patient, however, developed PF most likely triggered by the formation of anti-endothelial IgG autoantibodies, which functionally interfered with the PC-PS-TM system, in close association with the onset of CLL. Autoimmune complications are frequent in this hematological malignancy and affect up to 10% of all patients [2,7,8], but to the best of our knowledge this is the first report of autoimmunemediated PF in a CLL patient.

In our patient, AIHA, ITP and PF were the initial findings of the underlying lymphoproliferative disease. It is well-known that autoimmune complications in CLL can manifest at any disease stage [7] and might even precede overt CLL in a minority of patients [8]. Of note, especially non-hematological phenomena are linked to initial disease stages [2]. Nevertheless, vascular complications are rather uncommon in CLL [2], but have been reported in several cases [9- 11]. Although the most predominant finding was leukocytoclastic vasculitis [9-11], Yamac et al. reported a case of PF in a CLL patient almost 20 years ago [12].

While in the latter case [12], the pathophysiology of PF remained obscure, we identified anti-endothelial IgG autoantibodies (Figure 1C), which functionally interfered with the PC-PS-TM system on EA.hy926 endothelial cells (Figure 1E). Under physiological conditions, the endothelial PC-PS-TM restricts coagulation to the site of vessel wall injury [13], but severe acquired or inherited deficiencies of PC and PS, including formation of inhibitory PC or PS autoantibodies in rare occasions, have already been linked to PF [5,6,14,15]. In these patients, isolated skin manifestations of the lower extremities sparing the acral regions are common [16]. A similar distribution pattern was found in our patient at initial presentation (Figure 1A), further supporting our hypothesis of autoimmunemediated PF. Moreover, slightly elevated antinuclear antibody (ANA) titers compatible with an autoimmune epiphenomenon were measured (Table 1).

The pathophysiology of PF is complex and likely involves a plethora of mechanisms, including vascular congestion and disturbed endothelial integrity [17]. While severe sepsis and overt DIC are frequently observed [5,6,17], they were absent in the report from Yamac and colleagues [12] and in our patient at initial presentation (Table 1). It is thus tempting to speculate that the pathophysiology of PF in CLL patients might be distinct from other common conditions. Of note, we have recently demonstrated the serious consequences of the formation of a function-blocking anti-PS antibody in a woman with primary APS [18]. The patient suffered from overt DIC, recurrent arterial and venous thrombosis, and a highly painful livid skin erythema triggered by antiphospholipid antibody-mediated coagulation activation. Moreover, a multitude of procoagulant mechanisms have been delineated for anti-endothelial antibodies, such as release of pro-inflammatory cytokines, upregulation of adhesion molecules or induction of TF [19]. Interestingly, elevated von Willebrand factor levels were measured in our patient (Table 1). It is thus at least conceivable that additional IgG-related effects with activation of endothelial cells contributed to the procoagulant state, and that local or low-grade systemic coagulation activation was sufficient to trigger PF.

Although we could not identify the specific target of the antiendothelial antibodies, we ruled out direct interference with TM (Supplementary Figure 2A). Moreover, the function of both, PC and PS, was intact as evidenced by the inhibitory effect of soluble TM on thrombin generation in patient plasma (Supplementary Figure 2B). In addition, both proteins were in the respective reference ranges (Table 1). Thus, we suggest the formation of anti-endothelial autoantibodies, which functionally interfered with the anticoagulant PC-PS-TM system independently of PS, PC and TM. Interestingly, recent studies have identified an increased risk for venous and arterial thrombosis in patients with anti-endothelial antibodies directed against the endothelial protein C receptor (EPCR) [20,21]. Moreover, in a 74-year-old woman, the idiopathic development of such an antibody was associated with thrombotic skin necrosis [22]. Routine laboratory assessment of PC, PS and TM was unremarkable in this patient [22]. It is thus tempting to speculate that the EPCR might have been a potential target of the anti-endothelial IgG autoantibodies in our patient.

Current treatment guidelines still recommend corticosteroids in patients with isolated CLL-associated autoimmune cytopenia (AIC) as first line therapy, but specific recommendations for treatment of other CLL-associated autoimmune phenomena are lacking [2,3]. Secondline treatment options include anti-CD20 monoclonal antibodies, such as rituximab, or immunosuppressive agents, like cyclophosphamide [2,3]. Several studies have also indicated promising efficacy and safety for the Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib, in treatment of both, corticosteroid-responsive and refractory AIC [23- 25], but at the time point of our case presentation, these data have not yet been available, and ibrutinib was not approved for treatmentnaïve CLL patients. Our patient, however, showed at least temporarily a good clinical response to immunosuppressive therapy with both, corticosteroids and cyclophosphamide, in combination with PET. We also discussed escalation of immunosuppressive therapy by the addition of rituximab, but the patient died before rituximab could be initiated.

Taken together, autoimmune-mediated PF may occur in CLL patients in rare occasions. Rapid identification and initiation of adequate therapy are of utmost clinical importance in these patients. In addition to therapeutic anticoagulation and local wound management, antibody depletion by immunosuppressive therapy and PET might be a promising therapeutic approach.

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