Emicizumab, the bispecific antibody to factors IX/IXa and X/Xa, potentiates coagulation function in factor XI-deficient plasma in vitro.

Essentials Emicizumab mimics factor (F)VIIIa cofactor function, augments the intrinsic tenase activity. We assessed the emicizumab-driven hemostatic function in FXI-deficient plasmas. Emicizumab improved the coagulation potentials in severe FXI-deficient plasma. Emicizumab may provide a possibility for clinical application in patients with FXI deficiency. SUMMARY: Background Patients with factor (F)XI deficiency commonly present with markedly prolonged activated partial thromboplastin times (APTT), although bleeding phenotypes are heterogeneous. Emicizumab, a bispecific monoclonal antibody to FIX/FIXa and FX/FXa, mimics FVIIIa cofactor function on phospholipid (PL) surfaces. Antibody reactions were designed, therefore, to augment mechanisms during the propagation phase of blood coagulation. Aim To assess emicizumab-driven hemostatic function in FXI-deficient plasmas. Methods and Results Standard ellagic acid (Elg)/PL-based APTTs of different FXI-deficient plasmas (n = 13; FXI activity, < 1 IU dl-1 ) were markedly shortened dose dependently by the presence of emicizumab. To further analyze the effects of emicizumab, clot waveform analysis (CWA) in FXI-deficient plasmas with emicizumab, triggered by tissue factor (TF)/Elg demonstrated improvements in both clot times, reflecting the initiation phase, and coagulation velocity, which represents the propagation phase. Emicizumab also enhanced the TF/Elg-triggered thrombin generation in FXI-deficient plasmas dose-dependently although the degree of enhancement varied in individual cases. Thrombin generation with either FVII-deficient plasma or FIX-deficient plasma treated with anti-FXI antibody showed little or no increase by the co-presence of emicizumab, suggesting that the accelerated thrombin generation in FXI-deficient plasmas by emicizumab should depend on the FIXa-involved coagulation propagation initially triggered by FVIIa/TF. The ex vivo addition of emicizumab to whole blood from three patients with severe FXI deficiency demonstrated modest, dose-dependent improvements in Ca2+ -triggered thromboelastograms (NATEM mode). Conclusion Emicizumab appeared to improve coagulation function in severe FXI-deficient plasma, and might provide possibilities for clinical application in patients with FXI deficiency.

Routine measurements of factor VIII activity and inhibitor titer in the presence of emicizumab utilizing anti-idiotype monoclonal antibodies.

Essentials Emicizumab (Emi) affects the APTT-based assays of factor (F)VIII activity and inhibitor titer. A mixture of two anti-Emi monoclonal antibodies (mAb) effectively neutralized the Emi activity. Anti-Emi mAbs completely eliminated the influence of Emi on FVIII activity and inhibitor titer. The inclusion of anti-Emi mAbs in routine FVIII assays would be useful for Emi-treated patients. SUMMARY: Background Emicizumab is an anti-factor (F)IXa/X bispecific monoclonal antibody (mAb), mimicking the factor (F)VIIIa cofactor activity. Emicizumab does not require activation by thrombin and its shortening effect on the activated partial prothrombin time (APTT) is more pronounced than that of factor (F)VIII. APTT-based FVIII activity (FVIII:C) and FVIII inhibiter titer measurements are influenced by the presence of emicizumab. Aim To establish a reliable APTT-based assay to measure FVIII in the presence of emicizumab. Methods Plasmas from hemophilia A (HA) patients without or with inhibitors were studied using one-stage FVIII:C and Bethesda inhibitor assays. Two recombinant anti-idiotype mAbs to emicizumab (anti-emicizumab mAbs) were prepared, rcAQ8 to anti-FIXa-Fab and rcAJ540 to anti-FX-Fab. Results The combined anti-idiotype mAbs (2000 nm each) eliminated the effects of emicizumab on APTTs of HA plasmas without or with inhibitor by competitive inhibition of antibody binding to FIX(a)/FX(a). Measurements of FVIII coagulation activity in HA plasmas without inhibitor were overestimated in the presence of emicizumab (1 µm = ~150 µg mL-1 ) at all reference levels of FVIII. The addition of anti-emicizumab mAbs to the assay mixtures completely neutralized the emicizumab and facilitated accurate determination of FVIII:C. Anti-FVIII inhibitor titers were undetectable in the presence of emicizumab in HA plasmas with inhibitor or normal plasmas mixed with anti-FVIII neutralizing antibodies. These effects of emicizumab were completely counteracted by the addition of the anti-idiotype mAbs, allowing accurate assessment of inhibitor titers. Conclusion The in vitro inclusion of anti-emicizumab mAbs in the standard one-stage coagulation assays prevented interference by emicizumab and enabled accurate measurements of FVIII:C and inhibitor titers.

Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti-factor IXa/factor X bispecific antibody emicizumab.

Essentials The activated partial prothrombin time (aPTT) cannot predict the activity of emicizumab (Emi). Adjusted clot waveform analyses using a prothrombin time (PT)/aPTT initiator were developed. Activity of Emi in the co-presence of factor VIII or bypassing agents was quantified. This assay is useful for assessing coagulation potential in Emi-treated hemophilia A. SUMMARY: Background Emicizumab is an anti-activated factor IX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophilia A (HA) patients treated with emicizumab. Aim To evaluate the global coagulation potential of emicizumab. Methods Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted-|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process. Results Ad|min1| obtained with mixed-trigger reagent (PT/APTT/buffer, 1 : 15 : 135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2-0.4 IU dL-1 equivalent FVIII per 1 µg mL-1 emicizumab). Ex vivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration-dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose-dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose-dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII. Conclusion The PT/APTT reagent-triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab-treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

BACKGROUND: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. OBJECTIVES: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. METHODS: A non-human primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. RESULTS: A single bolus of 1 or 3 mg kg-1 ACE910 showed hemostatic activity comparable to that of 10 U kg-1 (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. CONCLUSIONS: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

BACKGROUND: We previously reported that a humanized anti-factor IXa/X bispecific antibody, hBS23, mimics the function of FVIII even in the presence of FVIII inhibitors, and has preventive hemostatic activity against bleeding in an animal model of acquired hemophilia A. After further molecular engineering of hBS23, we recently identified an improved humanized bispecific antibody, ACE910, for clinical investigation. OBJECTIVES: To elucidate the in vivo hemostatic potency of ACE910 by examining its effect against ongoing bleeds, and to determine its pharmacokinetic parameters for discussion of its potency for prophylactic use. METHODS: A nonhuman primate model of acquired hemophilia A was established by injecting anti-primate FVIII neutralizing antibody. When bleeds emerged following an artificial bleed-inducing procedure, either ACE910 or recombinant porcine FVIII (rpoFVIII) was intravenously administered. rpoFVIII was additionally administered twice daily on the following 2 days. Bleeding symptoms were monitored for 3 days. A pharmacokinetic study and multiple-dosing simulations of ACE910 were also performed. RESULTS: A single bolus of 1 or 3 mg kg⁻¹ ACE910 showed hemostatic activity comparable to that of 10 U kg⁻¹ (twice daily) rpoFVIII against ongoing bleeds. The determined ACE910 pharmacokinetic parameters included a long half-life (3 weeks) and high subcutaneous bioavailability (nearly 100%). The simulation results based on pharmacokinetic parameters indicated that the above hemostatic level could be maintained with once-weekly subcutaneous administration of ACE910, suggesting the possibility of more effective prophylaxis. CONCLUSIONS: ACE910 may offer an alternative on-demand treatment option for patients with hemophilia A, as well as user-friendly and aggressive routine supplementation.

Interactions between residues 2228-2240 within factor VIIIa C2 domain and factor IXa Gla domain contribute to propagation of clot formation.

Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 domain (residues 2228-2240) interacts with the FIXa Gla domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228-2240. The peptide 2228-2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 µM). This effect was significantly greater than that obtained by peptide 1804-1818 (IC50; ~180 µM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228-2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228-2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 domain (residues 2228-2240) with the FIXa Gla domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.

[Median re-sternotomy for aortic valve re-replacement assisted by video-assisted thoracic surgery].

Cardiac reoperation via a median re-sternotomy is associated with a high risk of injury to cardiac structures and the great vessels, and may result in massive bleeding. We report a case of aortic valve re-replacement, and severe adhesion was suspected between the sternum and the left brachiocephalic vein by preoperative computed tomography (CT) scans. To avoid injury to the vein, the adhesive tissue was dissected under video-assisted thoracic surgery (VATS). Then median re-sternotomy was performed safely, and the aortic valve was replaced again. The patient's postoperative course was uneventful. Since sternal adhesions are checked and dissected visually, concomitant VATS might be a very useful option after previous cardiac surgery.

Mechanisms of factor VIIa-catalyzed activation of factor VIII.

BACKGROUND: Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood. OBJECTIVES: In the present study, we investigated the precise mechanisms of FVIIa/TF-catalyzed FVIII activation. RESULTS: FVIII activity increased ~4-fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nM), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg(740) and Arg(372) more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg(336) was evident at ~1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu-Gly-Arg-active-site-modified FVIIa (K(d), ~0.8 nM) with a higher affinity for the HCh than for the LCh (K(d), 5.9 and 18.9 nm). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF-catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF-mediated reactions. CONCLUSIONS: The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a 'priming' mechanism for the activation of FVIII in the initiation phase of coagulation.

Factor XI contributes to thrombus propagation on injured neointima of the rabbit iliac artery.

BACKGROUND: Thrombus formation through the activation of tissue factor (TF) and factor (F) XI is a critical event in the onset of cardiovascular disease. TF expressed in atherosclerotic plaques and circulating blood is an important determinant of thrombogenicity that contributes to fibrin-rich thrombus formation after plaque disruption. However, the contribution of FXI to thrombus formation on disrupted plaques remains unclear. METHODS: A mouse monoclonal antibody against FXI and activated FXI (FXIa) (XI-5108) was generated by immunization with activated human FXI. Prothrombin time (PT), activated partial thromboplastin time (APTT), bleeding time, and ex vivo platelet aggregation in rabbits were measured before and after an intravenous bolus injection of XI-5108. We investigated the role of FXI upon arterial thrombus growth in the rabbit iliac artery in the presence of repeated balloon injury. RESULTS: The XI-5108 antibody reacted to the light chain of human and rabbit FXI/FXIa, and inhibited FXIa-initiated FXa and FXIa generation. Fibrin-rich thrombi developed on the injured neointima that was obviously immunopositive for glycoprotein IIb-IIIa, fibrin, TF, and FXI. Intravenous administration of XI-5108 (3.0 mg kg(-1)) remarkably reduced thrombus growth, and the APTT was significantly prolonged. However, PT, bleeding time and platelet aggregation were not affected. CONCLUSIONS: These results indicate that plasma FXI plays a potent role in thrombus growth on the injured neointima. Inhibition of plasma FXI activity might help to reduce thrombus growth on ruptured plaques without prolonging bleeding time.

[Acute type A aortic dissection combined with surgical treatment with acute myocardial infarction].

Acute myocardial infarction, as a result of coronary malperfusion caused by acute type A aortic dissection, has been identified as one of significant factors relating to operative mortality. This complication could be diagnosed with a combination of electrocardiography and echocardiography in acute phase. However, the indication of coronary angiography and/or intervention has been controversial as it is time-consuming and renders additional stress to a critical patient requiring an emergency operation. We report a case of myocardial infarction successfully treated with percutaneous transluminal coronary angioplasty (PTCA) at first, after that, recognition of dissection of aorta necessitated subsequent surgical therapy. In this particular case, coronary intervention in advance proved to be mandatory.

Cold induces shifts of voltage dependence in mutant SCN4A, causing hypokalemic periodic paralysis.

BACKGROUND: The authors reported a mutation, P1158S, of the human skeletal muscle sodium channel gene (SCN4A) in a family with cold-induced hypokalemic periodic paralysis (hypoKPP) and myotonia. OBJECTIVE: To identify mechanisms of temperature dependency in this channelopathy. METHODS: Using the amphotericin B perforated patch clamp method, sodium currents were recorded at 22 and 32 degrees C from the wild-type (WT) and P1158S mutant SCN4A expressed in tsA201 cells. Computer simulation was performed, incorporating the gating parameters of the P1158S mutant SCN4A. RESULTS: P1158S mutant SCN4A exhibited hyperpolarizing shifts in voltage dependence of both activation and inactivation curves at a cold temperature and a slower rate of inactivation than the WT. Computer simulation reproduced the abnormal skeletal muscle electrical activities of both paralysis at a low potassium concentration in the cold and myotonia at a normal potassium concentration. CONCLUSIONS: Both paralysis and myotonia are attributable to the biophysical properties of the SCN4A mutation associated with hypoKPP. This is the first report of an SCN4A mutation that exhibits temperature-dependent shifts of voltage dependence in sodium channel gating.

Soybean and milk proteins modified by transglutaminase improves chicken sausage texture even at reduced levels of phosphate.

Consumer demands for poultry processed meats have increased due to low fat content. In this experiment, chicken sausages were manufactured with various biopolymers prepared from soybean protein, casein, whey protein isolate (WPI), mixtures of soybean protein and casein, and soybean protein and WPI. The extent of various biopolymer formations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography. Cross-linking soybean protein and casein or WPI by transglutaminase provided biopolymers with improved heat stability and emulsifying property. Shear force of chicken sausages were measured to evaluate the addition of biopolymer on the hardness in the presence of 0.05 or 0.2% sodium tripolyphosphate (STPP). The texture of chicken sausages was improved by the addition of such biopolymers even in the presence of 0.05% STPP. These results suggested that chicken sausage texture was improved by the formation of network structures that contribute to hardness of sausage gels with the addition of biopolymers. Thus, addition of biopolymers in the manufacture of chicken sausages may permit reduction in phosphate content without loss in texture.

[A case report of Ebstein's anomaly treated with Hetzer's procedure].

A 27-year-old male who had been diagnosed with Ebstein's anomaly was admitted with uncontrollable congestive heart failure. The echocardiogram revealed severe tricuspid valve incompetence and the electrocardiogram showed atrial fibrillation. He underwent Hetzer's repair procedure for tricuspid valve incompetence and Minzioni's right atrial isolation technique to restore sinus rhythm. His congestive heart failure quickly disappeared and sinus rhythm was restored after operation. He was discharged 3 weeks postoperatively and remains well 22 months after his operation. Hetzer's technique for tricuspid valve repair in Ebstein's anomaly restructures the valve mechanism at the level of the true tricuspid anulus by using the most mobile leaflet for valve closure without plication of the atrialized chamber. We conclude that Hetzer's procedure is an effective operation for Ebstein's anomaly.

Surgical treatment of abdominal aortic aneurysms located close to the visceral arteries: report of three cases.

The standard surgical treatment for abdominal aortic aneurysms (AAA) is in situ replacement of the infrarenal aorta, which is associated with a low mortality rate. On the other hand, thoracoabdominal aortic aneurysms (TAA) remain a formidable challenge and the complications that can occur may be severe including neurologic dysfunction and renal failure. We report herein three cases of patients with AAA located very close to the visceral arteries, for which in situ replacement of the infrarenal aorta was not feasible due to severe inflammation and adhesion. Therefore, aortic stump closure and in situ bypass grafting was performed to avoid reconstruction of the visceral arteries. No major complications or operation-related deaths occurred. Thus, while in situ replacement is usually recommended over bypass grafting for patients whose aneurysms are located very close to the visceral arteries, aortic stump closure and in situ bypass grafting should be considered as a more effective surgical option.

Penetrating atherosclerotic ulcer at the proximal aorta complicated with cardiac tamponade and aortic valve regurgitation.

A 56-year-old man had a penetrating atherosclerotic ulcer originating in the proximal ascending aorta, which is an unusual case of penetrating aortic ulcer complicated with the aortic valve regurgitation and cardiac tamponade. This hemodynamically unstable patient was successfully treated by conservative management to control his blood pressure and was also monitored closely with follow-up imaging studies.

Antibody fragments as inhibitors of Japanese radish acid phosphatase.

VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.

Simultaneous measurements of lactate and blood flow during hypoxia and recovery from hypoxia in a localized region in the brain of the anesthetized rabbit.

We observed simultaneous changes in lactate level and regional blood flow (rBF) in the brain of the anesthetized rabbit by using localized proton magnetic resonance spectroscopy (1H MRS) and laser Doppler flowmetry. The volume of interest of 0.5 ml for 1H MRS contained mostly thalamic nuclei. During hypoxia peak area for lactate increased up to 57% of that from N-Acetylaspartate. While the rBF increased during hypoxia up to 260% of the control, oxygen delivery (rBF x arterial oxygen content) decreased. In the normoxic recovery period following hypoxia, the rBF recovered slowly and a consequent overshoot of oxygen delivery was observed. The multiple and stepwise linear regression analyses revealed that the averaged decrease in oxygen delivery during hypoxia was the most significant independent variable for the increase in lactate during hypoxia (correlation coefficient; r2 = 0.68) and also that the increase in lactate during hypoxia was the most significant independent variable for the time for half-recovery of rBF (r2 = 0.75). These results suggest that the increase in lactate during hypoxia is due to the deficiency of oxygen delivery and that the increase in lactate during hypoxia prolongs the period of enhancement of rBF during recovery from hypoxia.

Iliac artery dissection originating in a true aneurysm. A case report.

The spontaneous dissection of a peripheral artery not involving the aorta is rare and usually does not arise from a true aneurysm. This is the first report of a spontaneous external iliac artery dissection originating in a true aneurysm.

[A case report of huge bronchogenic cyst originated in the atrial septum].

Bronchogenic cysts of the heart are extremely rare neoplasma comprising only 1.3% of all the primary cardiac and pericardial tumors. We experienced a case of bronchogenic cyst originated in the interatrial septum that caused the marked swelling of the septum. An asymptomatic woman of 43 years of age was evaluated because of cardiac enlargement on the chest X-ray film. The examinations including cardiac catheterization indicated a left atrial tumor broadly based on the atrial septum. Surgical removal was attempted, and the tumor was revealed to be a cyst involving the whole atrial septum. The histological diagnosis was bronchogenic cyst. The surgery included resection of the cyst together with the septum that was reconstructed with Dacron patch. Postoperative course was smooth, and the patient has returned to normal daily life without any sign of recurrence for two years.