洛基维特单抗治疗皮肤肥大细胞增多症患犬的瘙痒

王帆

Lokivetmab therapy for pruritus in a dog with cutaneous mastocytosis

洛基维特单抗治疗皮肤肥大细胞增多症患犬的瘙痒

翻译：徐晋

Background – Cutaneous mastocytosis (CM) is a rare disease of dogs characterized by rash, pruritus and proliferation of mast cells in the skin. Oral H1 antihistamines are recommended as the treatment to control pruritus.

Hypothesis/Objective – To describe the effective treatment of pruritus associated with CM with lokivetmab in one dog.

Animal – A 4-year-old, spayed female cross-bred dog presented with severely pruritic, erythematous to pigmented macules and papules involving the ventral abdomen, interdigital skin, perivulval area and both pinnae; the pruritus had been unresponsive to treatment with antihistamines, prednisone and ciclosporin.

Methods and materials – Complete blood count and serum biochemistry, abdominal ultrasound, blood smear and skin cytological evaluation, PCR, histopathological and immunohistochemical examination of skin biopsies.

Results – Skin cytological evaluation revealed high numbers of uniform, heavily granulated mast cells;histopathological findings showed focal dermal proliferations of well-differentiated, uniform mast cells consistent with a low-grade mast cell tumour (MCT). Clinical staging revealed that the disease was confined to the skin. Mutations of c-kit exon 8 and 11 were not detected. Treatment was initiated with anti-canine-interleukin (IL)-31 monoclonal antibody lokivetmab; antihistamines were continued. The dog’s pruritus resolved within seven days and was maintained in remission over 15 months with once monthly lokivetmab injections; the skin lesions improved but did not resolve.

Conclusion and clinical importance – Lokivetmab treatment was effective in resolving and maintaining pruritus remission in this dog with widespread cutaneous mast cell disease. Whether CM in dogs represent a separate entity that should be distinguished from a low-grade MCT requires further investigation.

背景：皮肤肥大细胞增多症（CM）是犬的一种罕见疾病，其特征为皮疹、瘙痒和皮肤肥大细胞增生，推荐口服 H1抗组胺药作为控制瘙痒的治疗。

试验假设/目的：描述一个用洛基维特单抗有效治疗犬CM相关瘙痒的案例。

试验动物：一只4岁、雌性绝育混血犬出现重度瘙痒、红斑至色素沉着斑和丘疹，累及腹侧腹部、趾间皮肤、外阴周围区域和双耳廓；瘙痒对抗组胺药、泼尼松和环孢素治疗无反应。

方法与材料：全血细胞计数和血清生化、腹部超声、血涂片和皮肤细胞学、PCR、皮肤活检组织病理学和免疫组织化学检查。

试验结果：皮肤细胞学评价显示大量均匀、颗粒丰富的肥大细胞；组织病理学结果显示真皮中分化良好、均匀的肥大细胞局灶性增生，与低等级的肥大细胞瘤（MCT）一致。临床分期提示病变局限于皮肤。未检测到c-kit外显子8和11的突变。使用犬白细胞介素（IL）-31单抗洛基维特单抗开始治疗；期间抗组胺药继续使用。该犬的瘙痒症状在7天内消退，并通过每月一次洛基维特单抗注射在15个月内持续缓解；皮肤病变改善，但未消退。

结论和临床意义：洛基维特单抗治疗可有效缓解广泛性皮肤肥大细胞疾病患犬的瘙痒症状并维持其缓解效果。犬CM是否作为区别于低等级MCT的独立存在需进一步研究。

Introduction

简介

Cutaneous mastocytosis (CM) is a rare disease in humans characterized by accumulation of neoplastic mast cells in the skin. Because of the release of mast cell mediators, patients with CM can experience severe pruritus. Rare cases describing a disease in dogs that resembles human CM have been reported and they have been referred to as ‘mastocytosis’ to differentiate them from ‘true’ mast cell tumours (MCT). Oral H1 antihistamines successfully controlled pruritus in these animals.  

皮肤肥大细胞增多症（CM）是一种罕见的疾病，在人类的特点是以肿瘤性肥大细胞在皮肤聚集。由于肥大细胞介质的释放，CM患者可出现严重的瘙痒。现已报道了描述犬中类似人 CM 疾病的罕见病例，并将其称为“肥大细胞增多症”，以与“真正的”肥大细胞瘤（MCT）区分。在这些动物中，经口给予H1抗组胺药成功控制了瘙痒。

History

病史

A 4-year-old, 21 kg, spayed female cross-bred dog was presented to the University of Georgia Veterinary Teaching Hospital Dermatology Service with a three year history of progressive skin lesions and pruritus. When the dog was 1-year-old, the owner had observed multifocal cutaneous papules on the dog’s inguinal and perivulval area which progressed to involve the ventral abdomen and axillae. Histopathological findings were consistent with urticaria pigmentosa-like disease, a form of CM described in humans. Since then, the dog had been treated consecutively with oral antihistamines (diphenhydramine, 2 mg/kg two times daily; famotidine, 1 mg/kg once daily), prednisone (1 mg/kg tapered over 14 days, Roxane Laboratories; Columbus, OH, USA) and ciclosporin (5 mg/kg once daily for two months, Atopica, Elanco Animal Health; Greensboro, NC, USA) without improvement of pruritus or skin lesions.

一只4岁、体重21 kg的雌性已绝育混血犬因三年来进行性皮肤病变和瘙痒就诊于佐治亚大学动物医学教学医院皮肤科。该犬1岁时，主人在犬的腹股沟和外阴周围区域观察到多灶性皮肤丘疹，进行性累及腹侧腹部和腋窝。组织病理学结果与色素样荨麻疹疾病一致，这是在人医上CM的一种表现形式。此后，连续给予该犬口服抗组胺药（苯海拉明，2 mg/kg，每日两次；法莫替丁，1 mg/kg，每日一次）、泼尼松（1 mg/kg，14 天内逐渐减量）和环孢素（5 mg/kg，每日一次，持续两个月），瘙痒和皮肤病变无改善。

Physical findings

体格检查

Upon presentation to the authors, physical examination revealed no abnormalities apart from the skin lesions and severe pruritus, which the owner reported as 9 of 10 on the pruritus Visual Analog Scale (pVAS). Dermatological examination revealed multifocal to coalescing, pale to erythematous macules and papules involving both axillae and pinnae, the abdominal and interdigital skin, and the perivulval area (Figure 1a–c). In some areas, lesions progressed to confluent plaques and occasionally exhibited pigmentation. Palpation of the papules resulted in erythema and swelling of the skin (Darier’s sign; Figure 1d).

作者接诊时，体格检查发现除皮肤病变和重度瘙痒外无其他异常，主人报告的瘙痒视觉模拟评分 (pVAS) 为 9/10。皮肤检查：多灶性至聚结状、苍白至皮肤发红斑点及丘疹，累及双腋窝及耳廓、腹部及趾间皮肤、外阴周围（图1a-c）。在某些区域，病变进展为融合性斑块，有的表现出色素沉着。触诊丘疹部位出现皮肤发红和肿胀（丹宁征；图1d）。

Clinical staging and cytological evaluation

临床分期和细胞学评估

Clinical staging was performed to rule out systemic disease progression. Fine needle aspirates from a cutaneous papule revealed large numbers of uniform, heavily granulated mast cells (Figure 2). The dog’s CBC, biochemistry profile and an abdominal ultrasound were unremarkable. No circulating mast cells were detected on a blood smear. Given the lack of obvious lymph node, liver or spleen involvement, and the unremarkable CBC, a bone marrow aspirate was not considered meaningful and the disease was considered to be confined to the skin.

进行临床分期以排除系统性疾病进展。皮肤丘疹细针抽吸显示大量均匀、颗粒丰富的肥大细胞（图2）。该犬CBC、生化和腹部超声检查无明显异常。血涂片上未观察到循环中肥大细胞。考虑到无明显淋巴结、肝脏或脾脏被累及的表现，以及CBC无明显异常，认为骨髓穿刺无意义，疾病局限于皮肤。

Histopathological evaluation

组织病理学评价

Two papular skin lesions were submitted to the Michigan State University Veterinary Diagnostic Laboratory for a MCT prognostic panel and graded based on a two-tier grading scheme. The original skin biopsy from two years before also was submitted for comparison. Immunohistochemical evaluation for CD25 was included because it is routinely performed as a minor diagnostic criterion for human mastocytosis to differentiate reactive from neoplastic mast cells 1 (see Appendix S1). Tissue of anti- canine IgE induced skin lesions containing numerous non- neoplastic mast cells from a healthy research dog served as a control.

将两处丘疹性皮肤病变送至密歇根州立大学兽医诊断实验室MCT 预后小组，并根据两级分级法进行分级。同时附送两年前的皮肤活检原始样本进行比较。本次组织病理学检查纳入了CD25的免疫组化评价，因为其作为人肥大细胞增多症区分于肿瘤性肥大细胞的常规的次要诊断标准（见附录S1）。将来自健康试验犬的含有大量非肿瘤性肥大细胞的抗犬IgE诱导皮肤病变的组织作为对照。

The haematoxylin and eosin stained sections from the original skin biopsy and the newly collected lesions revealed focal and highly cellular, infiltrative population of very well-differentiated mast cells in the dermis (Figure 3a,b). The mitotic count was one in 10 high-power fields (×40 objective). Immunohistochemistry for KIT and Ki67 and silver staining for AgNORs was performed as described previously. Mast cells displayed weak cytoplasmic and rare stippled membrane-associated labelling for KIT (rabbit polyclonal anti-CD117 antibody, Agilent; Santa Clara, CA, USA) (Figure 3c). Staining for Ki67 (mouse monoclonal anti-Ki67 antibody, clone MIB-1, Agilent) showed strong nuclear labelling in an average of 15.2 cells per grid (Figure 4a). Staining for AgNOR showed an average of 1.29 positive AgNORs per nucleus (Figure 3d). The AgNOR x Ki67 score was low (19.61). The PCR of DNA extracted from formalin-fixed, paraffin-embedded skin revealed no c-kit mutations in exons 8 and 11. Modest numbers of mast cells displayed labelling for CD25 (mouse monoclonal anti-CD25 antibody, clone 4C9, Leica Biosystems Inc.; Buffalo Grove, IL, USA) (Figure 4b). CD25 staining also was observed in non-neoplastic mast cells of the control skin tissue (not shown).

原始皮肤活检样本和新采集病变样本的苏木精-伊红染色切片显示，真皮中存在局灶性和高度细胞浸润的分化良好的肥大细胞群（图3a，b）。高倍镜视野下（×40物镜）有丝分裂计数为 1/10。对KIT和Ki67的免疫组化和AgNORs的银染如前所述进行。肥大细胞显示出较弱的细胞质KIT标记和罕见的点状膜相关KIT标记（图3c）。Ki67染色显示在每个网格平均15.2个细胞中有较强的核标记（图4a）。AgNOR染色显示平均每个细胞核有1.29个AgNORs阳性标记（图3d）。AgNOR×Ki67 评分较低（19.61）。从福尔马林固定、石蜡包埋的皮肤中提取DNA的PCR显示外显子8和11无 c-kit 突变。中等数量的肥大细胞显示了CD25标记（图4b）。在对照犬皮肤组织的非肿瘤性肥大细胞中也观察到CD25标记（未展示）。

The cytomorphological, histopathological and immuno-histochemical findings were consistent with a low-grade MCT. Histopathological grading (low-grade) and c-kit mutation status (negative), respectively, were similar in the original skin biopsies obtained two years before.

细胞形态学、组织病理学和免疫组织化学结果与低等级MCT一致。与两年前采集的原始皮肤活检组织相比，组织病理学分级（低等级）和c-kit突变状态（未突变）均相似。

Treatment and outcome

治疗与转归

The dog was treated with monthly subcutaneous injections of lokivetmab (40 mg, anti-canine IL-31 mono-clonal antibody, Cytopoint, Zoetis; Parsippany, NJ, USA); diphenhydramine and famotidine were continued to counteract possible signs of mast cell degranulation. The dog’s pruritus resolved within seven days (pVAS score one of 10) and during a follow-up period of 15 months, clinical signs were in remission with once monthly lokivetmab injections. Although the skin lesions persisted, the flare episodes involving diffuse abdominal erythema with enlargement of skin lesions completely resolved.

该犬每月皮下注射一次洛基维特单抗（40 mg，犬IL-31单抗，赛妥敏）继续给予苯海拉明和法莫替丁以对抗肥大细胞脱颗粒可能造成的症状。瘙痒在7天内消退（pVAS 评分为 1/10），在15个月的随访期间，通过每月一次洛基维特单抗注射，临床症状在缓解。尽管皮肤病变持续存在，但涉及腹部弥漫性红斑伴皮肤病变扩大的发作完全消退。

Discussion

讨论

Although the stand-alone cytological and histopathological findings indicated a low-grade MCT, the clinical presentation of erythematous maculopapular lesions appearing at a young age, the long-term indolent behaviour, the absence of cutaneous nodules or masses typically seen with MCTs, and the apparent restriction of mast cell infiltration to the skin suggested a variant of CM in this dog.

尽管独立的细胞学和组织病理学结果提示为低等级MCT，但在年轻时出现红斑斑状丘疹病变的临床症状、长期惰性表现、无皮肤结节或肿块（MCT常见），以及肥大细胞浸润明显局限于皮肤，这些表明该犬所患为CM变异。

Based on the patterns of skin lesions, the WHO recognizes three major clinical manifestations of CM in humans: maculopapular type, diffuse CM and solitary mastocytoma of the skin. Maculopapular CM, historically termed “urticaria pigmentosa”, is the most common form of CM in humans characterized by typical brown discolouration of the disseminated maculopapular lesions. Rare reports about CM in dogs exist and revealed heterogeneous clinical presentations; dogs exhibited maculopapular lesions, plaques or nodules that were brownish, pale or erythematous. The reports commonly used the umbrella term “urticaria pigmentosa-like” disease when the lesions were nonpigmented. The present case had pale to erythematous maculopapular/plaque lesions with occasional pigmentation; therefore, based on the recent human CM findings, the dog was likely to be affected by a maculopapular form.

根据皮肤病变的模式，WHO记录了人类CM 的三大临床表现：斑丘疹型、弥散性CM和皮肤单发性肥大细胞瘤。斑丘疹型CM，过去称“色素性荨麻疹”，是人类最常见的CM形式，其特征为弥散性斑丘疹病变的典型棕色变色。关于犬CM现有罕见报道，并表现出临床特征的多样性：斑丘疹病变、斑块或结节，呈褐色、苍白或红斑。病变无色素沉着时，报道常用涵盖性术语“色素样荨麻疹”疾病。本病例有苍白至发红的斑丘疹/斑块病变，偶见色素沉着；因此，根据最新的人类CM研究成果，该犬可能为斑丘疹型。

Activating c-kit mutations in exons 8, 9, 10, 11 and 17 occur only in approximately one third of paediatric CM. A similar incidence of c-kit mutations is reported in canine MCTs, but these occur more commonly in high-grade tumours. Mutations of c-kit in human mast cells are distinct from dogs because they often affect the intracellular domain of the receptor, whereas in dogs they are found primarily in the juxtamembrane domain. This molecular difference could suggest a separate disease entity in dogs than in humans and further investigations of different signalling properties and whole-genome transcriptional profiles are needed. Although a report of CM in a young laboratory beagle revealed a mutation in exon 11 of c-kit, there were no mutations in c-kit found in the exons 8 and 11 amplified in the present case.

激活外显子8、9、10、11和17的c-kit基因突变仅发生在大约三分之一的儿童CM中。据报道，犬 MCT中c-kit 突变的发生率相似，但这些突变更常见于高等级肿瘤。人肥大细胞中c-kit的突变与犬不同，因为它们常影响受体的胞内结构域，而犬主要发生于近膜结构域。这种分子差异表明犬相对人类可能有一个独立的疾病存在，需要进一步研究不同的信号传导特性和全基因组转录谱。尽管关于一只年轻实验比格犬CM的报道揭示了c-kit外显子11的突变，在本病例扩增的外显子8和11没有发现c-kit突变。

Cutaneous mastocytosis occurs more commonly in children and, in contrast to adults, can regress spontaneously. Similarly, occurrence of skin lesions in five of six reported dogs, including the dog in the present case, was first documented in <1-year-old dogs. Spontaneous regression was reported in some dogs but not observed up to the time of writing this case.     

皮肤肥大细胞增多症更常见于儿童，与成人不同，可以自发消退。同样，在已报道的6只犬中，有5只在1岁前首次记录到出现皮肤病变（包括本例）。其中部分犬自发消退，但本病例患犬直至撰写本文时仍未观察到自发消退。

The treatment of mastocytosis involves control of the immediate, possibly severe signs regardless of the subtype. H1-antihistamines are commonly used for the reduction of pruritus whereas H2-antihistamines should prevent systemic effects. Previous canine CM reports showed good to excellent response to H1 and H2 blockers and resolution of lesions; in one dog lesions persisted but antihistamine controlled pruritus and recurrent flares. In humans, rapid resolution of maculopapular CM skin lesions is seen with topical glucocorticoids. Although systemic glucocorticoids are routinely integrated into the treatment of MCTs in dogs, data on their use in canine CM is scarce. Two dogs with CM showed excellent response to a combination of prednisone and antihistamines. In the present case, there was no resolution of pruritus and flares with any of these drugs.

无论何种亚型，肥大细胞增多症的治疗包括控制即刻的、可能是严重的症状。H1-抗组胺药常用于减轻瘙痒，而H2-抗组胺药用来预防全身反应。先前的报道显示CM患犬对H1和H2 阻断剂的反应良好至极佳，病变消退；其中1只犬病变持续存在，但抗组胺药控制了瘙痒和复发。在人医，外用糖皮质激素可使斑丘疹型CM的皮肤病变迅速消退。尽管全身给予糖皮质激素常规用于犬MCT的治疗，但其用于犬CM的数据很少。2只CM患犬对泼尼松和抗组胺药联合治疗有极好的反应。在本病例中，使用上述药物后瘙痒和潮红均未见消退。

Interleukin-31 is produced mainly by activated T cells, but also by mast cells, and is a critical pruritogenic cytokine associated with atopic dermatitis(AD) in humans and dogs. Pruritus also affects the majority of humans with systemic mastocytosis and a positive correlation between serum IL-31 levels and pruritus is found in these patients. Lokivetmab, an antibody against canine IL-31, is approved to treat pruritus associated with AD in dogs. Lokivetmab binds to circulating IL-31, inhibiting its binding to the IL-31 receptor on sensory nerves. In this dog with CM, Lokivetmab provided quick onset and resolved pruritus without any adverse effects. Maculopapular lesions persisted, but there were no flares during the treatment.

白细胞介素-31主要由活化的T细胞产生，但也可由肥大细胞产生，是与人和犬特应性皮炎（AD）相关的关键致痒细胞因子。瘙痒也影响到大多数系统性肥大细胞增多症患者，在这些患者中发现血清IL-31水平与瘙痒呈正相关。洛基维特单抗是一种抗犬IL-31抗体，获批用于治疗犬AD相关瘙痒。洛基维特单抗与循环中IL-31结合，抑制其与感觉神经上的IL-31 受体结合。在本病例中，洛基维特单抗快速起效，缓解瘙痒，且无任何不良反应。斑丘疹病变持续存在，但治疗过程中无复发。

In summary, this young dog presented with multifocal, cutaneous mast cell proliferations with an indolent behaviour. Morphologically, the lesions were consistent with low-grade MCTs but clinically the disease resembled human CM. This highlights how critical the integration of clinical presentation is when interpreting microscopic findings. Future studies should include more detailed comparisons at the molecular level to help clarify whether cutaneous mastocytosis truly represents a separate entity from “classic” canine MCTs. Anti-IL-31 therapy appears to be promising in controlling pruritus associated with mast cell disease in dogs.  

总之，该年轻犬表现为多灶性皮肤肥大细胞增生，并具有惰性特征。形态学上，病变符合低等级MCT，但临床上该病类似人类CM。这就强调了在解释显微镜检查结果时，结合临床表现是多么关键。未来的研究应该包括分子水平上更详细的比较，以帮助阐明皮肤肥大细胞增多症是否真正代表了与“经典的”犬 MCT不同的独立存在。抗IL-31治疗似乎有希望控制犬肥大细胞疾病相关的瘙痒。

Figure 1. Photographs of skin lesions of a 4-year-old, female spayed mixed breed dog with multifocal proliferations of uniform mast cells confined to the skin. Multifocal, pale pink macules and papules involving the abdominal skin (a, b). Lesions were occasionally dark pigmented (c) and urticated upon palpation (Darier’s sign, d).

图1. 一只4岁、雌性绝育混血犬的皮肤病变照片，该犬有局限于皮肤的多灶性均匀的肥大细胞增生。累及腹部皮肤的多灶性淡粉色斑点和丘疹（a，b）。病变偶见着色深暗（c），触诊呈荨麻疹样（丹宁征，d）。

Figure 2. Photomicrograph of a fine needle aspirate of cutaneous papules located at the ventral abdominal skin of a dog with cutaneous mastocytosis. Numerous uniform,well-differentiated, heavily granulated mast cells are visible on a pale eosinophilic background with numerous free magenta mastcell granules.Wright-Giemsastain,×50objective.

图2. 皮肤肥大细胞增多症患犬腹侧腹部皮肤丘疹的细针抽吸显微照片。在白色的嗜酸性背景下可见许多均匀、分化良好、颗粒丰富的肥大细胞，背景中有许多游离的品红着染的肥大细胞颗粒，瑞士-吉姆萨染色，×50物镜。

Figure 3. Photomicrographs of a cutaneous papule on the ventral abdominal skin of a dog with cutaneous mastocytosis. (a, b) Focally, the dermis is expanded by a highly cellular, fairly well delineated, infiltrative proliferation of very well differentiated mast cells. Haematoxylin & eosin, 94 (a) and 940 (b). (c) Proliferative mast cells display weak cytoplasmic and rare stippled membrane-associated labelling for KIT. 940. (d) Labelling for AgNOR shows an average of 1.29 positive AgNORs per nucleus.

图3. 皮肤肥大细胞增多症患犬腹侧腹部皮肤丘疹的显微照片。(a,b)真皮因高度细胞性、轮廓清楚、分化良好的肥大细胞浸润性增生而局部膨胀，苏木精-伊红染色，×4 (a)和×40（b）。 (c) 增殖性肥大细胞显示出较弱的细胞质KIT标记和罕见的点状膜相关KIT标记，×40。 (d) AgNOR染色显示平均每个核有1.29个AgNORs阳性标记。

Figure 4. Photomicrographs of a cutaneous papule on the ventral abdominal skin of a dog with cutaneous mastocytosis. (a) Immunohistochemically labelling for Ki67 shows strong nuclear

labelling in low numbers of cells. ×40. (b) Modest numbers of mast cells display diffuse weak cytoplasmic and membranous labelling for CD25.×40.

图4. 皮肤肥大细胞增多症患犬腹侧腹部皮肤丘疹的显微照片。（a）Ki67的免疫组化标记显示少量细胞中强的核标记，×40。（b）中等数量的肥大细胞显示出弱的弥散性细胞质和细胞膜CD25标记，×40。

bateer578

又一次
高级了，

Patrik

洋芋那个土豆

vet刘

非常新的研究，太感谢了