犬乳头瘤病毒2型相关的犬甲床内翻性鳞状乳头瘤 ...

王帆

Claw bed inverted squamous papilloma associated with canine papillomavirus type 2 in a dog

犬乳头瘤病毒2型相关的犬甲床内翻性鳞状乳头瘤

Ana R. Resendes1,2 | Karen E. Trainor3 | Monali Bera2 | Ryan Chuang Fu Cheng4 | Jennifer Luff

 

Abstract

A claw bed inverted squamous papilloma (ISP) presented clinically as a swollen digit in a dog. Canine papillomavirus (CPV) type 2 was amplified by PCR and localised to the papilloma's epidermis using in situ hybridisation. This is the first report demonstrating a claw bed ISP caused by CPV.

摘要

一只犬的甲床内翻性的鳞状乳头瘤在临床表现为趾部肿胀。通过PCR扩增检测确定为犬乳头瘤病毒(CPV)2型，并使用原位杂交技术，定位于表皮层的乳头瘤。这是首次报道由CPV引起的甲床内翻性鳞状乳头瘤。

 

 

 

INTRODUCTION

介绍

Canine papillomavirus (CPV) types 1, 2 and 6 and an unknown PV have been found to be associated with inverted papillomas (IPs). Furthermore, CPV2 is the main genotype associated with cutaneous endophytic papillomas. Claw bed inverted squamous papilloma (ISP) is a benign solitary neoplasm of the digit described in dogs, arising from the germinative epithelium of the claw and of unknown aetiology. Inverted squamous papillomas (ISPs) differ from IPs arising in other parts of the body because of their origin and clinical presentation. They are single and present as a swollen digit, sometimes ulcerated and often associated with an absent, broken or malformed claw. Radiographs usually show moderate to marked destruction of the third phalanx as a result of pressure lysis. Although they are thought to be PV-induced, their association with CPV has not been reported previously. Histologically, the neoplasm is analogous to the haired skin counterpart, consisting of a well-delineated, cup-shaped mass filled with dense keratin.

犬已知与内翻性乳头瘤(IPs)相关的病毒有乳头瘤病毒(CPV) 1型，2型和6型以及一种未知的PV。此外，与皮肤内生型乳头瘤相关的基因型主要为CPV2。甲床内翻性鳞状乳头瘤(ISP)是一种良性单发性肿瘤，起源于爪的生发上皮细胞，病因不明。内翻性鳞状乳头瘤(ISP)的起源和临床表现与其他部位的IPs不同。单发，临床表现为趾部肿胀，有时溃疡，常伴有甲缺失、断甲或甲畸形。x线片通常显示第三趾骨因压力松解而有中度至显著的破坏。虽然它们被认为是PV引发的，但以前并没有报道过它们与CPV的关系。组织学上，肿瘤类似于有毛皮肤肿瘤，包括一个轮廓清晰的杯状肿块，充满致密的角蛋白。

 

CASE REPORT

病例报道

A 10-year-old intact male dog was presented with swelling of the 4th digit of the right hind leg, at the level of the second and third phalanges. The surfaces of the digit and paw pad were free of lesions. A radiograph showed lysis of the third phalanx and periosteal reaction involving the second phalanx. The digit was amputated, placed in 10% neutral buffered formalin and submitted for histopathological examination. Histologically, in a midsagittal section from the decalcified digit, there was a well-circumscribed, endophytic papilloma extending through the subungual tissue below the claw bed and matrix, extending proximally to the third phalanx. The neoplasm had a cystic empty cavity lined by hyperplastic stratified squamous epithelium, displaying orderly keratinisation and forming papillary projections which extended inward and were supported by fine fibrovascular stalks (Figure 1a). The wall presented a scalloped border. Multifocally, keratinocytes in the stratum spinosum and granulosum showed vacuolar degeneration. At the stratum spinosum, numerous keratinocytes were swollen, with lightly basophilic cytoplasm and enlarged nuclei with lightly amphophilic inclusion bodies. Marginalised chromatin filled the nuclei (Figure 1b). There were keratinocytes with clear perinuclear halos and pyknotic nuclei (koilocytes). Anisocytosis and anisokaryosis was minimal, and no mitotic figures were noted. Proximally, the papilloma extended up to the third phalanx and replaced its normal architecture with trabeculae of woven bone. Moderate numbers of macrophages, plasma cells, lymphocytes and fewer neutrophils infiltrated the adjacent dermis. PCR was performed as described previously on the formalin-fixed paraffin-embedded(FFPE) tissue using three sets of degenerate primers MY09/MY11, FAP59/FAP64 and FAP6085/FAP64.5–7 A 265 bp fragment obtained using the FAP6085/ FAP64 primer set was compared with the nucleotide collection at NCBI using Blast (http://blast.ncbi.nlm. nih.gov/Blast.cgi). The sequence was 100% identical to CPV2 (GenBank accession no. LC363559.1). In situ hybridisation (ISH) was performed using a previously designed probe (V-CPV2-E6E7) that targets E6/ E7 genes of CPV2 (NC_006564.1; Advanced Cell Diagnostics). In situ hybridisation (ISH) was performed on 5-μm-thick FFPE sections using the RNAscope 2.5 RED assay kit according to the manufacturer's recommended protocols. Successful hybridisation results in deposition of a red stain that is in direct correlation with the amount of CPV2 nucleic acid. A negative control was run concurrently using a predesigned probe to the bacterial gene DapB (ACD). One canine cutaneous papilloma was included as a positive control. Strong hybridisation signals for CPV2 were detected within epithelial cells of the papilloma. Basal keratinocytes within the papilloma exhibited dot-like nuclear and cytoplasmic hybridisation signals, mostly likely corresponding to viral mRNA expression where the viral load (DNA) is expected to be relatively low. Keratinocytes within the stratum spinosum exhibited strong diffuse nuclear and cytoplasmic hybridisation signals, most likely corresponding to increased vegetative replication of the viral genome within the upper layers of the epithelium (Figure 2). No hybridisation signals were detected within the surrounding normal epithelium nor within the negative control probe.

一只10岁未去势雄犬的右后肢第4趾的第2和第3趾骨区域肿胀。趾和爪垫表面无病变。x线片示第3趾骨溶解，第2趾骨骨膜反应。断患趾切除，置于10%中性福尔马林缓冲液中，进行组织病理学检查。组织学上，在脱钙趾的正中矢状切面上，有一个边界清楚的内生乳头瘤，穿过甲床和基质下方的甲下组织，向近端延伸至第3趾骨。肿瘤有一个内衬增生性复层鳞状上皮的囊性空腔，显示有序的角质化，形成向内延伸的乳头状突起，并由细纤维血管柄支撑(图1a)。壁呈扇形边界。棘层和颗粒层的多灶性角质形成细胞空泡样变性。棘层大量角质形成细胞肿胀，胞质轻度嗜碱性，细胞核增大，可见轻度双染性包涵体。染色质边缘化充满细胞核(图1b)。可见明显核周晕和固缩的角质形成细胞(空泡细胞)。轻度细胞大小不等和细胞核大小不等，未见有丝分裂象。近端，乳头瘤延伸至第3趾骨，并以编织骨小梁取代其正常结构。真皮周围有中等数量的巨噬细胞、浆细胞、淋巴细胞及较少的中性粒细胞浸润。我们使用三组简化引物MY09/MY11, FAP59/FAP64和FAP6085/ FAP64.5-7对福尔马林固定的石蜡包埋(FFPE)组织进行PCR。使用FAP6085/FAP64引物获得的265 bp片段与使用Blast。该序列与CPV2序列完全相同。原位杂交(ISH)使用预先设计的探针(V-CPV2-E6E7)靶向CPV2。使用RNAscope 2.5 RED试剂盒，根据制造商推荐的方案，对5μm厚的FFPE切片进行原位杂交(ISH)。成功的杂交可导致与CPV2核酸量直接相关的红色染色物沉积。用预先设计的细菌基因DapB (ACD)探针同时进行阴性对照。以1例犬皮肤乳头瘤作为阳性对照。在乳头瘤的上皮细胞中检测到CPV2的强杂交信号。乳头瘤内的基底角质形成细胞表现出点状的细胞核和细胞质杂交信号，最可能对应的是病毒mRNA的表达，病毒载量(DNA)预计相对较低。棘层内的角质形成细胞表现出强烈的弥漫性核和细胞质杂交信号，很可能对应于上皮上层内病毒基因组的营养复制增加(图2)。在周围的正常上皮内和阴性对照探针内均未检测到杂交信号。

 

The clinical presentation and the microscopic findings were consistent with a claw bed ISP with the added novel findings of viral cytopathic change and CPV detection. The neoplasm showed the major distinguishing features of this papilloma variant, such as its location below the claw bed, an endophytic growth pattern and the clinical presentation of a swollen digit without noticeable lesions on the surface of the digit. Histologically, this papilloma had most of the features of an ISP, such as endophytic and expansive growth that extended through the claw bed, and a squamous epithelial wall displaying orderly keratinisation and centripetally orientated papillary projections. The main histological differential diagnoses for claw bed ISPs include the claw bed inclusion cyst, claw bed squamous cell carcinoma (SCC) and subungual keratoacanthoma. Diagnosis of claw bed inclusion cysts is straightforward because they lack papillary ingrowths from the wall. Claw bed SSC has an invasive contour, cellular atypia and mitotic activity. Subungual keratoacanthomas usually have a central pore opening, a symmetrical yet complex wall lacking central papillary projections and a central core of amorphous keratin that is often highly glycogenated. By contrast, other skin IPs present as firm, cutaneous masses that extend above the surface with a small pore opening. Claw bed ISP do not protrude externally and lack a pore opening. Their growth is completely internal, causing only swelling of the digit as detected in this case. This can be explained by their origin in the claw bed matrix, which runs internally. Some cases of claw bed ISP show diffuse replacement of the claw bed by proliferative epithelium, with the conical papillae having a more parallel orientation to one another, and columns of soft keratin projecting into and partially replacing the hard keratin of the claw plate. Replacement of the claw bed and the claw plate was not present in this case at the level of the sections evaluated, although these features are not always found in claw bed ISPs. Other features include absent or abnormally thick, broken, soft claws with longitudinal ridges, which also were not found in this case. It could be that the absence of claw damage in this case is related to the lack of extension of the lesions to the claw plate.

临床表现和显微镜检查结果与甲床ISP一致，并增加了病毒细胞病变和CPV检测的新发现。肿瘤的主要特征是位于甲床以下，内生生长方式，临床表现为趾部肿胀，但趾表面无明显病变。组织学上，该乳头瘤具有ISP的大部分特征，如穿过甲床的内生和膨胀性生长，鳞状上皮壁显示有序的角化和向心性乳头状突起。甲床ISP的主要组织学鉴别诊断包括甲床包涵囊肿、甲床鳞状细胞癌和甲下角化棘皮瘤。甲床包涵囊肿的诊断很简单，因为没有从壁向内生长的乳头状突起。甲床SSC具有侵袭性轮廓、细胞异型性和有丝分裂象。甲下角化棘皮瘤通常有一个中央孔，一个对称但复杂的壁，缺乏中央乳头突出和一个无定形角蛋白的中央核，通常是高度糖基化。相比之下，其他皮肤IPs表现为紧致的皮肤肿块，在表面上有一个小的毛孔。甲床ISP不突出外部和缺乏一个毛孔开放。它们的生长完全是内在的，在这个病例中只引起手趾肿胀。这可以解释为它们起源于内部运行的甲床矩阵。部分病例表现为增生的上皮弥漫性取代甲床，锥状乳头相互平行，软角蛋白柱突入并部分取代爪板的硬角蛋白。在本例中，在评估的断面层面，并没有更换甲床和爪板，尽管这些特征并不总是在甲床ISP中发现。其他特征包括缺如或异常粗、断、软爪伴纵向脊，本例也未发现。在这个病例中，没有出现甲损伤，这可能与病变没有延伸至甲板有关。

 

Besides the viral cytopathic effects detected, including swollen keratinocytes with increased quantities of blue-grey cytoplasm, koilocytes and intranuclear inclusion bodies, CPV2 was sequenced from this ISP and CPV2 nucleic acid was found at all layers of papilloma epidermis using ISH. CPV1, 2 and 6 and an unknown PV have been detected in cutaneous IPs. Curiously, only CPV2 has been associated with digital and footpad papillomas, both endophytic and exophytic. PVs are usually site-specific, and lesions are often restricted to limited locations on the body. It is not known whether claw bed ISPs are associated with particular type(s) of CPVs as well and whether papilloma viruses are always necessary for the development of these neoplasms. What is known is that viral cytopathic effects are not commonly detected in claw bed ISPs and a viral cause has not been confirmed previously. Notwithstanding this, CPV may be detected either with or without associated cytopathic effects,1 which could potentially explain why papilloma viruses have not been pursued further in prior studies of claw bed ISPs. A single report of a subungual IP associated with papilloma virus virions is available, yet in this case, the lesion arose at the junction of the paw pad and the ventral aspect of the claw with a growth extending focally and disrupting the adjacent ventral claw. In contrast to the case reported here, the prior lesion was directly associated with a subungual cyst and an exophytic papilloma and for this reason was thought to have arisen in the skin adjacent to the paw pad and not from the claw bed.

除了检测到的病毒细胞病变效应，包括肿胀的角质形成细胞和数量增加的蓝灰色细胞质，空泡细胞和核内包涵体，CPV2测序从这个ISP和CPV2核酸发现乳头瘤表皮的所有层。在皮肤IPs中检测到CPV1, 2和6以及一种未知的PV。奇怪的是，只有CPV2与趾和足部乳头瘤相关，包括内生的和外生的。pv通常具有部位特异性，病变常局限于身体的有限部位。目前尚不清楚甲床isp是否也与特定类型的cpv相关，以及乳头瘤病毒是否总是这些肿瘤的发展所必需的。目前所知的是，病毒引起的细胞病变在甲床isp中并不常见，而且病毒的原因此前尚未得到证实。尽管如此，CPV可能被检出并伴有或不伴有相关的细胞病变效应1，这可能解释了为什么在之前对甲床isp的研究中，乳头瘤病毒未被进一步研究。目前已有一例与乳头瘤病毒病毒颗粒相关的甲下IP的单一报道，但在该病例中，病变发生在爪垫和爪腹交界处，生长物局灶性延伸并破坏邻近的爪腹。与本文报道的病例相反，先前的病变与甲下囊肿和外生型乳头瘤直接相关，因此认为起源于爪垫附近的皮肤，而不是甲床。

 

Malignant transformation into SCC is a concern in CPV-induced papillomas. CPV1 has been detected in papillomas that progressed to SCC at the lips, oropharyngeal mucosa, foot and bulbar conjunctiva in six dogs, and CPV2 was detected in interdigital and foot-pad papillomas that progressed to SCC in four dogs with bone marrow-transplanted X-linked severe combined immunodeficiency (XSCID). Although CPV DNA was not amplifiable from canine subungual SCCs in 28 cases, the possibility of malignant transformation of claw bed IP into a SCC should not be excluded, especially considering the oncogenic effects of CPV2. Lack of detection of papilloma virus in subungual SCCs could have been related to the use of archival material, low sensitivity of the primers, small amounts of viral DNA in the lesions, or the degradation of CPV DNA from older tissues. Furthermore, even in cases with evidence of viral cytopathic effects, papillomaviral DNA might not be detected, suggesting that failure to detect the virus in archived material can occur.

cpv诱导的乳头瘤恶性转化为SCC是一个值得关注的问题。在6只犬的口唇、口咽黏膜、足部和球结膜的乳头瘤进展为鳞状细胞癌中检测到CPV1，在4只患骨髓移植x连锁重度联合免疫缺陷(XSCID)的犬的趾间和足部乳头瘤进展为鳞状细胞癌中检测到CPV2。虽然在28例犬甲下SCC中未扩增出CPV DNA，但考虑到CPV2的致癌作用，不应排除甲床IP恶性转化为SCC的可能性。甲下scc中未检出乳头瘤病毒可能与使用档案材料、引物灵敏度低、病变中有少量病毒DNA或来自旧组织的CPV DNA被降解有关。此外，即使在有病毒细胞病变效应证据的病例中，也可能未检出乳头瘤病毒DNA，这提示可能未检出存档材料中的病毒。

 

In conclusion, this report documents the first definitive case of claw bed ISP caused by CPV infection.

总之，本报告记录了第一例由CPV感染引起的甲床性ISP。

 

 

 

 

 

 

FIGURE 1 (a) Photomicrograph of the second digit from the right hind leg. Below the claw plate (✷) and the claw bed, there is an endophytic growth consisting of an empty cavity lined by stratified squamous epithelium with inward-projecting papillae. There is pressure lysis of the third phalanx (arrows). (b) At the stratum spinosum, numerous keratinocytes were swollen, with lightly basophilic cytoplasm, and with enlarged nuclei, some with lightly amphophilic inclusion bodies (inset, arrows) and marginalised chromatin.

图1 (a)右后腿第二指的显微照片。在爪板(✷)和爪床的下方，有一个由复层鳞状上皮内衬的伴有向内乳头突出生长的空腔。第三指骨(箭头)有挤压溶解。(b)在棘层，大量角质形成细胞肿胀，胞质轻度嗜碱性，细胞核增大，一些有轻度两亲性包涵体(插图，箭头)和边缘染色质。

 

 

 

 

 

FIGURE 2 Canine papillomavirus (CPV2) nucleic acid hybridisation by in situ hybridisation (ISH). Note that a strong hybridisation signal, consistent with viral nucleic acid (red), is present only within the papilloma; Inset: all layers of the papilloma reveal hybridisation signals for CPV2.

图2犬乳头瘤病毒(CPV2)核酸原位杂交(ISH)。注意强杂交信号，与病毒核酸一致(红色)，仅存在于乳头瘤内;插图:乳头瘤的所有层显示CPV2杂交信号。

杨先生