Elsevier

Human Pathology

Volume 45, Issue 1, January 2014, Pages 119-126
Human Pathology

Original contribution
Merkel cell polyomavirus DNA sequences in peripheral blood and tissues from patients with Langerhans cell histiocytosis,☆☆

https://doi.org/10.1016/j.humpath.2013.05.028Get rights and content

Summary

Langerhans cell histiocytosis (LCH) is a group of granulomatous disorders in which abnormal Langerhans cells proliferate as either a localized lesion in a single bone or disseminated disease involving two or more organs or systems. Because the different LCH forms exhibit significantly elevated levels of inflammatory molecules, including pro-inflammatory cytokines and tissue-degrading enzymes, we investigated for a possible viral trigger in LCH pathogenesis. We looked for Merkel cell polyomavirus (MCPyV) in peripheral blood cells and tissues using quantitative real-time PCR and immunohistochemistry staining with anti-MCPyV large T-antigen antibody. Our findings revealed elevated amounts of MCPyV DNA in the peripheral blood cells of 2 of 3 patients affected by LCH with high-risk organ involvement (RO+) and absence of MCPyV DNA in the blood cells in all 12 LCH-RO− patients (P = .029). With lower viral loads (0.002-0.033 copies/cell), an elevated number of MCPyV DNA sequences was detected in 12 LCH tissues in comparison with control tissues obtained from patients with reactive lymphoid hyperplasia (0/5; P = .0007), skin diseases not related to LCH in children younger than 2 years (0/11; P = .0007), or dermatopathic lymphadenopathy (5/20; P = .0002). The data, including frequent but lower viral loads and low large-T antigen expression rate (2/13 LCH tissues), suggest that development of LCH as a reactive rather than a neoplastic process may be related to MCPyV infection.

Introduction

Langerhans cell histiocytosis (LCH) is a neoplastic lesion characterized by uncontrolled clonal proliferation of Langerhans cells (LCs) in a tissue environment containing other lymphoid cells [1], [2], [3]. The disease differs widely in clinical presentation, from a localized lesion as a single-system (SS-LCH) disease to a severe disseminated multisystem form (MS-LCH) [4]. The latter form is frequent in children younger than 2 years, whereas SS-LCH is more common in children older than 2 years [5].

The liver, spleen, and bone marrow are considered high-risk organs for LCH, whereas skin, bone, lymph nodes, gastrointestinal tissue, pituitary gland, and central nervous system are considered low-risk organs [4]. Therefore, LCH is classified clinically as either affecting at least one high-risk organ (RO+) or involving only organs without high risk (LCH-RO−) [4]. Although most patients with LCH-RO+ develop MS-LCH, in some patients having only one high-risk organ involved, the clinical course is milder, with symptoms similar to those observed in SS-LCH [6], [7].

Although numerous studies have attempted to classify the LCH subtypes anatomically, no clear-cut consensus has yet been reached. For example, mutations in the proto-oncogene BRAF were found in 57% of a series of 61 cases, but there was no significant relation between the mutations and the clinical course of the disease [3]. An interleukin (IL)-17A autocrine LCH model of the severity of the disease was proposed [8], yet conflicting data remain, creating what often is described as the IL-17A controversy [9], [10]. More recently, our group proposed an IL-17A endocrine model, showing a specific pattern of expression of the IL-17A receptor (IL-17RA) in relation to LCH subtype [11]. In fact, the IL-17A/IL-17RA autocrine/endocrine loop appears important in host defense, in that IL-17A boosts the pro-inflammatory reaction against viral aggression [12], [13]. In the same context, we previously reported higher expression of phosphatase SHP-1 (also known as Tyrosine-protein phosphatase non-receptor type 6) [14], which by itself might promote Toll-like receptor–activated production of antiviral molecules such as interferon type I in abnormal LCH cells [15].

The LCH subtypes are associated with the release of inflammatory molecules such as pro-inflammatory cytokines and tissue-degrading enzymes [8], [11], [14], [16], [17] that are produced by LCs in contact with pathogens [18]. The most characteristic lesions observed in patients with LCH are skin maculopapular excrescences that imply involvement of an unknown dermotropic virus [4]. One such agent inducing LCH proliferation might be the common dermotropic Merkel cell polyomavirus (MCPyV) [19], [20], [21], the major pathogenic agent of Merkel cell carcinoma (MCC) of the skin [22], [23]. Histologically, LCs are located above the middle of the prickle cell layer of the epidermis, whereas the Merkel cells are mostly sited in the basal layer of the epidermis. It was previously proposed that LCs capture external pathogens by elongation of dendrites beyond the tight junction barrier and function as antigen-presenting cells [24]. External pathogens might be recognized primarily by LCs or by a precursor LC present in the skin and not by the Merkel cells, as presently alleged.

In this report, elevated numbers of MCPyV DNA sequences were detected in the blood in two of three patients clinically classified as LCH-RO+. Altogether, the findings could be an important additional argument supporting the view that LCs behave as candidate sanctuary cells for asymptomatic MCPyV despite active antibody synthesis and that this pathogenic agent is involved in the development of LCH.

Section snippets

Patients, peripheral blood, and LCH tissue samples

This study was approved by the Institutional Review Board of Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan, and the Faculty of Medicine, Tottori University, Yonago, Japan.

Peripheral blood samples were obtained from 15 patients affected by LCH (6 SS-LCH, 9 MS-LCH; 3 LCH-RO+, 12 LCH-RO−). A total of 13 tissue samples from patients with LCH (7 SS-LCH, 6 MS-LCH; 1 LCH-RO+, 12 LCH-RO−) also were analyzed. As controls, 5 tissue samples from

MCPyV DNA in PBMCs from patients with LCH

The MCPyV DNA sequences were detected in PBMCs from patients with LCH-RO+ in 2 of the 3 analyzed samples, but there were no DNA sequences in any of the 12 samples corresponding to LCH-RO− (P = .029). Comparisons of MCPyV DNA sequences in PBMCs of patients with SS-LCH and of patients with MS-LCH did not differ significantly (0/6 versus 2/9; P = .49) (Table 1).

MCPyV DNA sequences in LCH tissues versus tissues of RLH, non-LCH skin diseases, and DLA

The viral loads of MCPyV detected by Q-PCR analysis in LCH tissues are shown in Table 2. MCPyV DNA sequences were detected in 12 of 13

Discussion

Our previous data indicated that Q-PCR was the most sensitive method for the detection of MCPyV, superior to single PCR and immunohistochemistry staining [19], [23]. The Q-PCR for MCPyV-LT also was more sensitive than immunohistochemistry using the CM2B4 antibody (see Table 2). In this report, we describe the presence of MCPyV DNA sequences in two blood samples (see Table 1) as well as in four LCH tissues samples obtained from children younger than 2 years (Table 2). The viral DNA load in blood

Conclusion

This is to our knowledge the first report of a relation between MCPyV infection and LCH. We suggest that MCPyV triggers an inflammatory process associated with LCH activity and subtype. The frequent but lower viral loads detected by Q-PCR and low positive immunoreactivity rate using the anti-MCPyV-LT antibody in 2 of 13 of LCH tissue samples suggest that development of LCH is more a reactive rather than a neoplastic process, which may be relevant to MCPyV infection. We propose that LCH subtypes

Acknowledgment

The authors thank all patients, parents, and physicians who participated in this study.

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      Citation Excerpt :

      However, the type of productively infected skin cells and whether or not differentiation of such cells may contribute to completion of the productive cycle remains unknown. Likewise, considering the frequent detection of viral sequences in other tissues from healthy patients (e.g., respiratory tract, urine, lymphoid tissue and blood) [40–44], it is unclear whether the skin also represents the primary reservoir of long term persistence. Given the recently reported role of the BKV-encoded viral miRNA in mediating persistent infections, it is an intriguing possibility that similar mechanisms may allow MCPyV to persist in dedicated tissues or cell types, either via restricted low-level particle production that allows continuous re-infection or via the establishment of a latency state.

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    Competing interests: The authors declare no competing financial interests.

    ☆☆

    Funding/Support: This work was partly supported by a Grant-in-Aid for Scientific Research (C) 23590426 from the Japanese Ministry of Education, Science, Sports and Culture and by 2010 and 2011 research grants from the Japan LCH Study Group.

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