Mirka Schmid

Inflammatory Monocytes Are a Reservoir for Merkel Cell Polyomavirus

Merkel cell polyomavirus (MCPyV) is a recently discovered virus that is implicated in the oncogenesis of Merkel cell carcinoma (MCC). The route of dissemination and the reservoir(s) of MCPyV within the human body have not yet been identified. In this study we describe two patients with multiple MCPyV-positive inflammatory and neoplastic skin lesions at different anatomic sites. Patient 1 was suffering from psoriasis for many years and was diagnosed with MCC 7 years before this study. Patient 2 had developed numerous non-melanoma skin cancer lesions under post-transplant immunosuppression. In both patients, MCPyV DNA was detected in whole blood and in urine using PCR and direct sequencing of PCR products. When we analyzed different blood compartments, we found MCPyV exclusively in cell-free serum and in blood monocytes, but not in lymphocytes or granulocytes. Upon separate analysis of resident (CD14(lo)CD16(+)) and inflammatory (CD14(+)CD16(-)) monocytes, we detected MCPyV exclusively in inflammatory, but not in resident monocytes. Our findings raise the possibility that MCPyV persists in inflammatory monocytes and spreads along the migration routes of inflammatory monocytes. This points to intervention strategies to contain MCPyV. Moreover, blood or urine tests may serve as ancillary tests to confirm MCPyV infection in a clinical setting.

Human herpesvirus type 6 and cytomegalovirus in AIDS-associated Kaposi's sarcoma: No evidence for an etiological association☆

Epidemiological studies indicate that acquired immune deficiency syndrome (AIDS)-associated Kaposis sarcoma (KS) may be caused by an infectious, preferentially sexually transmitted agent. Herpesviruses infections are common sexually transmitted diseases in homosexual men, who are also the main risk group for developing Kaposis sarcoma. To evaluate a possible role of human herpesvirus-6 (HHV-6) and cytomegalovirus (CMV) in the development of AIDS-associated KS, we investigated cutaneous AIDS-associated KS in 26 AIDS patients using the polymerase chain reaction (PCR) and immunohistochemistry (IHC) to detect the presence of HHV-6 and CMV. Human herpesvirus-6 was detected in nine of 26 Kaposis sarcoma specimens (all cases were HHV-6 subtype B) and in eight of 27 normal skin specimens from human immunodeficiency virus (HIV) seropositive and HIV seronegative patients (one case was HHV-6 subtype A and seven cases were HHV-6 subtype B). In two of four patients showing HHV-6 in KS of the skin, the virus also was detected in other investigated tissues, such as heart, lung, liver, kidney, and adrenals. Cytomegalovirus was detected only in AIDS-associated KS (seven of 26 KS specimens) and not in normal skin tissues of HIV-seropositive and HIV-seronegative patients. Cytomegalovirus was detected in other organs of those patients showing CMV in Kaposis sarcoma. Our data indicate that the presence of HHV-6 and CMV in AIDS-associated KS most likely reflects disseminated viral infection. Human herpesvirus-6 and CMV may be cofactors but not the only causative agents for the development of AIDS-associated KS.

Borrelia in granuloma annulare, morphea and lichen sclerosus: a PCR-based study and review of the literature

Background: Morphea, granuloma annulare (GA) and lichen sclerosus et atrophicans (LSA) have also been suggested to be linked to Borrelia infection. Previous studies based on serologic data or detection of Borrelia by immunohistochemistry and polymerase chain reaction (PCR) reported contradictory results. Thus, we examined skin biopsies of morphea, GA and LSA by PCR to assess the prevalence of Borrelia DNA in an endemic area and to compare our results with data in the literature.

Hepatitis C and G viruses in B‐cell lymphomas of the skin

Background:  The etiology of B‐cell lymphoproliferative disorders (LPDs) of the skin has still to be elucidated. So far, Borrelia sp. has been identified as the causative agent of some cases of B‐cell LPDs of the skin. Apart from bacterial pathogens, recent studies suggested that also flaviviruses, in particular hepatitis C (HCV) and G (HGV) viruses, may be involved in the pathogenesis of B‐cell non‐Hodgkins lymphomas (NHLs). Most studies were performed in patients with known HCV infection, but the overall frequency of HCV‐ and HGV‐RNA in tumoral tissue of primary cutaneous B‐cell lymphomas (CBCLs) is unknown.

Detection and typing of human papillomavirus in biopsy and cytological specimens by polymerase chain reaction and restriction enzyme analysis: A method suitable for semiautomation

Screening for high‐risk human papillomavirus (HPV) types allows the detection of women at a high risk of cervical squamous carcinomas, thereby defining a subset of patients targeted for more intensive screening and follow‐up. Thirty‐four cervical biopsy specimens and isolated cells from cervical smears of normal women or women diagnosed with high‐grade intraepithelial lesion (HGSIL) were screened for the presence of HPV by in situ hybridization (ISH) and/or by polymerase chain reaction (PCR). The exact HPV type was determined using a novel restriction typing method. The detection of HPV was facilitated greatly by the use of a PCR‐enzyme‐linked immunosorbent assay (ELISA)‐based method. HPV was detected by PCR in 32% of the biopsy specimens, whereas only 23% had a positive staining by ISH. In one case, a double infection was detected by ISH as well as by PCR. In two cases, the presence of HPV was detected by both methods but the exact type was different. Analyzing cells isolated from cervical smears by the PCR‐ELISA technique or by PCR followed by agarose gel electrophoresis, HPV was detected only in patients with HGSIL and not in the control group. The PCR system is more sensitive than conventional ISH, and the PCR‐ELISA system presented in this study is efficient in screening large series of cytological samples. Furthermore, this system allows exact HPV typing on the microtiter plate. These innovations may allow the application of HPV detection and typing as a routine screening method to identify patients with a high risk of developing cervical neoplasia.

HHV-8 DNA Sequences in the Peripheral Blood and Skin Lesions of an HIV-Negative Patient with Multiple Eruptive Dermatofibromas: Implications for the Detection of HHV-8 as a Diagnostic Marker for Kaposi’s Sarcoma

Background: Multiple eruptive dermatofibroma (MEDF) is a rare disorder seen in immunocompromised patients, simulating Kaposi’s sarcoma (KS). Whereas KS is strongly associated with human herpesvirus 8 (HHV-8), the virus has never been detected in MEDF until now. Objective: To present a patient with MEDF who showed no signs of immunodeficiency but was seropositive for HHV-8 antibodies and demonstrated HHV-8 DNA both in the peripheral blood and lesional skin of MEDF. Methods: Clinical, histological and serological investigations were performed as well as polymerase chain reaction (PCR) studies and in situhybridization (ISH). Results: A 35-year-old white man with suspected KS was referred for evaluation of multiple pigmented nodules and patches. Biopsies revealed features of dermatofibroma, superficial fibrosing dermatitis and scar. One of the nodular lesions harbored HHV-8 DNA sequences. A faint amplification product was detected in the superficial fibrosing dermatitis lesion, while no HHV-8 sequences were found in normal skin and scar. Whole-blood samples and serum were positive for HHV-8. None of the skin lesions shown to harbor HHV-8 DNA sequences by nested PCR displayed a signal for HHV-8 RNA by ISH. Repetitive peripheral blood examinations did not reveal any serum antibodies against or antigens of HIV. Serum antibodies against the HHV-8 capsid antigen orf 65.2 were detected. Conclusion: Results of PCR studies and ISH indicate that the presence of HHV-8 in the lesional tissue was probably blood-borne due to viremia and not due to viral replication in tumor cells. The presence of HHV-8 is not fully restricted to KS. The differential diagnosis of KS and its simulators should be based on an integrative analysis of all available clinicopathological and molecular data and should not rely exclusively or predominantly on the presence or absence of HHV-8.

Detection of Merkel cell polyomavirus and human papillomaviruses in Merkel cell carcinoma combined with squamous cell carcinoma in immunocompetent European patients.

Background: About 10% of patients with Merkel cell carcinoma (MCC) suffer from an associated squamous cell carcinoma (SCC). In European patients, Merkel cell polyomavirus (MCPyV) is detectable in 60%–88% of the MCC tumors. In combined lesions, MCPyV was not detectable so far. Methods: We investigated 2 combined tumors of MCC and SCC for the presence of MCPyV and human papillomavirus (HPV) by polymerase chain reaction and immunohistochemistry. Results: In both lesions, MCPyV DNA was found, and in 1 case, HPV DNA was also detected. This is the first report of a coinfection with HPV and MCPyV in combined MCC–SCC tumors. Conclusions: The results underline the hypothesis of cocancerogenesis of 2 oncogenic viruses in nonmelanoma skin cancer. Technical reasons and a low viral copy number of MCPyV hampering immunohistochemical detection may be responsible for the negative results in the literature.

Apoptosis in CD30-positive lymphoproliferative disorders of the skin.

Abstract:  The spectrum of CD30‐positive cutaneous lymphoproliferative disorders (CD30+ CLPD) includes lymphomatoid papulosis (LyP), primary cutaneous CD30+ large T‐cell lymphoma (LTCL) and rare borderline patients. Despite their malignant histopathology, CD30+ CLPD exhibit a low‐grade malignant course with an excellent prognosis and a characteristic tendency for spontaneous regression. Apoptosis of tumour cells is considered a principal mechanism of tumour regression. We examined the proliferation and apoptosis rates as well as the expression of apoptosis‐related proteins in various clinical entities, tumour cell lines and evolutional (evolving and regressing) stages of CD30+ CLPD. Skin biopsies of LyP (n = 20) and LTCL (n = 19) and five CD30+ lymphoma cell lines were analysed by means of immunohistochemistry and Western blotting in order to evaluate the proliferation (Ki67), apoptosis (FragEl) and expression of Bax, Bcl‐x, C‐kit and Mcl‐1. A significantly higher apoptotic index (AI) was found in LyP (AI = 12.5%) than in LTCL (AI = 3.1%, P < 0.005). Bax was expressed by the majority of tumour cells in all forms of CD30+ CLPD and CD30+ cell lines. However, no Bax expression was found in tumour cell lines derived from systemic CD30+ lymphomas, which lack spontaneous regression and display an aggressive clinical course. No significant correlation was found between the expression of apoptosis‐related proteins and the tumour type and evolutional stage of CD30+ CLPD. We conclude that the higher AI in LyP may contribute to the regression of LyP lesions and the excellent prognosis of the disease. Pro‐apoptotic protein Bax is expressed at high levels in CD30+ CLPD and may play a crucial role in mediating apoptosis of tumour cells.

Study of HHV‐8 DNA sequences in archival biopsies from lesional skin of Kaposi's sarcoma, various mesenchymal tumors and related reactive conditions

Background: HHV‐8 has been identified as the causative agent of Kaposis sarcoma (KS) and some lymphoproliferative disorders. In addition, there are anecdotal reports on the presence of HHV‐8 in other tumors, especially cutaneous epithelial and mesenchymal neoplasms. The aim of the study was to ascertain the value of identification of HHV‐8 viral DNA sequences in routinely processed, formalin‐fixed, paraffin‐embedded tissues for the diagnosis of Kaposis sarcoma and other mesenchymal tumors.

Detection of Merkel Cell Polyomavirus in Epidermodysplasia-Verruciformis-Associated Skin Neoplasms

Background: Epidermodysplasia verruciformis (EV) is a rare genodermatosis that is characterized by susceptibility to infection with specific human papillomavirus (HPV) genotypes. Among polyomaviruses, the novel Merkel cell polyomavirus (MCPyV) has been found in different epithelial skin neoplasias. Objective: To examine whether EV is associated with cutaneous MCPyV infection. Methods: We used MCPyV-specific PCR to study skin neoplasms of 6 congenital EV patients and of 1 patient with acquired EV. Results: In all congenital EV patients, MCPyV DNA was found in carcinomas in situ, in invasive squamous cell carcinomas and in common warts. In 4 of these patients, the MCPyV-positive skin lesions were from different anatomic locations. In addition, 1 immunosuppressed patient suffering from acquired EV harbored MCPyV DNA in 2 common warts. In contrast, 7 normal skin samples tested negative for MCPyV DNA. Only 2 out of 24 carcinomas in situ (8.3%) and 2 out of 30 common warts (6.7%) from immunocompetent individuals were positive for MCPyV DNA. Conclusions: The strong association of EV-associated skin neoplasms with MCPyV suggests a unique susceptibility of EV patients to infections with MCPyV. Both MCPyV and EV-HPV may act as synergistic oncogenic cofactors in the development of EV-associated skin neoplasms.