記住我
ORIGINAL ARTICLE
Year : 2022 | Volume
: 40
| Issue : 1 | Page : 28-33
A 10-case series of Merkel cell carcinoma in tropical Taiwan: Focusing on clinical outcomes and the interaction of oxidative stress and Merkel cell polyomavirus infections
Chia-Jui Su1, Jui Lan2, Chih-Hung Lee1
1 Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan
2 Department of Pathology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
Correspondence Address:
Prof. Chih-Hung Lee
No. 123, Dapi Road, Niaosong District, Kaohsiung City 83301
Taiwan
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/ds.ds_12_22
Background: There are limited data discussing Merkel cell carcinoma (MCC) clinicopathological characteristics in Asian patients. Furthermore, the association between two key pathogenic factors, ultraviolet (UV) radiation and Merkel cell polyomavirus (MCPyV), remained unclear. Objectives: This study aimed to study the clinicopathological features in Taiwanese patients and to discuss the interaction between MCPyV infection and UV radiation-induced oxidative stress. Methods: We retrospectively reviewed the clinical features, pathological morphology, treatment, and outcomes of ten patients with histologically proved MCC. Eight specimens were tested for MCPyV large T-antigen. Five specimens with positive MCPyV large T-antigen were tested for 8-hydroxy-2-deoxyguanosine (8-OHdG). Results: Eight (80%) patients were male, and the median age at diagnosis was 81 years. The most common primary tumor site was extremities (8 patients, 80%), with the rest located on the head and neck region (2 patients, 20%). Intermediate type was the most common pathology subtype, with more than 70% showing reactivity to MCPyV large T-antigen. Four tumors out of 5 (80%) showed 8-OHdG positivity. Conclusion: The study showed similar demographics with previous studies in western countries but had different tumor location frequencies when compared with northeast Asia. The high frequency of UV signature oxidative stress marker, 8-OHdG in MCPyV large T-antigen-positive tumors may indicate the correlation between UV radiation and MCPyV infection. Further studies with larger numbers of patients may be required to verify its significance.
Keywords: 8-hydroxy-2-deoxyguanosine, Merkel cell carcinoma, Merkel cell polyomavirus, polyomavirus infections, ultraviolet radiation
Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine cancer that was first described by Toker as trabecular cell carcinoma of the skin in 1972.[1] By the time of 1980, the term MCC was adopted due to the discovery that trabecular cell carcinoma cells share similar histological and structural features with Merkel cells in the epidermis. Clinical manifestations of MCC tumors often present as asymptomatic, lack of tenderness, and expanding rapidly. Some risk factors are associated with the development of MCC, such as those with immunosuppressive status, age older than 50, and sun-exposed site on a person with fair skin.[2]
The United States has observed a steady increase of incidence in MCC since 1995 which may have been related to the progression of diagnostic tools such as cytokeratin-20 and raising awareness of MCC.[3],[4] Stang et al. analyzed 11,028 cases from 21 countries for the period 2003–2007. Japan is the only Asian population in the study, which showed a lower incidence compared with other non-Asian countries.[5]
MCC has a high relapsing rate and poor prognosis. The overall 5-year survival rate for MCC was 64% as compared with 92% for melanoma, according to the American Cancer Society.[6],[7] Surgical resection and radiation therapy are the mainstay of treatments currently, but many emerging immunotherapies such as immunotherapy with programmed cell death protein-1 and ligand-1 inhibitors are evolving rapidly and improving the prognosis of MCC.[8],[9]
The pathogenesis of MCC remains elusive. The association of immunosuppression in MCC and its good response to immunotherapy suggest the importance of immunosurveillance on the development of MCC. In addition, the frequent occurence of MCC in the sun-exposed skin highlights the role of ultraviolet (UV) in the tumorigenesis of MCC. Previous studies indicated that chronic sun exposure, UVA photochemotherapy, and concurrence of other UV-associated cutaneous malignancies contribute to the risk of developing MCC.[3],[10] In 2008, Merkel cell polyomavirus (MCPyV) was identified integrating into MCC genome and led to tumorigenesis.[11] Further, in a series of work in Tottori University, Japan, that demonstrated morphological differences between MCPyV-positive and MCPyV-negative tumors, tumors without MCPyV antigen had more nuclear polymorphism and their cytoplasm is more abundant.[12],[13],[14] Goh et al. proposed that MCC may develop either from the accumulation of mutation caused by UV radiation or the incorporation of MCPyV oncoprotein-induced oncogenesis.[15] However, the interplay of UV radiation and viral gene integration is yet to be clarified. UV-signature damages, such as 8-hydroxy-2-deoxyguanosine (8-OHdG), have been linked to other UV-associated cutaneous malignancy.[16] The expression of 8-OHdG, coupled with the presence of MCPyV large T-antigen, may be able to determine the interactions between UV damages and viral infection in MCC.
In contrast to the ample data in western countries, there are limited studies of MCC in Asia. Shinogi et al. published the largest epidemiologic study in Japan so far, in which they described different sex ratio and tumor differentiation pattern as compared with western countries.[17] However, in Taiwan, there were only 40 MCC cases reported in the past two decades.[18],[19] The southern part of Taiwan has subtropical climate and high UV radiation level all year round, which has been recognized as a major MCC risk factor. In the current study, we aim to investigate the demographics, clinical presentations, pathological features, and UV induced oxidative stress in a southern Taiwan medical center.
MethodsData collection
We identified 11 patients who had the pathological diagnosis of MCC between 2000 and 2020. One patient was excluded due to incomplete and unclear medical record. Patient characteristics, clinical features of the lesion (i.e., site, tenderness, and color), initial clinical impression at the time of biopsy, treatments, recurrence, survival time, and outcomes were reviewed. The patients were staged with the 8th American Joint Committee on Cancer staging system. The study is approved by the Institutional Review Board of the affiliated hospital (20210053B0). The patient consent is waived by IRB.
Histology
The histopathology of MCC tumors was reviewed by at least two board-certified pathologists to identify the cell morphology and histology. The histological patterns of MCC were categorized into intermediate type, small cell type, and trabecular type.
Immunofluorescence and immunohistochemistry
Immunofluorescence for detecting MCPyV large T-antigen (clone sc-27645, 1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) was analyzed using BOND-III fully automated IHC and ISH staining system (Leica, Wetzlar, Germany) in the department of anatomic pathology.
For 8-OHdG, we performed the immunohistochemical staining at the laboratory in the Department of Dermatology. We used de-identified skin sample of Bowen's disease from the tissue bank as a positive control.
The samples were first deparaffinized in three changes of xylene, 5 min each, transferred to 100% alcohol, for 3 min, and then transferred through 95%, 70%, and 50% alcohol, respectively, for 3 min each. The slides were then rinsed with ddH2O, retrieved in Tris-EDTA buffer, pH 9.0 at 85°C for 2 h, and finally rinsed with PBS. We blocked endogenous peroxidase activity and permeability by incubating sections in 3% H2O2/0.3% triton X-100/PBS solution at room temperature for 30 min and rinsed with PBS. DNA denaturation was completed by incubating sections in 70 nM NaOH/70% EtOH at room temperature for 4 min and rinsing with PBS. The slides proceeded to be blocked with 1% BSA in PBS at room temperature for 1 h and drained off the blocking buffer. In the next step, we applied 8-OHdG antibody (clone A5830, Merck Millipore, Merck Millipore, Burlington, MA, USA) diluted at 1:400, to the slides then incubated them at 4°C overnight. The next day, we washed the slides with PBST for three changes 20 min each and applied appropriately diluted secondary antibody-AF568 for 8-OHdG at room temperature for 60 min. We washed the slides with PBST for three changes 20 min each and rinsed with PBS. The 8-OHdG slides were stained with DAPI for 30 min and rinsed with PBS and water. Subsequently, the slides were counterstained with hematoxylin for 5 min and rinsed with PBS and water. All slides were air-dried, mounted, and ready for observation.
Statistical analysis
Overall survival (OS) was defined as the date from diagnosis to death by any causes or to the date of the last follow-up visit. The estimated 5-year survival rate was produced and analyzed with International Business Machines (IBM) Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, New York: IBM Corp.
ResultsMale and senile predominance in Merkel cell carcinoma
Ten MCC patients were identified. Eight (80%) patients were male, and only two (20%) patients were female. The median age at diagnosis was 81 years. The age ranged from 61 to 93 years, with 80% of patients older than 65 years.
Main distribution on extremities and initial clinical impression
The most common primary tumor site was extremities (8 patients, 80%) with the rest located on the head and neck region (2 patients, 20%). Four patients' lesions were described as flesh-colored, firm, and asymptomatic nodules; 1 had an ulcerative tumor; 1 had an easy-bleeding tumor; the rest 4 patients did not have a clear clinical description of the tumors. [Figure 1] demonstrates some of the patients' cutaneous manifestation. Two patients (20%) had an initial clinical diagnosis of squamous cell carcinoma and sarcoma, respectively. Two patients were initially diagnosed with benign skin disease, while the rest six patients' diagnoses were unclear.
Figure 1: (a) A flesh-colored nodule beside a surgical scar on a 62-year-old male patient's shin, later proven to be local recurrence. (b) A smooth protruding nodule on the cheek with peripheral ulceration and overlying crusts in an 87-year-old female patient. (c) A flesh-colored nodule with telangiectasia on an 80-year-old male patient's arm.Majority of patients had locoregional disease upon presentation
Number of patients (percentage) in stage I, II, III, and IV was 4 (40%), 2 (20%), 3 (30%), and 1 (10%), respectively. In total, nine patients (90%) had locoregional disease and 1 had metastatic disease (10%) at diagnosis [Table 1].
Intermediate type is the most common pathology subtype with high MCPyV immunoreactivity
Intermediate type accounted for 50% of all the patients, followed by small cell type (20%) and trabecular type (10%) [Figure 2]. Immunofluorescence showed that seven patients (70%) were positive for MCPyV large T-antigen [Figure 3], while only one patient was negative. Two patients' specimens were not examined due to unavailability.
Figure 2: Pathological subtypes of Merkel cell carcinoma tumors: (a) Intermediate type, (b) small cell type, and (c) trabecular type.
Figure 3: Merkel cell polyomavirus large T-antigen was identified by immunohistochemical staining in this representative section of Merkel cell carcinoma.High 8-hydroxy-2-deoxyguanosine positivity among Merkel cell polyoma virus-positive specimens
We accessed to five specimens of MCC tumors [patient No. 1, No. 5, No. 6, No. 7, and No. 10 in [Supplementary Table 1]] with MCPyV large T-Ag positivity for 8-OHdG IHC. All these tumors were located at sun-exposed site; 3 on lower legs, 1 on the forearm, and 1 on upper arm. Four tumors out of 5 (80%) had 8-OHdG positivity [Figure 4].
Figure 4: 8-hydroxy-20-deoxyguanosine is expressed in Merkel cell carcinoma. (a) Isotype control of a Bowen's disease specimen; (b) positive control of a Bowen's disease specimen; (c) and (d) positive staining of 8 hydroxy-2-deoxyguanosine in a representative Merkel cell carcinoma specimen.All patients had surgical excision, most of them combined with radiotherapy, and some received chemotherapy and immunotherapy
All ten patients (100%) received surgical excision, including 1 (10%) having Moh's surgery. Three (30%) did not have a proper margin. Seven patients (70%) had adjuvant radiotherapy. Four patients (40%) underwent chemotherapy, 3 of them received the regimen of etoposide and cisplatin, and 1 had cyclophosphamide. Four patients (40%) adopted immunotherapy; 2 with pembrolizumab and 2 with avelumab. The follow-up time ranged from 3 to 67 months, and the median follow-up time was 28 months [Supplementary Table 1]. The estimated 5-year OS was 32% [Figure 5].
DiscussionIn the current study, we aim to investigate MCC patients in southern (tropical) Taiwan regarding the clinical features, epidemiology, pathogenesis, and treatment. Moreover, we performed immunohistochemical staining to clarify the correlation between UV radiation and MCPyV viral gene integration in MCC tumorigenesis.
Our patient demographics were generally consistent with the large-scale study in the United States in terms of age over 65 years and male predominance.[3] When compared with local research in Taiwan, the median age of our patient group was older than the two previous studies but shared the same gender predilection.[18],[19] The most common site of MCC tumors was on extremities in our study, and the rest of the tumors grew on the head and neck region. This was consistent with the MCC characteristics that it often appears on sun-exposed area of the body. When compared with the largest Asian series in Japan, however, they had more female patients and the most common tumor site was the head and neck region.[17] In the current study, we found that only two patients were listed malignant diagnoses at first clinical impression and neither of them was thought to be MCC. Similar underdiagnosis was brought up in the study conducted by Heath et al.; more than half of the lesions were given a benign diagnosis in their study.[2] The lack of awareness about MCC may contribute to the erroneous diagnosis and influence the upcoming management. Three patients in our study did not have a proper margin of excision due to a tentative clinical impression other than MCC.
The classic histological findings of MCC include small blue round cells with scant cytoplasm, stippled chromatin, and inconspicuous nucleoli. Three histomorphologic subtypes were identified: intermediate type, small cell type, and trabecular type. Among the three subtypes, the intermediate type is the most common one.[20] Nevertheless, the histologic subtypes did not exhibit robust prognostic implications. Tumor thickness, the presence of lymphovascular invasion, and tumor architecture (nodular or infiltrative) were found to be independent predictors of survival.[21] Half of our patients were categorized as intermediate type, and we did not observe a significant difference in the outcomes between histologic subtypes.
We identified seven tumors carrying MCPyV large T-antigen from eight available specimens. Moshiri et al. found that in contrast to MCC patients with virus-positive tumors, virus-negative MCC patients had significantly increased risk of disease progression and death.[22] Histologically, Iwasaki et al. and Kuwamoto et al. found that the tumors without MCPyV infection had higher nuclear polymorphism and more abundant cytoplasm than their MCPyV-positive counterparts.[12],[14] Given the limitation of case number with only one patient showing MCPyV large T-antigen negativity, we did not observe significant association of viral antigen status with either the patients' prognosis or nuclear polymorphism in our study.
The bimodal tumorigenesis was proposed in previous research after analyzing mutational landscape of MCC tumors.[15] In multiple epidemiological studies from China, Japan and Korea uncovered high MCPyV seropositivity in general population.[23],[24],[25] However, the high prevalence of MCPyV infection does not match the low incidence of MCC. This discrepancy sparked a question whether UV radiation affects the integration of viral gene. The hypothesis is supported by a study in which the researchers found MCPyV small T-antigen mRNA level is increased following in vivo UV irradiation.[26] In our study, we performed 8-OHdG immunohistochemical stain on five tumor specimens with positive MCPyV large T-antigen to evaluate whether the two etiologies have intersection. The original tumors were all located on sun-exposed areas; 3 on lower legs, 1 on upper arm, and 1 on the forearm. Four tumors out of 5 have 8-OHdG positivity. The high 8-OHdG positivity may suggest that UV radiation and viral gene integration are interactive in the tumorigenesis rather than independent events. Nonetheless, the IHC stain of 8-OHdG may not reflect the cumulative insults and damages of UV radiation. Moreover, other environmental factors such as tobacco smoke, asbestos fibers, heavy metals, and polycyclic aromatic hydrocarbons also contribute to 8-OHdG production and become the confounding factors in our study.[27] Another conflicting evidence lies in the study by Mori et al., in which they found no significant difference was detected in TP53 UV-signature mutations between MCPyV-positive and -negative groups.[13] This issue remains to be studied further in the future.
Most of our patients received a standard care with surgical resection and adjuvant radiotherapy. The importance of radiotherapy was highlighted in earlier studies; timely adjuvant radiotherapy lowers the local and regional recurrence rate.[28],[29]
One of our patients, a 62-year-old man with a primary tumor on his left lower leg received wide excision and sentinel lymph node biopsy but delayed the radiotherapy due to poor wound healing. Merely, 3 months after his surgery, there were three new skin lesions appeared around the primary tumor site and later proven to be MCC recurrence. In the latest National Comprehensive Cancer Network guideline, chemotherapy is only suggested in certain patients with contraindications to immunotherapy and not routinely recommended for adjuvant therapy. Immunotherapy with programmed death-ligand 1 inhibitors such as avelumab or pembrolizumab has been applied to patients with advanced disease and showed good disease control.[8],[9] Nonetheless, the durability of immunotherapy remains an issue; in the avelumab clinical trial, up to 80% of the patients lost response to the agent after 12 months.[30] Two patients in our study adopted immunotherapy with pembrolizumab and 2 with avelumab, 2 of them gained good disease control, 1 had local recurrence 4 months after commencement of immunotherapy, and 1 died 2 months after treatment.
The estimated overall 5-year survival was only 32%, much lower than previously reported cases in Taiwan which was around 65%.[18] Several reasons may be associated with the poor outcome. First, our patients were much older and had many comorbidities, which led to mortalities. Two out of five patients died during the follow-up period did not die from MCC metastasis but sepsis and shock. Moreover, many patients with local regional disease were diagnosed later in the whole follow-up window, which produced the discrepancy that local-regional disease had shorter survival time. At last, our study has a small number with merely 10 patients, which is prone to statistical errors.
ConclusionOur study expanded the database of MCC patients in Taiwan despite the limitation of small sample size and retrospective nature. Moreover, we found that high 8-OHdG positivity in tumors with positive MCPyV large T-antigen may indicate correlations of UV oxidative stress and MCPyV infection in the tumorigenesis.
Financial support and sponsorship
The study is supported by Chang Gung Memorial Research Program (CMRPG8K0292, CMRPG8K0293, and CORPG8L0211).
Conflicts of interest
Prof. Chih-Hung Lee, an editorial board member at Dermatologica Sinica, had no role in the peer review process of or decision to publish this article. The other authors declared no conflicts of interest in writing this paper.
References
留言 (0)