Human Pathology (2012) 43, 980–985
www.elsevier.com/locate/humpath
Original contribution
Extracutaneous Merkel cell carcinomas harbor polyomavirus DNA Dario de Biase BSc, PhD a , Moira Ragazzi MD a , Sofia Asioli MD b , Vincenzo Eusebi MD, FRCPath a,⁎ Department of Hematology and Oncological Sciences “L. & A. Seragnoli,” Section of Anatomic Pathology at Bellaria Hospital, University of Bologna, 40139 Bologna, Italy b Department of Biomedical Sciences and Human Oncology, University of Turin, 10126 Torino, Italy a
Received 29 June 2011; revised 22 August 2011; accepted 24 August 2011
Keywords: Merkel cell carcinoma; Merkel cell polyomavirus; Lymph node; Salivary glands
Summary Merkel cell carcinoma is a neuroendocrine tumor, with characteristic morphological and immunohistochemical features. Originally reported as primary carcinoma of skin, it has been described in numerous other sites such as lymph nodes, oral cavity, breast, vaginal walls, and salivary glands. Recent studies have revealed in cutaneous Merkel cell carcinomas a clonally integrated polyomavirus, named Merkel cell polyomavirus. The aim of the present study was to verify the presence of Merkel cell polyomavirus in 5 cases of primary Merkel cell carcinomas of lymph nodes and 1 case of parotid gland to investigate similarities or differences among Merkel cell carcinomas from various sites. Cases studied were 5 primary Merkel cell carcinomas in lymph nodes, 1 in the parotid gland, and 12 in the skin. Twelve cases of primary and metastatic small cell carcinoma of the lung were also investigated. Immunohistochemistry for keratin 20, chromogranin, synaptophysin, and thyroid transcription factor 1 was performed in all cases. Viral DNA was studied using polymerase chain reaction assay and the products evaluated in agarose gel and sequenced. Cytokeratin 20 and Merkel cell polyomavirus were detected in all cases of primary Merkel cell carcinoma irrespective of their site of origin. On the contrary, all cases of pulmonary small cell carcinoma were negative for both Merkel cell polyomavirus and cytokeratin 20. It appears that cutaneous and extracutaneous Merkel cell carcinomas share similar histologic, immunohistochemical, and molecular features. This is further evidence that Merkel cell carcinomas are a multiorgan carcinoma and that Merkel cell polyomavirus might play a role in the pathogenesis of this neoplasm. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Merkel cell carcinoma (MCC) was originally reported in 1972 by Toker [1] as trabecular carcinoma of the skin. The discovery of electron-dense neurosecretory granules in the
⁎ Corresponding author. E-mail address:
[email protected] (V. Eusebi). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.08.014
tumor cells established its neuroendocrine nature, and origin from Merkel cells (MCs) was postulated [2]. Histologically, MCC is an intradermal tumor composed of small- to medium-sized globoid cells with scant eosinophilic cytoplasm and round to oval nuclei. The latter show finely dispersed chromatin encircled by a well-evident nuclear membrane. The cells are usually arranged in sheets or nests often showing a noncohesive lymphoma-like pattern [3]. The mitotic index is high, and many atypical mitoses are seen [3]. Tumor necrosis is frequent.
Extracutaneous MCCs polyomavirus DNA MCC is typically immunoreactive for chromogranin, synaptophysin, and cytokeratin 20 (CK20), the latter giving a characteristic “dot-like” paranuclear signal [4]. Cytogenetic studies evidenced loss of long arm of chromosome 10 [5] and trisomy of chromosome 6 in approximately 50% of cutaneous cases [6]. In 2008, Feng et al [7] sequenced a novel polyomavirus, named Merkel cell polyomavirus (MCPyV), in most cases of MCC. Since the original report of Feng et al, several independent groups have confirmed the presence of MCPyV DNA in a large proportion, ranging from 40% to 100% of MCCs cases [8-13]. Tumors identical to MCC of the skin in terms of histology, immunohistochemistry, and cytogenetics have been reported in several organs including breast [14,15], salivary glands [16,17], oral mucosa [18] and vaginal wall [19] as well as lymph nodes [20,21]. The aim of this study was to investigate the presence of MCPyV in extracutaneous MCCs of lymph nodes and parotid gland.
2. Material and methods 2.1. Case selection Eighteen MCCs were collected, 16 from the routine files of the Section of Anatomic Pathology of the University of Bologna at Bellaria Hospital and 2 from the consult case files of one of us (V. E.). Six were extracutaneous MCCs, 5 primary in lymph nodes and 1 in parotid glands, and 12, MCCs of the skin. In addition, 12 consecutive pulmonary small cell carcinomas (SCCs) were selected from the routine files of the same institution. Criterion for the definition of primary extracutaneous MCCs was absence of any other primary tumor elsewhere in spite of extensive clinical search [20]. All cases were studied histologically and immunohistochemically. In addition, molecular analysis for MCPyV DNA detection was performed.
981 Table 1
Antibodies used for immunohistochemistry
Antibody
Dilution
CK20
Prediluted Ventana Benchmark a Chromogranin Prediluted Ventana Benchmark a Synaptophysin Prediluted Ventana Benchmark a TTF-1 Prediluted Ventana Benchmark a
CC1 a,b CC1 a,b CC1 a,b CC1 a,c
Ventana-Benchmark, Tucson, AZ. Detected by “Ultraview universal DAB detection system” (Ventana-Benchmark). c Detected by “Universal alkaline phosphatase red detection system” (Ventana-Benchmark). b
sponding H&E sections. Tumor cells were dissected manually with a sterile blade, and DNA was extracted using the RecoverAll kit (Ambion, Inc, Austin, TX), following the manufacturer's instructions. The DNA was eluted in TE (tris-EDTA) buffer and quantified utilizing Quant-iT dsDNA-HS kit (Invitrogen, Milan, Italy). A polymerase chain reaction (PCR) was performed using the FastStartTaq kit (Roche, Milan, Italy) to double check the presence of viral DNA. Primers used for MCPyV typing have been previously described [9]. Amplicons obtained were 107 base pairs (bp), preventing the loss of viral DNA detection due to DNA fragmentation [11]. Furthermore, multiplexed primers set (consisting of 100, 200, 300, and 400 bp products) [22] and β2-microglobulin gene (length 165 bp) were used as amplification control. The PCR products were run in a 3% agarose gel (Lonza, Rockland, ME) in the presence of GelStar intercalating dye (Lonza, Rockland, ME). To verify PCR products, MCPyV sequence analysis was performed in agreement with the CEQ2000 Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Fullerton, CA). The products were loaded on a CEQ2000 automatic sequencer (Beckman Coulter).
3. Results
All cases were formalin fixed and paraffin embedded (FFPE). Sections were stained with hematoxylin-eosin (H&E). Immunohistochemistry was performed on 3-μmthick sections cut from selected paraffin blocks. All specimens were stained using an automatic immunostainer (Ventana Benchmark, Tucson, AZ). The antibodies used together with their antigen retrieval are reported in Table 1.
3.1. Clinical features
DNA of neoplastic cells was extracted from FFPE blocks. Molecular analysis was performed on 5 consecutive 10-μm sections. Neoplastic areas were selected from the corre-
Antigen retrieval/ detection system
a
2.2. Histology and immunohistochemistry
2.3. MCPyV DNA analysis
Source
3.1.1. Extracutaneous MCCs Clinical features of extracutaneous MCCs are summarized in Table 2. The male-female ratio was 1:1 in the 6 patients with extracutaneous MCC (5 in lymph nodes and 1 of parotid gland). The age of patients with MCC of lymph nodes ranged from 65 to 85 years (mean, 74.3 years). The lymph nodes involved were inguinal (4 cases) and crural (1 case). Tumor sizes ranged from 2.9 to 14 cm (mean, 5.5 cm). The patient with MCC of parotid gland was 64 years old, and tumor size was 4 cm.
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D. de Biase et al.
Table 2
Clinical features of extracutaneous MCCs a
Case
MCC site
Sex
Age
Tumor size (cm)
FU (mo )
1 2 3 4 5
LN, crural LN, inguinal (L) LN, inguinal (L) LN, inguinal LN, inguinal (R)
M F F M F
79 67 65 85 77
6.5 14 4.5 2.9 5.5
6
Parotid (L)
M
64
4.0
A&W (14) A&W (30) AWD (23) A&W (37) Recent case A&W (4) DOD (12)
Abbreviations: LN, lymph node; (L), left side; (R), right side; A&W, alive and well; AWD, alive with disease; DOD, dead of disease. a FU started from the date of surgery.
At follow-up (FU), the patient with the parotid tumor (case 6) died of laterocervical lymph node metastatic disease after 12 months after diagnosis. No primary tumor was ever found. Cases 1 and 2 were alive and well after 14 and 24 months, respectively, when they were lost to FU. Case 3 presented a recurrent tumor in the same inguinal site after 6 months and was still alive 30 months from initial diagnosis when lost to FU. Case 4 had a local nodal recurrence after a month and is alive and well after 36 months. Case 5 is a recent patient (4 months). 3.1.2. Cutaneous MCCs Clinical features of cutaneous MCCs are summarized in Table 3. Patients with cutaneous MCC were 5 women and 7 men, with age ranging from 64 to 89 years (mean, 79.3 years). Tumor sizes ranged from 1 to 6 cm (mean, 2.68 cm). Lesions were located on the face (4 cases); in thigh (3 cases); and in right foot, leg, groin, back, and buttock. 3.1.3. Lung small cell carcinomas The small cell carcinoma (SCC) of the lung included 9 primaries and 3 brain metastases from 5 women and 7 men, ranging in age from 53 to 76 years (mean, 62.75 years).
3.2. Histology, immunohistochemistry, and MCPyV detection 3.2.1. Extracutaneous MCCs All 6 cases showed histologic features consistent with MCC (Fig. 1A, B, and E). CK20 was expressed in all cases (Fig. 1C and F) with positive cells ranging from 10% to 99% in different cases (mean, 61%). All cases were positive for chromogranin (Fig. 1D); cell positivity ranged from 20% to 99% (mean, 63.75%). All but 1 case (case 1) were positive for synaptophysin (mean percentage of positive cells was 88.25%; range, 75%-99%); all cases were negative for TTF-1 (thyroid transcription factor-1) (Table 4). MCPyV DNA was detected in all 5 cases of lymph node primary MCC and in the parotid gland MCC case (Fig. 2 and Table 4).
Viral DNA as sequenced corresponded to that deposited in the NCBI (National Center for Biotechnology Information) database for MCPyV DNA. 3.2.2. Cutaneous MCCs Histologically, all cases were consistent with typical features of MCC. At immunohistochemistry, all 12 cases stained for CK20 with percentages of positive cells ranging from 25% to 99% (mean, 86.3%). Chromogranin positivity ranged from 10% to 99% (mean, 87.1%). Of 12 cases, 11 were positive for synaptophysin (range, 70%-99%; mean, 83.7% of positive cells). All cases were negative for TTF-1 (Table 4). In all cases of cutaneous MCCs, the presence of MCPyV DNA was observed (Fig. 2B and E and Table 4). 3.2.3. Lung SCCs All cases of SCCs of the lung were negative for CK20 and positive for synaptophysin. Of 12, 7 (58.3%) were positive for chromogranin, and 6 (50%) of 12, for TTF-1. All cases of SCC of the lung, either primary or metastatic, were negative for MCPyV (Fig. 2C).
4. Discussion MCC is a malignant primary cutaneous neoplasm showing neuroendocrine differentiation [23] that shows characteristic morphological and immunohistochemical features. Tumors sharing the same features have been reported in other sites such as lymph nodes [20,21], parotid gland [16,17], oral cavity [18], breast [14,15], and vagina [19]. Trisomy of chromosome 6, the most frequent cytogenetic alteration reported in 47% of cutaneous MCCs, has been also detected in 50% of nodal MCCs [21]. Whether extracutaneous MCCs are true primary tumors rather than metastases from regressed skin lesions is still a matter of debate [20,24]. Similarly to all cases of MCC located outside the skin, the 6
Table 3
Clinical features of cutaneous MCCs
Case
MCC site
Sex
Age (y)
Tumor size (cm)
7 8 9 10 11 12 13 14 15 16 17 18
Cheek (L) Chin (R) Thigh (R) Nose Foot (R) Thigh (L) Thigh (R) Dorsum Leg (R) Chin Abdomen Buttock (R)
F F M M F M M F M F M M
81 89 67 88 88 81 64 77 74 81 70 86
1.7 1.8 NE 3 2.3 5 3 6 1 1.5 2 3.8
Abbreviations: F, female; M, male; NE, not evaluable.
Extracutaneous MCCs polyomavirus DNA
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Fig. 1 A, Primary MCC of lymph node (case 1, H&E). B, Characteristic round nuclei in MCC of lymph node (case 5, H&E). C, CK20 positivity in primary MCC of lymph node (case 1). D, Chromogranin positivity in primary MCC of lymph node (case 5). E to F, H&E and CK20 positivity in primary MCC of parotid gland (case 6).
extracutaneous MCC cases reported here were not associated with a primary tumor of the skin, and no signs of regressed lesion were found despite extensive investigation. Care was taken to exclude any primary neoplasm located elsewhere. Metastases from pulmonary SCC can simulate MCC especially when located in lymph nodes, and the differential diagnosis is mainly based on clinical and immunohistochemical grounds. CK20 is a consistent MCC marker expressed in 100% of cases [25] especially when typical paranuclear dots are present but is occasionally expressed in cases of both pulmonary and extrapulmonary SCCs [4,25]. On the contrary, TTF-1 stains up to 90% of pulmonary SCCs [26,27] and 42% of extrapulmonary SCCs [25], whereas
MCCs are all virtually negative [28]. All MCC cases selected (cases 1-18), including primary extracutaneous and cutaneous MCCs, in addition to sharing typical histologic features of MCC [3], were positive for CK20 and negative for TTF-1, and 15 (83.3%) of 18 showed typical keratin-positive paranuclear dots. On the contrary, all cases of pulmonary SCC expressed TTF-1, whereas CK20 was negative highlighting the immunohistochemical differences between the 2 groups of tumors. It has been demonstrated that cutaneous MCCs are positive for a specific type of polyomavirus, named MCPyV [7]. All extracutaneous MCCs as well as all skin MCCs in this series showed presence of MCPyV. On the contrary,
984 Table 4 MCCs
D. de Biase et al. Molecular and immunohistochemical results of Case MCPyV CK20 Synapto CHR TTF(%) (%) (%) 1
Extracutaneous 1 2 3 4 5 6 Total Cutaneous 7 8 9 10 11 12 13 14 15 16 17 18 Total
√ √ √ √ √ √ 100% √ √ √ √ √ √ √ √ √ √ √ √ 100%
99 10 99 60 20 65 61 25 99 99 99 50 80 90 90 90 85 90 90 86.3
– 75 99 80 99 99 88.25 80 99 99 85 – 80 99 70 70 70 99 85 83.7
95 20 70 60 99 30 63.75 40 99 90 85 99 99 99 99 90 85 10 85 87.1
– – – – – – / – – – – – – – – – – – – /
Abbreviations: MCPyV, MCPyV DNA detected by PCR; CK, cytokeratin; Synapto, synaptophysin; CHR, chromogranin.
all SCCs analyzed here were consistently negative. These features are in keeping with the data of the literature where MCPyV DNA was not detected in SCC [8], whereas in MCC, it has been found in a range of 40% to 100% of cases [8-13]. The wide range of positivity for MCPyV DNA is probably due mainly to methodological artifacts. In fact, primers that were used in several reports lead to long amplicons (178-440 bp). The length of the latter might not
always be effective in revealing the presence of viral DNA in FFPE tissues because DNA in formalin-fixed tissues is frequently fragmented [11]. In the present work, we have used primers amplifying short amplicons that are more sensitive for revealing fragments of DNA. In this series, only 4 cases of MCC were located in sun-exposed areas. It has been demonstrated that lowest incidence of MCPyV is seen in these same areas, whereas the highest is seen in MCC from other areas [29]. This would also be in keeping with the high incidence of MCPyV in all of our cases, which were mostly from non–sun-exposed areas, including the extracutaneous cases. The association of viruses with neoplasms is well documented including hepatitis B and C viruses in hepatocellular carcinoma, Epstein-Barr virus in nasopharyngeal carcinoma, certain strains of human papillomavirus in cervical cancer, and Kaposi sarcoma-associated herpesvirus in Kaposi sarcoma. MCPyV in MCC might be an additional viral strain to include in the above list, and in addition, it has been increasingly demonstrated in other tumors such as squamous cell carcinomas [30]. As MCPyV can be found in the skin adjacent to the latter tumor [30], it seems that MCPyV is not confined to MCC only, but it can also affect primarily the epidermis. To the best of our knowledge, this is the first report concerning the presence of MCPyV DNA in primary extracutaneous MCC. Therefore, it appears that extracutaneous MCCs are similar to MCCs of the skin not only from morphological, immunohistochemical, and cytogenetic point of view but also because both show MCPyV DNA. All these features probably indicate that MCC is a multiorgan neoplasm not confined only to the skin. It also seems possible that MCPyV might play a key role in MCC development.
Fig. 2 Agarose gels showing MCPyV positivity in primary MCCs of lymph node and parotid gland (A) and skin (B). C, SCCs were negative for MCPyV DNA. Multiplex primers set used as DNA quality control in primary MCCs of LN and parotid gland (cases 1-6) (D) and of skin (cases 7-18) (E). MW indicates molecular weight (100, 200, 300, 400 bp); LN, lymph node; DNA ctrl, β2-microglobulin gene used as amplification control.
Extracutaneous MCCs polyomavirus DNA
Acknowledgments D.dB. was the recipient of Vanini-Cavagnino grant from the Centro Interdipartimentale di Ricerca sul Cancro “G. Prodi,” University of Bologna.
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